Characterization of proteins involved in Bacillus subtilis spore formation and germination Bidisha Barat Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biological Sciences David L. Popham, Chair Clayton C. Caswell Birgit E. Scharf Ann M. Stevens April 28, 2020 Blacksburg, Virginia Keywords: Bacillus subtilis, spore, germination Copyright CC BY-NC-SA 2020, Bidisha Barat
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Characterization of proteins involved in Bacillus subtilis spore formation and
germination
Bidisha Barat
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy In
Biological Sciences
David L. Popham, Chair Clayton C. Caswell
Birgit E. Scharf Ann M. Stevens
April 28, 2020 Blacksburg, Virginia
Keywords: Bacillus subtilis, spore, germination
Copyright CC BY-NC-SA 2020, Bidisha Barat
Characterization of proteins involved in Bacillus subtilis spore formation and
germination
Bidisha Barat
ABSTRACT
Members of the Bacillus genus, when faced with unfavorable environmental conditions
such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the
formation of an endospore. In some instances, the spore serves as the infectious agent of an
associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax.
Spores are resistant to a variety of unfavorable environmental conditions including traditional
decontamination techniques. Spore resistance is due to the formation of specialized structures
that contribute to spore dormancy through several mechanisms, including maintenance of the
dehydrated state of the spore core. Spore germination is a rapid process resulting in the
irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative
outgrowing bacterium. The exact mechanism by which individual proteins function in the
germination pathway remains unknown. In this study, we have focused on the roles of putative
ion transporters and other germination-active proteins in affecting spore formation and
germination.
Metal ions can activate enzymes during the sporulation process and/or be factors in spore
resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA,
YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These
proteins may play roles in the accumulation of ions during sporulation and/or the release of ions
during germination. Multiple mutants altered in the putative ion transporter genes were
generated, and the effects of these mutations were analyzed. All strains containing a yloB
deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase
dark during microscopy, indicating the formation of unstable spores. Studies were conducted to
quantify the amounts of individual ions in phase-bright spores using atomic emission
spectroscopy and to analyze the rate at which ions are released from germinating spores. The
transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the
yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive,
and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is
nonfunctional.
To better understand the underlying mechanisms of germination, a high-throughput
genetic screening method called transposon sequencing was used. This analysis identified genes
that had not been previously implicated in germination. To investigate their functions, a number
of functional assays of all the Ger mutant strains were performed that indicated a delay in stage
I of germination. The mutant strains showed significant reduction in germination efficiency with
L-valine: about 50% of the population failed to initiate germination suggesting a defect in the
GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion
followed by quantitative western blot analyses to determine abundance of GerA in mutant
strains. The mutants were classified based upon normal or decreased gerA transcription and
normal or reduced GerA protein. Further work involves understanding the functions of the
identified genes and their correlation to the GerA receptor.
Insight into ion transporters of spore-forming bacteria and understanding the
germination apparatus may lead to promising new applications, detection methods, or
therapeutics for spores, and may allow the development of better spore decontamination
procedures.
Characterization of proteins involved in Bacillus subtilis spore formation and
germination
Bidisha Barat
GENERAL AUDIENCE ABSTRACT
Bacillus subtilis is an ubiquitous bacterium that is capable of forming endospores when
faced with unfavorable environmental conditions. Spores are highly resistant to heat, radiation,
lack of nutrients, desiccation and oxygen deprivation. They lack metabolism, which effectively
keeps them in a state of suspended animation until germinated. They may remain stable and
viable in this state for extremely long periods of time. Several important pathogenic bacteria are
spore formers. This leads to difficulty in their environmental eradication and the treatment of
patients. Germination allows spores to resume metabolism and reestablish a vegetative state.
Certain key molecules activate the germination process. Each species of spore-forming bacteria
has a specific set of these molecules called germinants that will enable the spore to exit its
dormant state. The work presented focuses on the understanding of the germination apparatus
of Bacillus subtilis, which may provide a model to understand the germination of other spore
formers and help to improve methods of decontamination.
vi
DEDICATION
This dissertation is dedicated to my tremendously supportive and loving parents, Vaswati and
Indranil Barat who have given me invaluable educational opportunities and for their endless
motivation. Thank you for believing in me and being there for my successes, my failures and
always encouraging me to strive for excellence.
vii
ACKNOWLEDGEMENTS
First, I would like to thank my advisor, Dr. David L. Popham and Dr. Birgit E. Scharf for
interviewing me via Skype during the summer of 2014 and giving me the opportunity to come to
USA and pursue my doctoral studies at Virginia Tech.
Dr. Popham, I cannot thank you enough for your patience and all the advice and
knowledge that you have shared with me over the years. I really appreciate your open-door
policy and your readiness to answer all my doubts during the countless number of times that I
have walked in, fretting over an experiment. I have always wondered how you juggle so many
things with ease, from research, writing grants, teaching to helping us with prelim practice,
feedback on our manuscripts, presentations and always replying promptly to our emails. You
have taught me to troubleshoot wisely, draw clear pictures to understand the experiment in
depth and develop as an independent scientist and I could not have asked for a better mentor.
I would also like to thank my other committee members, Dr. Ann M. Stevens, Dr. Birgit E.
Scharf and Dr. Clay C. Caswell for all your advice and support during my graduate studies. Your
words of encouragement and challenging questions always kept me motivated.
To all the past and present members of the Popham lab, all of you have made this
experience a little less daunting and a lot more fun. Dr. Yan Chen, although I never had the
opportunity to meet you, thank you for laying the foundation for my first project in the lab. Sean
Mury, thank you for welcoming me so easily and making the lab so lively! I will always cherish
our weekly lunch buffets, conversations about India and your experiences along with all your lab
hacks!
viii
Dr. Cameron V. Sayer, thank you for teaching me how to survive as a graduate student!
You have taught me how to be calm and not stress over a failed experiment and thank you for
always answering the numerous questions that I asked you! You were an amazing colleague and
friend and I am thrilled for the exciting research life you have ahead.
Matthew Flores, you have made the past year in the lab highly enjoyable! You are wise
beyond your years and thank you for having both science and non-science related conversations
with me! I wish you all the best for the rest of your graduate career and envision a great future
for you.
To all the undergraduate researchers, thank you for giving me the opportunity to be a
mentor and learning together. Isabelle Wal, thank you for being my first undergraduate mentee
and keeping the conversations alive!
To the rest of my friends and colleagues of the Microbiology program and teaching labs,
thank you for making graduate school fun. You are all destined for great things. A big thank you
to Holly Bartholomew, Dr. Manisha Shrestha, and Dr. Katie M. Broadway for the brunch sessions
and for being a great support system.
A special thank you to Kinanka Ghosh for always being there and listening to my rantings.
Thank you for your words of encouragement that got me through my moments of self-doubt.
A huge thank you to my friends in Blacksburg who made weekends fun and became my
family away from home. Last but not the least, thank you to all my friends and family who
supported me throughout this journey from afar.
ix
TABLE OF CONTENTS
ABSTRACT ii
GENERAL ABSTRACT v
DEDICATION vi
ACKNOWLEDGEMENTS vii
LIST OF FIGURES xiii
LIST OF TABLES xv
CHAPTER 1: Introduction and Literature Review 1
Bacillus subtilis sporulation 2
Spore structure 4
Gene regulation during sporulation 5
Spore germination 6
Ions in the spore core 9
Ca2+ 10
Mn2+ 11
K+ 12
Mg2+ 12
Cu2+, Fe3+, Zn2+ 13
Ion release during germination 13
Ion transporters in sporulation and germination 14 Transposon sequencing (Tn-seq) 17 Study objectives 17 References 21
CHAPTER 2: Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores 30
Attributions 31
Abstract 32 Importance 33
x
Introduction 34
Results 36
Proteins identified in dormant spore membrane preparations 36
Proteins identified in germinated spore membrane preparations 36
Novel membrane proteins identified in spore membrane fractions 37
Membrane proteins under control of sporulation-specific sigma factors 38
Similarities between the spore membrane proteomes in the two Bacillus species 38
Membrane protein changes during spore germination 39
Growth and sporulation of strains lacking putative ion transporters present in spore membranes 40
Quantification of metal ions in cellular compartments during sporulation 42
Germination rate and release of ions during germination 43
Discussion 44
Materials and Methods Spore preparation 49
Preparation of spore membrane fractions 50
SDS-PAGE, Trypsin digestion, and peptide fractionation 51
Mass spectrometry and protein identification 52 Generation of mutants lacking putative ion transporter genes 54
Analysis of sporulation properties 55 Separation of mother cell and forespore fractions 55
Quantification of ions using atomic emission spectroscopy 55
Acknowledgements 57
References 58
CHAPTER 3: Identification of L-valine-Initiated-Germination-Active Genes in Bacillus subtilis using Tn-seq 76
Attributions 77
Abstract 78
Introduction 79
xi
Material and Methods 81
Strain constructions 81
Spore preparation 82
Sequencing of Tn insertion sites 82
Germination assays 83
Assay of gerA transcription 85
Western blotting 85
Results 86
Identification of mutant strains with slowed or reduced germination 86
Characterization of germination mutant strains 88
Expression of the GerA receptor in mutant strains 90
Discussion 91
Acknowledgements 96 References 97
CHAPTER 4: Role of YlbC and YlbB in GerA-mediated spore germination in Bacillus subtilis 112
Attributions 113
Abstract 114
Introduction 115
Results 117
Germination rates of deletion strains 117
Microscopic analysis of germination 118
Polar effects of ylbB mutations 118
Expression of the GerA receptor 119
Overexpression of ylbB 120
Materials and Methods 120
Strain constructions 122
Spore preparation 122
Germination assays 122
Assay of gerA and ylbC transcription 122 Western blot analysis 123 Microscopy 123
xii
Discussion
124
References 127
CHAPTER 5: Final Discussion 141
References 147
APPENDIX A: Supplementary Materials for Chapter 2 148
APPENDIX B: Supplementary Materials for Chapter 3 152
APPENDIX C: Supplementary Materials for Chapter 4 168
References 172
xiii
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Spore structure 25
Figure 1.2 Bacillus subtilis sporulation 26
Figure 1.3 Cascade of sigma factors 27
Figure 1.4 Spore germination 28
Figure 1.5 Organization of GerA receptor 29
CHAPTER 2
Figure 2.1 Predicted membrane-spanning domains of B. anthracis and B. subtilis spore membrane proteins 62
Figure 2.2 Mutant strains lacking yloB produce many phase-dark spores 63
Figure 2.3 Ca2+ content of forespore and mother cell in sporulating cells 64
Figure 2.4 Ion contents of purified phase-bright spores 65
Figure 2.5 Germination of purified phase-bright spores 66
Figure 2.6 Release of ions by germinating spores 67
CHAPTER 3
Figure 3.1 Germination rates of B. subtilis strains 102
Figure 3.2 Phase-contrast microscopy of germinating B. subtilis spore populations 103
Figure 3.3 Expression of a gerA-lacZ transcriptional fusion 104
Figure 3.4 GerAC is reduced in the spores of several B. subtilis mutant strains 105
xiv
CHAPTER 4
Figure 4.1 Germination rates of B. subtilis strains 130
Figure 4.2 Phase - contrast microscopy of germinating B. subtilis spore populations 131
Figure 4.3 Expression of ylbC-lacZ transcriptional fusion 132 Figure 4.4 Expression of gerA-lacZ transcriptional fusion 133
Figure 4.5 Quantitative anti-GerAC western blots of ylbB, ylbB::kan and yhcV::kan (A-C) with graphical representation 134
Figure 4.6 Germination rates of B. subtilis strains with ylbB overexpression 135 Figure 4.7 Hypothetical model A 136 Figure 4.8 Hypothetical model B 137
xv
LIST OF TABLES
CHAPTER 2
Table 2.1 B. anthracis and B. subtilis spore germination proteins identified in spore membrane proteomes 68
Table 2.2 Proteins detected in both Bacillus species spore membrane proteomes 69
Table 2.3 Validation of membrane protein quantification 71
Table 2.4 Changes in Bacillus spore membrane protein detection following germination 72
Table 2.5 Production of heat resistant spores of B. subtilis strains lacking putative ion transporters 74
Table 2.6 Production of heat resistant spores by B. subtilis ion transporter mutants with different Ca2+ concentrations 75
CHAPTER 3
Table 3.1 Genes in which Tn insertions altered germination 106
Table 3.2 Genes without previously known germination role identified by Tn-Seq and in spore membrane proteome 108
Table 3.3 Phenotypic properties of B. subtilis strains 109
Table 3.4 Response of B. subtilis strains to varied germinants 110
Table 3.5 Overexpression of gerA suppresses germination defect of multiple mutants 111
CHAPTER 4
Table 4.1 Response of B. subtilis strains to 10 mM L-valine 138
Table 4.2 ylbC-lacZ expression of sporulating cells 139
Table 4.3 Germination response of B. subtilis strains to 10mM L-valine 140
1
Chapter 1
Introduction and Literature Review
2
Bacillus subtilis sporulation:
Bacillus subtilis is a Gram-positive, aerobic, rod-shaped, and ubiquitous bacterium with a
doubling time of about 20 minutes when grown in rich medium at 37°C. The best genetically
characterized Gram-positive bacterium, it is used as model organism due to its natural
competency and easy genetic manipulation. B. subtilis is often used as the organism of choice for
industrial and pharmaceutical applications. This can be attributed to its high-level secreted
enzyme production, genetic engineering amenability and large-scale fermentation properties
without any production of toxic by-products.
Many members of the Bacillus genus and closely related genera such as Clostridium are
capable of forming endospores when nutrients in the environment are depleted. Under normal
conditions, these organisms grow vegetatively through symmetric binary fission of a cell resulting
in the production of two identical daughter cells. However, when the environment is not
conducive to growth, these organisms undergo sporulation wherein an asymmetric cell division
results in the formation of a larger, mother cell and a smaller developing endospore which is
eventually released and can be easily dispersed by wind [1, 2]. The entire sporulation event is a
well-coordinated process that takes approximately 6-8 hours and requires a large energy
investment (Figure 1.2). Spores of these species are dormant with little or no metabolic activity
and are resistant to various stress factors such as heat, UV radiation and toxic chemicals [2].
Transcription factors called sigma factors control the process of sporulation by undergoing a
coordinated sequence of activation and inactivation [4]. Electron microscopy can be used to
characterize the various stages (0-VII) of morphological changes during spore formation [5, 6].
3
Upon starvation, stage I of sporulation is characterized by the development of the axial
filament. The axial filament is formed by the condensation and aligning of the two copies of the
chromosome, immediately following DNA replication, along the long axis of the cell [7] [8]. Stage
II is characterized by an asymmetric septum formation near a polar position. The septum splits
the cell into a larger mother and smaller forespore component, each of which receives a copy of
the chromosome [3, 9]. The smaller developing forespore contains only about 30% of a
chromosome during this time; SpoIIIE, which is a DNA translocase, pumps in the remaining
chromosome [3-6]. Following the formation of the septum, the forespore is engulfed by the
larger mother cell and becomes enclosed by two membranes of opposing polarity during stage
III [1, 7, 10]. One membrane is attained from the mother cell and the other one is acquired from
the forespore. Once engulfed, the forespore forms a free protoplast inside the mother cell [9].
During stage IV, the spore cortex peptidoglycan is then deposited in between the inner and outer
membrane of the engulfed forespore [11]. The germ-cell wall is also synthesized during stage IV
[1, 3, 5]. Stage V is characterized by the creation of proteinaceous spore coats around the spore
cortex and relative dehydration of the spore core, which confers some resistance properties to
the developing spore [1, 3, 8]. In certain species, the exosporium is also synthesized outside the
coats during this stage. Stage VI is the maturation stage wherein the spore attains its remaining
resistance properties and dehydration, and develops refractility whilst inside the mother cell [3,
12]. Dipicolinic acid (DPA[pyridine-2,6-dicarboxylic acid]) synthesized within the mother cell is
transported and accumulates within the forespore [2]. DPA chelates calcium ions, leading to a
very high concentration of Ca2+-DPA within the spore core during stages V and VI of sporulation
[2]. DPA is not found in vegetative cells and accumulates late in sporulation making up 5-15% of
the total dry weight of the spore [12, 13]. Spores lacking DPA are extremely unstable and
4
germinate spontaneously [2]. Lastly, during stage VII, the mother cell is lysed, releasing the
mature spore into the environment, thereby completing the process [1, 3, 14] . When suitable
environmental conditions return, the dormant spore undergoes germination with a resumption
of metabolic activities [1]. Mature spores that are metabolically dormant contain very low
amounts of cellular high energy compounds such as ATP or NADH but their quantities rise once
germination is initiated [15, 16] .
Spore structure:
The structure of the spore is unique and differs considerably from that of a vegetative cell
(Figure 1.1). The outside of some spores is covered by a large, loose fitting structure termed the
exosporium. This is composed of proteins and glycoproteins but its function is not well defined
[17]. The exosporium may play a role in spore-host interactions based on the pathogenic nature
of some exosporium-containing spores [9]. Below the exosporium lies a multilayered structure
composed of a variety of proteins termed the spore coat. The spore coat provides no resistance
to heat or radiation, however, spores lacking spore coats are more sensitive to lysozyme and
certain chemicals such as hydrogen peroxide [9]. Underneath the spore coat is the outer
membrane of the spore which is important during spore formation but its exact role in the
dormant spore is unknown as it does not have a considerable protective role against heat,
radiation or chemicals and likely does not serve as a significant permeability barrier [3, 18] . Below
this membrane is the spore cortex which consists of peptidoglycan [9]. A notable difference is
the presence of muramic acid lactam (MAL) in the spore cortex peptidoglycan which is absent in
the peptidoglycan of a vegetative cell [12]. MAL is important as it is recognized by lytic enzymes
that degrade the cortex during spore germination. Inside the cortex lies the germ cell wall
peptidoglycan, which does not contain MAL and thus remains intact from the hydrolyzing action
5
of germination lytic enzymes [19]. The germ cell wall becomes the cell wall of an outgrowing
spore resulting in the generation of rod-shaped vegetative cells [11]. The inner membrane is
located below the germ cell wall [20]. It serves as a barrier that is impermeable to small
(molecular weight >100 g/mol) hydrophilic molecules [21, 22] and contains the germinant
receptor proteins [21]. The spore core, containing DNA, RNA, DPA, cations and various enzymes
is surrounded by the inner membrane. Due to the dehydrated state of the spore core, it is likely
that there is no enzymatic activity in the spore core [2, 12].
Gene regulation during sporulation:
Upon sensing unfavorable conditions, the change from vegetative growth to sporulation
involves a two-component signal transduction system, that begins with the phosphorylation of
the master regulator of sporulation, Spo0A, via a phosphorelay system [1]. Sensor kinases KinA,
KinB and KinC undergo autophosphorylation using ATP and the phosphate is transferred to
Spo0F, a response regulator. Spo0F-P transfers the phosphate to Spo0B and eventually to Spo0A.
Spo0A-P regulates the transcription of nearly 500 genes including the spoIIA operon encoding σF,
the spoIIG operon encoding σE and the spoIIE gene involved in activation of σF in the forespore
[1, 4]. The mother cell and the developing forespore undergo separate developmental routes
upon initiation of sporulation involving five distinct RNA polymerase sigma (σ) factors [1] that
undergo a coordinated sequence of activation and inactivation as shown in Figure 1.3.
At the onset of sporulation, gene expression is controlled by σH which is activated by
Spo0A-P [4, 23, 24]. σE and σF are synthesized prior to septation but remain inactive until after
septum formation and then control the mother cell and forespore gene expression, respectively.
An anti-sigma factor, SpoIIAB is responsible for maintaining σF in an inactive state although σF is
produced in an active state. σE is produced as a pro-sigma factor and needs proteolytic processing
6
for its activity, signaled by σF-directed expression of spoIIR [4, 23, 24]. Genes transcribed by σE
control the prevention of further asymmetric division, engulfment of the forespore, spore coat
assembly initiation, and σK production. Activity of σE is also linked to the engulfment of the
forespore. Other sporulation-specific sigma factors that are involved in the transcription of late
stage sporulation genes are σG and σK, which are forespore-specific and mother cell-specific
respectively. σK is produced as a pro-sigma factor (pro-σK). σG is initially held inactive by SpoIIAB
and is activated by SpoIIIA upon completion of engulfment. Genes transcribed by σG function in
pairing gene expression of late forespore and mother cell and preparing the spore for
germination. Pro-σK is processed to the active state by a protein encoded by spoIVB, which is
transcribed post activation of σG. σK transcribes genes involved in spore coat formation and spore
maturation [1, 4, 23, 24].
Spore germination:
When favorable conditions return to the environment, nutrients that are termed
germinants are sensed by the spores resulting in activation of germination and return to
vegetative growth [15, 16, 25, 26]. Germinants include nutrients such as L-alanine, sugars
(glucose and fructose), purine nucleosides or a combination. Most of the spores will germinate
within 5 minutes of exposure to germinants [26, 27]. In addition, spores will also germinate in
response to non-nutrients such as calcium dipicolinic acid, lysozyme, high pressure and salts,
although it is likely that steps of the germination pathway may be bypassed if germination is
triggered in this fashion [26, 27]. For germination to occur, the germinant must travel across the
spore coat and cortex to contact germination receptors (GR) located within the inner membrane
[16, 22].
7
Once germinants have contacted the GR receptors, Stage I of spore germination is
initiated, which involves release of certain core components of the spore such as H+, monovalent
cations, DPA and its bound divalent cations, causing water to rush in and partially hydrate the
core [15] [36]. During the expulsion of H+ from the core, the pH of the core increases from 6.5 to
7.7 which is essential for resumption of metabolism once the spore is rehydrated enough for
enzyme action [28]. During Stage II, the spore cortex is then broken down by the germination-
specific lytic enzymes (GSLEs) which causes the spore core to swell by further water uptake, germ
cell wall expansion and return to a completely hydrated state (Figure 1.4). Small acid-soluble
proteins (SASP), bound to the spore DNA for protection from stress factors, are also degraded
[14, 15] . At this point the spore is no longer dormant and has lost the majority of its resistance
characteristics [26, 27]. Eventually, there is spore outgrowth coupled with resumption of
metabolic activity. Under a light microscope, germination can be observed as a change from a
phase bright appearance to a phase dark appearance. This can also be observed as a decrease in
the optical density of spore cultures at 600 nm [27].
B. subtilis spores have three known functional germinant receptors in the inner
membrane that sense certain nutrient compounds and activates spore germination. These
germinant receptors may be functioning as ion channels themselves or their activation may
provide a signal to other proteins involved in germination [16][29]. The germinant receptor
proteins generally consist of three subunits (A, B and C), each encoded in a tricistronic operon
(gerA, gerB, and gerK operons), which are expressed in the forespore during late stage of
sporulation under the control of σG [29]. The gerA operon encodes three proteins, GerAA with
probable hydrophilic and hydrophobic domains, GerAB which is a putative integral membrane
protein and part of the single-component polyamine/amino acid/organocation transporter
8
superfamily and GerAC which is a predicted lipoprotein is associated with the forespore
membrane via a lipid anchor (Figure 1.5) [21, 30]. The B subunit is likely involved in germinant
binding [14]. The GerA nutrient receptor is the most abundant in B. subtilis spores and responds
to L-valine and L-alanine, while the GerB (GerBA, GerBB and GerBC) and GerK (GerKA, GerKB and
GerKC) nutrient receptors cooperate to respond to a combination of L-asparagine, D-glucose, D-
fructose and K+ ions (AGFK) [31]. Each B. subtilis spore contains nearly 2500 total germinant
receptors with ~1100 molecules of GerAA and GerAC and ~700 molecules of GerBC and GerKA
subunits [32]. Spore germination can potentially be inhibited by deficiencies in germinant
receptors and accompanying proteins such as Ca2+-dipicolinic acid channels and lytic enzymes
[33]. Spores fail to germinate with nutrients upon deletion of all three germinant receptors, but
they maintain an unknown mechanism of slow spontaneous germination. There may also be
some interaction between the germinant receptors and SpoVAD (~6,500 molecules per spore)
and SpoVAE, that are involved in releasing Ca2+-DPA during germination [34]. The signal
transduction mechanism from the germinant receptors to other spore proteins to begin
germination is not completely understood [35]. In Bacillus species, GerD is another inner
membrane protein involved in germination though it shares no homology with any known
protein, nor does it bear resemblance to Ger receptor proteins. Recent work indicates that GerD
is a ~20 kDa protein that may be a lipoprotein with a diacylglycerol associated to a particular
cysteine residue [36]. The transcription of gerD is also under the control of σG [37]. Each B. subtilis
spore contains ~3500 molecules of GerD [32]. In B. subtilis, GerD may be involved in signal
transduction from the nutrient receptors to downstream germination components, by the
colocalization of GerD and the germinant receptors within a cluster termed the germinosome in
the spore’s inner membrane [35, 38].
9
There is variation in the substrate specificity, affinity, and numbers of germinant
receptors between different Bacillus species and strains. There is some evidence indicating that
individual germinant receptors and subunits interact with each other and show synergy [34].
Single amino acid changes can eliminate the dependence of GerB on GerK [39]. Studies have
shown that overexpression of any individual GR, such as GerA, increases the rate of spore
germination with L-alanine or L-valine and decreases rates of germinations via GerB and GerK
[31, 40]. These results suggest that the overexpression of one germinant receptor may lead to a
decrease in the level of other germinant receptors resulting in decreased rate of germination.
Another explanation may be that all the germinant receptors are competing for a low
concentration of a signaling molecule that is present downstream in the germination pathway,
thus the overexpressed germinant receptor interferes with the accessibility of the signaling
molecule [34, 40, 41].
Ions in the spore core:
Metal ions potentially act as catalytic activators of some enzymes essential for the
sporulation process [42]. The mineral composition of the medium influences the variability of
ions incorporated into spores. Usually divalent cations present in the spore core are associated
with DPA and there may be some additional minerals in the core. Calcium (Ca2+), manganese
(Mn2+), zinc (Zn2+), nickel (Ni2+) , iron (Fe3+) and copper (Cu2+) ions accumulate in developing
spores and affect the heat resistance of the spores [43][44]. The higher the spore levels of
divalent cations, the more wet heat resistant the spore. Resistance in spores imparted by cations
are in the following order: Ca2+ > Mn2+ > Mg2+> K+> Na+ > H+ [22]. Apart from affecting the amount
of water in the spore core, it is not evident why the spore wet heat resistance is affected by
mineralization of the spore core and the characteristic features of the mineral ions [22]. It may
10
be due to the interplay of spore macromolecules with a DPA-metal ion matrix that pervades the
spore core [45].
Ca2+
The most abundant cation in Bacillus spores is Ca2+ with the formation of calcium
dipicolinate (Ca2+-DPA) in the core. As spores mature, Ca2+ is accumulated and is considered to
be a prerequisite in the production of heat-resistant and refractile spores. A transporter possibly
accumulates Ca2+ in the cytosol of the mother cell, followed by facilitated diffusion of Ca2+ into
the core of the forespore, where the level of free Ca2+ is low due to the Ca2+-DPA complex
formation within the core [46, 47]. A Ca2+/H+ antiporter has been characterized in B. subtilis based
on Ca2+/H+ exchange driven by an electrical gradient over the plasma membrane in B. subtilis and
B. megaterium [48]. Ca2+ content in spores can be reduced at higher concentrations of Zn2+, Mn2+
and Ni2+ due to competition for Ca2+ sites in the spores. However, the competing divalent ions do
not confer the same degree of heat resistance in spores as they are incorporated into the spores
with low efficiency [49].
During Stage II of germination, the hydrolysis of the spore cortex is accomplished through
the activity of the GLSEs that specifically recognize muramic-δ-lactam and may require Ca2+-DPA.
The two main categories of GSLEs are the spore cortex-lytic enzymes (SCLE), which degrade the
spore cortex peptidoglycan, and the cortical fragment-lytic enzymes (CFLE) that further break
down the partly degraded peptidoglycan [16, 29]. In B. subtilis, SleB, which is a lytic
transglycosylase, and CwlJ are involved in cortex degradation. SleB is present in the region
between the inner membrane and coat while CwlJ is found in the inner spore coats near the
cortex. The activity of both SleB and CwlJ could involve the presence of Ca2+ [16, 49, 50]. After
the release of Ca2+-DPA from the spore core, there is partial rehydration of core as well as
11
deformation of the cortex [16, 29]. A suggested mechanism is that the Ca2+ from the released
Ca2+-DPA results in the collapse of a loosely cross-linked peptidoglycan, which causes rehydration
of the core and activation of the GSLEs [50]. It was proposed that SleB acts only on this structural
modification of the cortex [50], however studies by Heffron et al showed that SleB is active on
both intact and fragmented cortex [51]. It has been seen that calcium ions alone cannot trigger
spore germination but Ca2+-DPA can. CwlJ is activated in response to the release of Ca2+-DPA
during spore germination or addition of exogenous Ca2+-DPA as a germinant [50].
B. cereus spores germinate poorly in response to nutrient germinants when they have
lower DPA content, which is linked to the calcium content [52, 53]. Also, rate of germination of
spores with low Ca2+-DPA is enhanced in response to alanine by the exogenous addition of
calcium along with the uptake of these calcium ions while germination is inhibited by calcium
channel blockers [50, 52]. In Clostridium perfringens, a GSLE known as SleM also requires divalent
cations such as Ca2+ and Mg2+ for its activity [50].
Mn2+
The requirement of Mn2+ for sporulation is critical as it seems to be a co-factor of SpoIIE
serine phosphatase involved in the formation of the polar septum of the spore [54]. A complex
of Mn-superoxide dismutase (MnSOD) increases the resistance of sporulating cells against
oxidative stress [55]. Mn2+, upon forming a complex with DPA provides protection from ionizing
radiation in spores [56]. Dormant spores contain 3-phosphoglyceric acid (3PGA) as an essential
energy reserve comprising 0.15-0.3% of the spore dry weight. The accumulation of 3PGA late
during sporulation in the developing spore occurs upon inhibition of the enzyme
phosphoglycerate mutase. Phosphoglycerate mutase is extremely pH sensitive and acidification
of the forespore during sporulation results in deactivation of the enzyme [57]. Mn2+ can bind to
12
the catalytically inactive phosphoglycerate mutase and may promote the conversion of inactive
phosphoglycerate mutase as well as induce a conformational change to a catalytically active
state. Phosphoglycerate mutase catalyzes the interconversion of 3PGA and 2 phosphoglyceric
acid, during spore germination which results in the generation of ATP [57]. Henriques et al
provided evidence that MnSOD may be involved in spore coat assembly in B. subtilis by
generating hydrogen peroxide. H2O2 is essential for the o,o-dityrosine cross-linking of CotG, an
outer coat structural protein [58]. During the growth and early sporulation phase in B. subtilis,
Mn2+ is accumulated in an exchangeable and free form that is later converted to a bound form
during the later stages of sporulation [59].
K+
K+ is essential for activation of certain enzyme systems required for protein synthesis as
well as maintaining ribosomal structures in a suitable functional configuration [43]. One of the
first detectable events in spore germination is the release of K+ ions and it is not firmly bound
inside sporulating cells, unlike calcium [14].
Mg2+
Mg2+ is considered to be involved in protein and nucleic acid synthesis and thus essential
for sporulation. Magnesium ions can bind to enzymes and alter their structure as well as generate
magnesium-substrate scaffolds, to which enzymes bind. Mg2+ is involved in protein synthesis
through specific catalytic roles. Magnesium ions are required for activation of enzymes involved
in replication of DNA (topoisomerase II, polymerase I) and transcription and is essential for the
stability of nucleic acids [60]. Mg2+ is also associated with ribosomes and activates amino acids
involved in mRNA attachment to ribosomes [60]. Mg2+ however does not contribute to heat
resistance but rather interferes with the Ca2+-DPA complex and reduces heat resistance. In B.
13
subtilis, the uptake of Mg2+ declines during sporulation probably due to the reduction in the
function of one of the two magnesium transport systems postulated during late sporulation [61].
Cu2+, Fe3+, Zn2+
In Clostridium spores, Cu2+ limits sporulation by reacting with essential macromolecules
that catalyze hydrolytic reactions resulting in the production of free radicals that are toxic to DNA
[62]. Cu2+ forms non-functional metal-protein complexes by interacting with the sulfhydryl
groups of spore proteins resulting in reduction in sporulation [63]. Fe3+ and Zn2+ are considered
to be essential for sporulation in B. megaterium spores, however their exact role has not been
ascertained [43].
Ion release during germination:
Germination of spores begins with a rapid change in the membrane permeability of the
spore coupled with the rapid uptake of water in the protoplast along with the passage of ions
and water across the spore layers and induction of cortex lytic enzymes [64]. Upon interaction
with germinants, germination of spores involves a flux of monovalent cations such as K+, H+, Na+
probably along with the release of anions. Around 80% of the spore’s Na+ and K+ is released
during the early steps of germination driven by the concentration gradient between the inside
and the outside of the spore. This efflux is followed by the reuptake of K+ based on an energy
dependent system [65]. Various possible Na+/H+-K+ antiporters have been identified such as GerN
in B. cereus and B. megaterium [66, 67]. The release of monovalent ions is followed by the release
of Ca2+-DPA complex from the spore core and associated divalent cations through one or more
channels [14, 65] .
14
Ion transporters in sporulation and germination:
The extrusion of sodium is an important detoxification process in bacterial cells as a high
internal concentration of sodium inhibits many metabolic activities. Na+/H+ antiporters export
Na+ in exchange for H+ driven by a proton electrochemical gradient [68]. ShaA is a Na+/H+
antiporter deemed to be involved in sodium extrusion in B. subtilis and is essential for initiation
of sporulation [69]. Disruption of ShaA results in decreased sporulation but has no effect on
vegetative growth under increased concentration of external sodium [69]. The defect in the early
stages of sporulation may result from an effect on posttranscriptional control of σH due to an
increase in the level of internal Na+. At an early stage of sporulation, the expression of both spoOA
and spoVG under the control of σH is affected by addition of NaCl as the level of σH protein
diminishes in the shaA mutant [69]. Under high concentration of internal Na+, σH may be unable
to form the holoenzyme due to unstable association with RNA polymerase [69].
In B. megatarium ATCC 12872, grmA encodes a Na+/H+ antiporter homologue that shares 47%
amino acid identity with a Na+/H+ antiporter of Enterococcus hirae, NapA. GrmA, a member of
the PA-2 family of membrane transport proteins involved in monovalent cation and proton
antiport is considered to be essential for germination in response to all germinants as grmA
mutants failed to release DPA or lose heat resistance [70].
GerN of B. cereus ATCC 10876 and GrmA of Bacillus megaterium share 58% amino acid
identity and NapA of Enterrococcus hirae shares 43% amino acid identity with B. cereus GerN [66,
70] . Mutation of gerN results in a significant defect in inosine- triggered germination. The defect
in germination seems to be at an initial stage before the spore loses heat resistance wherein the
inosine germination receptor may be functionally coupled to GerN. This suggests that germinant
receptors may be dependent on the function of different ion transport proteins. Both Na+/H+
15
antiport as well as Na+/H+-K+ antiport are catalyzed by an electrogenic ATP-dependent GerN
antiporter, the latter being more rapid. The expression of GerN still remains unknown, whether
it is expressed in vegetative cells of B. cereus or is sporulation specific [66, 67] .
B. cereus has a GerT protein that is homologous to GerN and shares 74% amino acid
identity [71]. GerT is expressed during late stages of sporulation and may be involved in Na+ efflux
due to the sensitivity to NaCl and alkali observed during spore outgrowth in the gerT mutant of
B. cereus [71]. Thus, GerT protein may play a significant role in resumption of growth in B. cereus
spores. During germination of B. cereus ATCC 10876, GerT is required for the remaining inosine
germination of a gerN mutant mediated by GerI [71, 72].
In C. perfringens, GerO and GerQ proteins display both structural and sequence homology
to transporters of monovalent cations which are suggested to be involved in Bacillus spore
germination. GerO is suggested to be Na+/H+-K+ antiporter while GerQ may be a putative Na+
transporter. The expression of both GerO and GerQ occurs in the mother cell during C.
perfringens sporulation. In a rich medium, GerO mutants as well as, to a lesser extent, GerQ
mutants show a defect in spore germination but not with the germinant dodecylamine, which
suggests that monovalent cation transporters may be involved in C. perfringens nutrient-
triggered germination of spores [73].
In spore-forming bacteria, transport of calcium and DPA plays an important role in heat
and radiation resistance of spores as well as response to germination-inducing compounds. Gene
expression during the sporulation process is affected by calcium inside the forespore. A
transcriptome approach by Oomes et al., revealed that the transcription of nearly 305 genes are
influenced by calcium level in sporulating B. subtilis cells [74]. Transcription of genes such as
spoVFA and spoVFB that are responsible for the synthesis of DPA, the sps operon involved in
16
spore coat polysaccharide synthesis as well as other germination genes are induced by Ca2+ [74].
The exact mechanism for the transport of calcium in spores has not yet been elucidated. Most
microorganisms use transport mechanisms such as Ca2+/H+ exchangers to move calcium through
secondary transport in the cell using the energy accumulated in the gradient generation of other
ions [75]. During spore formation, the development of calcium transport systems result in
significant quantities of calcium accumulating in spores [13, 43] . In B. subtilis, a putative P-type
Ca2+-transport ATPase is encoded by yloB which shows similarity to the endo(sarco)plasmic
reticulum (SERCA) and PMR1 Ca2+ transporters which are type IIA P-type ion-motive ATPases in
eukaryotes [76]. The YloB protein forms a phosphorylated intermediate that is Ca2+- dependent
and is expressed by sporulating B. subtilis cells only. Neither the production of viable B. subtilis
spores nor the growth of vegetative cells was affected by the mutation of yloB [76]. However, a
yloB mutation reduced spore resistance to heat and resulted in slower germination of B. subtilis
spores. As the mutant cells were capable of accumulating large amounts of calcium, YloB may be
involved in the transport of other cations such as Mn2+ apart from Ca2+ [76].
During sporulation in B. subtilis, the uptake of the 1:1 chelate of DPA and Ca2+ and its
release during germination is mediated by the heptacistronic spoVA operon encoded proteins
[77]. There is structural homology between the SpoVAD crystal structure from B. subtilis and
thiolase-fold containing enzymes that bind to small aromatic molecules. SpoVAD can specifically
bind with similar affinity to both DPA and Ca2+-DPA [77]. Mutations in the putative SpoVAD
binding pockets of DPA weaken its binding capacity for DPA and abolishes DPA uptake by
developing spores [77].
17
Transposon sequencing (Tn-seq):
Tn-seq, a robust and high throughput screen for the discovery of quantitative genetic
interactions in microorganisms through massively parallel sequencing can be used to identify the
plethora of genes that may play roles in spore germination [78]. Previously, genetic studies of
spore germination have focused mainly on germination mutants that were identified using
methods that require a very large change in germination efficiency (>10-fold), while Tn-Seq can
potentially identify genes that produce important but more subtle changes in germination
efficiency on a genome-wide scale.
A transposon mutagenized library, which contains restriction sites in the inverted repeats
flanking the resistance genes, is first subjected to desired test conditions. This is followed by DNA
extraction and digestion of DNA with restriction enzymes resulting in isolation of fragments
containing the entire transposon flanked by unique genomic sequences. Following this, adapters
are ligated to the fragments, which are PCR amplified and submitted for next generation
sequencing. High throughput sequencing reveals the genomic sequences encompassing the
transposon ends and transposon insertion site. All Tn insertions are mapped to the genome,
allowing quantification of the abundance of each insertion within the cell population.
Comparisons are then drawn between the sample sets and controls to determine the identity of
genes selected for under the tested experimental conditions [84].
Study Objectives:
The aim of this research was to characterize proteins involved in B. subtilis spore
formation and spore germination with a focus on metal ion transporters and additional
germination active proteins. There are many unanswered questions about spore germination,
18
specifically with respect to the regulation of signal transduction and the events that take place
immediately after germinant receptor activation.
In a previous study, proteomic analysis of the spore membrane resulted in the
identification of nearly 100 membrane proteins of which nearly thirty proteins had not been
previously identified in spores. Chapter 2 investigates the role of six proteins (ChaA, YcnL, YflS,
YloB, YugS, ZnuA) from this repertoire of newly identified spore membrane proteins that bear
resemblance to components of identified cation transport systems. These proteins could play
roles in accumulating ions during sporulation and/or rapidly releasing ions during germination.
In B. subtilis, ZnuA and its apparent B. anthracis ortholog, have been suggested to be involved in
zinc transport in vegetative cells [79]. ChaA, has been reported to be a Ca2+/H+ antiporter under
the control of a sporulation-specific sigma factor in the forespore, σG [80]. YloB, which is a P-type
Ca2+ transport ATPase, is expressed during sporulation. YloB may play a role in Ca2+ efflux during
spore germination as spores prepared from a yloB deletion mutant show a slower rate of
germination [76]. YcnL and YflS, may be involved in copper, malate and sodium transport [81,
82]. YugS belongs to an uncharacterized protein family 0053 (UPF0053) and may be involved in
divalent ion transport as several proteins from this family are involved in the transport of
magnesium and cobalt in other bacterial species [83].
Previously, the interaction of proteins during spore germination was studied on a small
scale, focusing on specific interactions one at a time. Chapter 3 describes a Tn-Seq approach that
allows analysis of genetic interactions on the genome scale to identify additional genes that may
play a more subtle role in spore germination. A Tn-insertion library was generated in our
laboratory B. subtilis wild type strain, PS832, using a modified magellan6 transposon insertion
library featuring 5.5 x 104 insertions in over 3,114 genes with ≥ 10 unique insertions per gene
19
[84]. The generated transposon library was subjected to germination conditions with L-valine and
separated into two fractions: a dormant or partially germinated spore population and a fully
germinated population, using a sodium ditriazoate gradient. Following the processing,
normalization and mapping of reads to the genome, comparisons were drawn between the
sample sets. A two-fold difference in reads between the partially germinated sample vs the
germinated sample was used to select the genes of interest. This threshold left 61 genes in total.
Known germination proteins and others known to have germination defects such as coat proteins
were excised from the list, leaving a total of 35 genes. From this point the list was cross
referenced against two inner spore membrane proteome data sets [85][86]. Of the 35 genes, 14
genes were detected in the spore inner membrane proteome of one or both studies. The majority
of the 14 genes are largely uncharacterized, some better annotated than others but none have
been previously studied in the context of spore germination. Putative functions of the genes
varied from general stress, lipid metabolism, to completely uncharacterized. The genes we have
identified likely function in a complementary or supplementary role, and thus deletion mutants
may not result in complete abolishment of germination. From our study, it was seen that most
of the mutant phenotypes were consistent with a decrease in GerA receptor function but the
4. Piggot PJ, Losick R. Sporulation Genes and Intercompartmental Regulation.483-517. 5. Kay D, Warren SC. 1968. Sporulation in Bacillus subtilis. Morphological changes.
Biochem J 109:819-24. 6. Ryter A. 1965. Morphologic Study of the Sporulation of Bacillus Subtilis. Ann Inst
Pasteur (Paris) 108:40-60. 7. Stragier P, Losick R. 1996. Molecular genetics of sporulation in Bacillus subtilis. Annu
Rev Genet 30:297-341. 8. Hilbert DW, Piggot PJ. 2004. Compartmentalization of gene expression during Bacillus
subtilis spore formation. Microbiol Mol Biol Rev 68:234-62. 9. Setlow P. 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation,
heat and chemicals. J Appl Microbiol 101:514-25. 10. Setlow P. 1995. Mechanisms for the prevention of damage to DNA in spores of Bacillus
species. Annu Rev Microbiol 49:29-54. 11. Buchanan CE, Henriques AO, Piggot PJ. 1994. Cell wall changes during bacterial
endospore formation, p 167-186. In Ghuysen J-M, Hakenbeck R (ed), Bacterial Cell Wall. Elsevier Science Publishers, New York, NY.
12. Russell AD. 1982. The destruction of bacterial spores. Academic Press, London. 13. Paidhungat M, Setlow B, Driks A, Setlow P. 2000. Characterization of spores of
Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512. 14. Setlow P. 2003. Spore germination. Curr Opin Microbiol 6:550-556. 15. Paidhungat M, Setlow P. Germination and Outgrowth.537-548. 16. Setlow P. 2014. Germination of spores of Bacillus species: what we know and do not
know. J Bacteriol 196:1297-305. 17. Lai EM, Phadke ND, Kachman MT, Giorno R, Vazquez S, Vazquez JA, Maddock JR,
Driks A. 2003. Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J Bacteriol 185:1443-54.
18. Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P. 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64:548-572.
19. Popham DL, Helin J, Costello CE, Setlow P. 1996. Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci USA 93:15405-10.
20. Paidhungat M, Setlow P. 2001. Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183:3982-3990.
21. Hudson KD, Corfe BM, Kemp EH, Feavers IM, Coote PJ, Moir A. 2001. Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol 183:4317-4322.
22. Gerhardt P, Marquis RE. 1989. Spore thermoresistance mechanisms, p 43-63. In Smith I, Slepecky RA, Setlow P (ed), Regulation of Prokaryotic Development. American Society for Microbiology, Washington, D.C.
23. Losick R, Stragier P. 1992. Crisscross regulation of cell-type-specific gene expression during development in B.subtilis. Nature 355:601-4.
24. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R. 2003. The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50:1683-701.
22
25. Foster SJ, Johnstone K. 1990. Pulling the trigger: the mechanism of bacterial spore germination. Mol Microbiol 4:137-141.
26. Gould GW, Hurst A. 1969. The bacterial spore. Academic Press, London; New York. 27. Moir A, Kemp EH, Robinson C, Corfe BM. 1994. The genetic analysis of bacterial spore
germination. J Appl Bacteriol 77:9S-16S. 28. Jedrzejas MJ, Setlow P. 2001. Comparison of the binuclear metalloenzymes
diphosphoglycerate-independent phosphoglycerate mutase and alkaline phosphatase: their mechanism of catalysis via a phosphoserine intermediate. Chem Rev 101:607-18.
29. Moir A, Corfe BM, Behravan J. 2002. Spore germination. Cell Mol Life Sci 59:403-409. 30. Jack DL, Paulsen IT, Saier MH. 2000. The amino acid/polyamine/organocation (APC)
superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146 ( Pt 8):1797-1814.
31. Atluri S, Ragkousi K, Cortezzo DE, Setlow P. 2006. Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor. J Bacteriol 188:28-36.
32. Stewart KA, Setlow P. 2013. Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis. J Bacteriol 195:3575-82.
33. Ghosh S, Scotland M, Setlow P. 2012. Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis. J Bacteriol 194:2221-7.
34. Yi X, Liu J, Faeder JR, Setlow P. 2011. Synergism between different germinant receptors in the germination of Bacillus subtilis spores. J Bacteriol 193:4664-71.
35. Li Y, Jin K, Ghosh S, Devarakonda P, Carlson K, Davis A, Stewart KA, Cammett E, Pelczar Rossi P, Setlow B, Lu M, Setlow P, Hao B. 2014. Structural and functional analysis of the GerD spore germination protein of Bacillus species. J Mol Biol 426:1995-2008.
36. Yon JR, Sammons RL, Smith DA. 1989. Cloning and sequencing of the gerD gene of Bacillus subtilis. J Gen Microbiol 135:3431-45.
37. Kemp EH, Sammons RL, Moir A, Sun D, Setlow P. 1991. Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis. J Bacteriol 173:4646-4652.
38. Vepachedu VR, Setlow P. 2007. Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores. FEMS Microbiol Lett 274:42-7.
39. Paidhungat M, Setlow P. 1999. Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine. J Bacteriol 181:3341-3350.
40. Cabrera-Martinez RM, Tovar-Rojo F, Vepachedu VR, Setlow P. 2003. Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. J Bacteriol 185:2457-64.
41. Paidhungat M, Setlow P. 2000. Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182:2513-2519.
42. Kolodziej BJ, Slepecky RA. 1962. A copper requirement for the sporulation of Bacillus megaterium. Nature 194:504-5.
43. Kolodziej BJ, Slepecky RA. 1964. Trace Metal Requirements for Sporulation of Bacillus Megaterium. J Bacteriol 88:821-30.
44. Levinson HS, Hyatt MT. 1964. Effect of Sporulation Medium on Heat Resistance, Chemical Composition, and Germination of Bacillus Megaterium Spores. J Bacteriol 87:876-86.
45. Leuschner RGK, Lillford PJ. 2000. Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores. Microbiology 146 ( Pt 1):49-55.
46. Seto-Young DL, Ellar DJ. 1981. Studies on calcium transport during growth and sporulation. Microbios 30:191-208.
47. Stewart BT, Halvorson HO. 1953. Studies on the spores of aerobic bacteria. I. The occurrence of alanine racemase. J Bacteriol 65:160-6.
23
48. Matsushita T, Ueda T, Kusaka I. 1986. Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis. Eur J Biochem 156:95-100.
49. Igura N, Kamimura Y, Islam MS, Shimoda M, Hayakawa I. 2003. Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure. Appl Environ Microbiol 69:6307-10.
50. YP DV. 2004. The role of calcium in bacterial spore germination. Microbes and Environments 19(3):199-202
51. Heffron JD, Sherry N, Popham DL. 2011. In vitro studies of peptidoglycan binding and hydrolysis by the Bacillus anthracis germination-specific lytic enzyme SleB. J Bacteriol 193:125-31.
52. Kamat AS, Lewis NF, Pradhan DS. 1985. Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination of Bacillus cereus BIS-59 spores. Microbios 44:33-44.
53. Keynan A, Murrell WG, Halvorson HO. 1962. Germination properties of spores with low dipicolinic acid content. J Bacteriol 83:395-9.
54. Schroeter R, Schlisio S, Lucet I, Yudkin M, Borriss R. 1999. The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C. FEMS Microbiol Lett 174:117-23.
55. Inaoka T, Matsumura Y, Tsuchido T. 1999. SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis. J Bacteriol 181:1939-43.
56. Granger AC, Gaidamakova EK, Matrosova VY, Daly MJ, Setlow P. 2011. Effects of Mn and Fe levels on Bacillus subtilis spore resistance and effects of Mn2+, other divalent cations, orthophosphate, and dipicolinic acid on protein resistance to ionizing radiation. Appl Environ Microbiol 77:32-40.
57. Chander M, Setlow B, Setlow P. 1998. The enzymatic activity of phosphoglycerate mutase from gram-positive endospore-forming bacteria requires Mn2+ and is pH sensitive. Can J Microbiol 44:759-67.
58. Henriques AO, Melsen LR, Moran CP, Jr. 1998. Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J Bacteriol 180:2285-91.
59. Eisenstadt E, Fisher S, Der CL, Silver S. 1973. Manganese transport in Bacillus subtilis W23 during growth and sporulation. J Bacteriol 113:1363-72.
60. Wolf FI, Cittadini A. 2003. Chemistry and biochemistry of magnesium. Mol Aspects Med 24:3-9.
61. Scribner H, Eisenstadt E, Silver S. 1974. Magnesium transport in Bacillus subtilis W23 during growth and sporulation. J Bacteriol 117:1224-30.
62. Williams RJP. 1981. The Bakerian Lecture, 1981: Natural Selection of the Chemical Elements. procroyasocilon2 Proceedings of the Royal Society of London Series B, Biological Sciences 213:361-397.
63. Loeb LA, James EA, Waltersdorph AM, Klebanoff SJ. 1988. Mutagenesis by the autoxidation of iron with isolated DNA. Proc Natl Acad Sci USA 85:3918-22.
64. Keynan A. 1978. Spore structure and its relations to resistance, dormancy, and germination. Spores VII American Society for Microbiology, Washington, DC:43-53.
65. Swerdlow BM, Setlow B, Setlow P. 1981. Levels of H+ and other monovalent cations in dormant and germinating spore of Bacillus megaterium. J Bacteriol 148:20-29.
66. Thackray PD, Behravan J, Southworth TW, Moir A. 2001. GerN, an antiporter homologue important in germination of Bacillus cereus endospores. J Bacteriol 183:476-482.
67. Southworth TW, Guffanti AA, Moir A, Krulwich TA. 2001. GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter. J Bacteriol 183:5896-903.
24
68. Padan E, Schuldiner S. 1994. Molecular biology of Na+/H+ antiporters: molecular devices that couple the Na+ and H+ circulation in cells. Biochim Biophys Acta 1187:206-10.
69. Kosono S, Ohashi Y, Kawamura F, Kitada M, Kudo T. 2000. Function of a principal Na(+)/H(+) antiporter, ShaA, is required for initiation of sporulation in Bacillus subtilis. J Bacteriol 182:898-904.
70. Tani K, Watanabe T, Matsuda H, Nasu M, Kondo M. 1996. Cloning and sequencing of the spore germination gene of Bacillus megaterium ATCC 12872: similarities to the NaH-antiporter gene of Enterococcus hirae. Microbiol Immunol 40:99-105.
71. Senior A, Moir A. 2008. The Bacillus cereus GerN and GerT protein homologs have distinct roles in spore germination and outgrowth, respectively. J Bacteriol 190:6148-52.
72. Clements MO, Moir A. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180:6729-6735.
73. Paredes-Sabja D, Setlow P, Sarker MR. 2009. GerO, a putative Na+/H+-K+ antiporter, is essential for normal germination of spores of the pathogenic bacterium Clostridium perfringens. J Bacteriol 191(12):3822-31.
74. Oomes SJ, Jonker MJ, Wittink FR, Hehenkamp JO, Breit TM, Brul S. 2009. The effect of calcium on the transcriptome of sporulating B. subtilis cells. Int J Food Microbiol 133:234-42.
75. Smith RJ. 1995. Calcium and bacteria. Adv Microb Physiol 37:83-133. 76. Raeymaekers L, Wuytack E, Willems I, Michiels CW, Wuytack F. 2002. Expression of
a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation. Cell Calcium 32:93.
77. Li Y, Davis A, Korza G, Zhang P, Li YQ, Setlow B, Setlow P, Hao B. 2012. Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis. J Bacteriol 194:1875-84.
78. Van Opijnen T, Bodi KL, Camilli A. 2009. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767-72.
79. Gaballa A, Helmann JD. 1998. Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis. J Bacteriol 180:5815-21.
80. Fujisawa M, Wada Y, Tsuchiya T, Ito M. 2009. Characterization of Bacillus subtilis YfkE (ChaA): a calcium-specific Ca2+/H+ antiporter of the CaCA family. Arch Microbiol 191:649-57.
81. Tanaka K, Kobayashi K, Ogasawara N. 2003. The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium. Microbiology 149:2317-29.
82. Chillappagari S, Miethke M, Trip H, Kuipers OP, Marahiel MA. 2009. Copper acquisition is mediated by YcnJ and regulated by YcnK and CsoR in Bacillus subtilis. J Bacteriol 191:2362-70.
83. Gibson MM, Bagga DA, Miller CG, Maguire ME. 1991. Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Mol Microbiol 5:2753-62.
84. Johnson CM, Grossman AD. 2014. Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis. Mol Microbiol 93:1284-301.
85. Zheng L, Abhyankar W, Ouwerling N, Dekker HL, van Veen H, van der Wel NN, Roseboom W, de Koning LJ, Brul S, de Koster CG. 2016. Bacillus subtilis Spore Inner Membrane Proteome. J Proteome Res 15:585-94.
86. Chen Y, Barat B, Ray WK, Helm RF, Melville SB, Popham DL. 2019. Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores. J Bacteriol 201(6).
25
Figure 1.1. Spore structure. Cartoon depicting the different structural components of the
dormant spore. The dehydrated core, which stores all the nucleic acids is surrounded by multiple
layers including an inner membrane, a germ cell wall, the cortex, an outer membrane, and
proteinaceous coats that contribute to spore resistance capacities and dormancy. An additional
exosporium layer is present in B. anthracis spores but this is not present in all species.
26
Figure 1.2. Bacillus subtilis sporulation. The events of sporulation occur when nutrients decrease
to a certain critical point in the environment of B. subtilis. Sporulation takes approximately eight
hours from onset to complete the assembly of a dormant spore. The beginning steps in
sporulation involve the segregation of two copies of the chromosome along an axial filament to
the two polar ends of the cell. A septum is formed asymmetrically towards one end of the cell
separating it into two compartments, each containing a copy of the chromosome. The larger
mother cell compartment engulfs the smaller forespore compartment. The forespore is then
surrounded by two cell membranes, and cortex peptidoglycan is deposited between the two
membranes. The inner and outer coat layers are built around the framework of the outer
forespore membrane and the spore undergoes maturation during which it achieves full
dormancy and resistance properties. Finally, the mother cell lyses to release the dormant spore.
27
Figure 1.3. Cascade of sigma factors. Simplified schematic depicting the crisscross regulation of
sigma factors involved in sporulation. Each sigma factor is required for activity of the next in a
temporal manner which keeps the mother cell and the forespore coordinated temporally.
28
Figure 1.4. Spore germination. Germination is initated when nutrients called germinants contact
Ger receptors located in the inner forespore membrane. During Stage 1 there is release of Ca2+-
DPA and other ions followed by partial rehydration of the core. Stage 2 features breakdown of
the cortex by cortex-lytic enzymes, coinciding with further rehydration of the core and
resumption of metabolic activities.
29
Figure 1.5. Organization of GerA receptor. The GerA receptor is encoded by the tricistronic gerA
operon. The operon contains three genes forming the three specific subunits GerAA, GerAB and
GerAC. Each of these subunits are required and they interact to form a functional GerA receptor.
The predicted membrane topologies of the proteins are indicated, with the core of the spore on
the bottom of the figure.
30
Chapter Two
Membrane Proteomes and Ion Transporters in Bacillus
anthracis and Bacillus subtilis Dormant and Germinating Spores
Yan Chen^, Bidisha Barat^, W. Keith Ray, Richard F. Helm, Stephen B. Melville, and David L. Popham#. Membrane proteomes and ion transporters in Bacillus anthracis and Bacillus subtilis dormant and germinating spores. Journal of Bacteriology. 2019 Mar 15;201(6): e00662-18.
Quantification of ions using atomic emission spectroscopy. Phase bright spores were
purified away from phase dark spores by centrifugation through a 50% sodium diatrizoate layer
as described (64) and were suspended at OD600 = 10 in 1 ml 200 mM Tris-HCl, pH 8.0 to remove
coat-associated ions. The spore suspension was rocked at room temperature for 20 minutes and
56
washed three times with fresh deionized water by centrifugation at 15,800 x g for 2 minutes.
Pellets were suspended in 1 ml of 6 M ultrapure HCl (Fisher Chemicals) and heated at 100°C for
10 minutes followed by centrifugation at 15,800 x g for 10 minutes. The supernatant was
collected, and the amounts of individual ions were quantified using atomic emission
spectroscopy.
Purified phase bright spores were heat activated (70°C for 20 minutes and cooling on ice)
and stimulated to germinate at a starting OD600 of 10 by addition of 10 mM L-alanine in 50 mM
NaPO4 buffer at pH 7.0. At different time intervals, samples were removed and centrifuged for 2
mins at 15,800 x g. The ion contents of germination exudate (supernatant) samples were
analyzed using atomic emission spectroscopy. Significant differences between values were
determined using unpaired two-tailed Student’s t-tests.
57
ACKNOWLEDGEMENTS
Research reported in this publication was supported by the National Institute of Allergy
and Infectious Disease of the National Institutes of Health under award number R21AI088298.
The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institutes of Health. The mass spectrometry resources used in this
work are maintained in part through funding by the Fralin Life Science Institute at Virginia Tech
and the Agricultural Experiment Station Hatch Program at Virginia Tech (CRIS Project Number:
VA-135981).
58
REFERENCES
1. Setlow P. 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat
and chemicals. J Appl Microbiol 101:514-525. 2. Moir A. 2006. How do spores germinate? J Appl Microbiol 101:526-530. 3. Setlow P. 2003. Spore germination. Curr Opin Microbiol 6:550-556. 4. Mallozzi M, Viswanathan VK, Vedantam G. 2010. Spore-forming Bacilli and Clostridia in
human disease. Future Microbiology 5:1109-1123. 5. Setlow P, Johnson EA. 2007. Spores and Their Significance. Food Microbiology:
Fundamentals and Frontiers, Third Edition:35-67. 6. Amuguni H, Tzipori S. 2012. Bacillus subtilis: a temperature resistant and needle free
delivery system of immunogens. Hum Vaccin Immunother 8:979-986. 7. Cutting SM, Hong HA, Baccigalupi L, Ricca E. 2009. Oral vaccine delivery by
recombinant spore probiotics. Int Rev Immunol 28:487-505. 8. Knecht LD, Pasini P, Daunert S. 2011. Bacterial spores as platforms for bioanalytical and
biomedical applications. Anal Bioanal Chem 400:977-989. 9. Cowan AE, Olivastro EM, Koppel DE, Loshon CA, Setlow B, Setlow P. 2004. Lipids in
the inner membrane of dormant spores of Bacillus species are largely immobile. Proc Natl Acad Sci USA 101:7733-7738.
10. Koshikawa T, Beaman TC, Pankratz HS, Nakashio S, Corner TR, Gerhardt P. 1984. Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers. J Bacteriol 159:624-632.
11. Popham DL, Heffron JD, Lambert EA. 2012. Degradation of Spore Peptidoglycan During Germination, p. 121-142. In Abel-Santos E (ed.), Bacterial Spores: Current Research and Applications. Caister Academic Press, Norwich, UK.
12. Indest KJ, Buchholz WG, Faeder JR, Setlow P. 2009. Workshop report: modeling the molecular mechanism of bacterial spore germination and elucidating reasons for germination heterogeneity. Journal of food science 74:R73-78.
13. Bergman NH, Anderson EC, Swenson EE, Niemeyer MM, Miyoshi AD, Hanna PC. 2006. Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J Bacteriol 188:6092-6100.
14. Eichenberger P, Fujita M, Jensen ST, Conlon EM, Rudner DZ, Wang ST, Ferguson C, Haga K, Sato T, Liu JS, Losick R. 2004. The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2:e328.
15. Eichenberger P, Jensen ST, Conlon EM, van Ooij C, Silvaggi J, Gonzalez-Pastor JE, Fujita M, Ben-Yehuda S, Stragier P, Liu JS, Losick R. 2003. The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327:945-972.
16. Liu H, Bergman NH, Thomason B, Shallom S, Hazen A, Crossno J, Rasko DA, Ravel J, Read TD, Peterson SN, Yates J, 3rd, Hanna PC. 2004. Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186:164-178.
17. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, Eichenberger P. 2006. The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358:16-37.
18. Abhyankar W, Beek AT, Dekker H, Kort R, Brul S, de Koster CG. 2011. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics 11:4541-4550.
19. Delvecchio VG, Connolly JP, Alefantis TG, Walz A, Quan MA, Patra G, Ashton JM, Whittington JT, Chafin RD, Liang X, Grewal P, Khan AS, Mujer CV. 2006. Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl Environ Microbiol 72:6355-6363.
59
20. Huang CM, Foster KW, DeSilva TS, Van Kampen KR, Elmets CA, Tang DC. 2004. Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores. Proteomics 4:2653-2661.
21. Jagtap P, Michailidis G, Zielke R, Walker AK, Patel N, Strahler JR, Driks A, Andrews PC, Maddock JR. 2006. Early events of Bacillus anthracis germination identified by time-course quantitative proteomics. Proteomics 6:5199-5211.
22. Kuwana R, Kasahara Y, Fujibayashi M, Takamatsu H, Ogasawara N, Watabe K. 2002. Proteomics characterization of novel spore proteins of Bacillus subtilis. Microbiology 148:3971-3982.
23. Lai EM, Phadke ND, Kachman MT, Giorno R, Vazquez S, Vazquez JA, Maddock JR, Driks A. 2003. Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J Bacteriol 185:1443-1454.
24. Mao L, Jiang S, Wang B, Chen L, Yao Q, Chen K. 2011. Protein profile of Bacillus subtilis spore. Curr Microbiol 63:198-205.
25. Swarge BN, Roseboom W, Zheng L, Abhyankar WR, Brul S, de Koster CG, de Koning LJ. 2018. "One-Pot" Sample Processing Method for Proteome-Wide Analysis of Microbial Cells and Spores. Proteomics Clin Appl:e1700169.
26. Zheng L, Abhyankar W, Ouwerling N, Dekker HL, van Veen H, van der Wel NN, Roseboom W, de Koning LJ, Brul S, de Koster CG. 2016. Bacillus subtilis Spore Inner Membrane Proteome. J Proteome Res 15:585-594.
27. Korza G, Setlow P. 2013. Topology and accessibility of germination proteins in the Bacillus subtilis spore inner membrane. J Bacteriol 195:1484-1491.
28. Paidhungat M, Setlow P. 2001. Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183:3982-3990.
29. Fort P, Errington J. 1985. Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis. J Gen Microbiol 131:1091-1105.
30. Tovar-Rojo F, Chander M, Setlow B, Setlow P. 2002. The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis. J Bacteriol 184:584-587.
31. Vepachedu VR, Setlow P. 2005. Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis. J Bacteriol 187:5677-5682.
32. Vepachedu VR, Setlow P. 2007. Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme. J Bacteriol 189:1565-1572.
33. Igarashi T, Setlow B, Paidhungat M, Setlow P. 2004. Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis. J Bacteriol 186:2984-2991.
34. Pelczar PL, Setlow P. 2008. Localization of the germination protein GerD to the inner membrane in Bacillus subtilis spores. J Bacteriol 190:5635-5641.
35. Chirakkal H, O'Rourke M, Atrih A, Foster SJ, Moir A. 2002. Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiology 148:2383-2392.
36. Stewart KA, Yi X, Ghosh S, Setlow P. 2012. Germination protein levels and rates of germination of spores of Bacillus subtilis with overexpressed or deleted genes encoding germination proteins. J Bacteriol 194:3156-3164.
37. Steil L, Serrano M, Henriques AO, Volker U. 2005. Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiol 151:399-420.
38. Serrano M, Corte L, Opdyke J, Moran CP, Jr., Henriques AO. 2003. Expression of spoIIIJ in the prespore is sufficient for activation of sigma G and for sporulation in Bacillus subtilis. J Bacteriol 185:3905-3917.
39. Winstedt L, von Wachenfeldt C. 2000. Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth. J Bacteriol 182:6557-6564.
60
40. Mattatall NR, Jazairi J, Hill BC. 2000. Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis. J Biol Chem 275:28802-28809.
41. Liu H, Sadygov RG, Yates JR, 3rd. 2004. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:4193-4201.
42. Gilchrist A, Au CE, Hiding J, Bell AW, Fernandez-Rodriguez J, Lesimple S, Nagaya H, Roy L, Gosline SJ, Hallett M, Paiement J, Kearney RE, Nilsson T, Bergeron JJ. 2006. Quantitative proteomics analysis of the secretory pathway. Cell 127:1265-1281.
43. Chen Y, Ray WK, Helm RF, Melville SB, Popham DL. 2014. Levels of Germination Proteins in Bacillus subtilis Dormant, Superdormant, and Germinating Spores. PloS one 9:e95781.
44. Patel VJ, Thalassinos K, Slade SE, Connolly JB, Crombie A, Murrell JC, Scrivens JH. 2009. A comparison of labeling and label-free mass spectrometry-based proteomics approaches. J Proteome Res 8:3752-3759.
45. Stewart KA, Setlow P. 2013. Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis. J Bacteriol 195:3575-3582.
46. Fabret C, Hoch JA. 1998. A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180:6375-6383.
47. Fukuchi K, Kasahara Y, Asai K, Kobayashi K, Moriya S, Ogasawara N. 2000. The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis. Microbiology 146 ( Pt 7):1573-1583.
48. Bernhards CB, Chen Y, Toutkoushian H, Popham DL. 2015. HtrC is involved in proteolysis of YpeB during germination of Bacillus anthracis and Bacillus subtilis spores. J Bacteriol 197:326-336.
49. Dalbey RE, Wang P, van Dijl JM. 2012. Membrane proteases in the bacterial protein secretion and quality control pathway. Microbiol Mol Biol Rev 76:311-330.
50. Lorca G, Winnen B, Saier MH, Jr. 2003. Identification of the L-aspartate transporter in Bacillus subtilis. J Bacteriol 185:3218-3222.
51. Sekowska A, Robin S, Daudin JJ, Henaut A, Danchin A. 2001. Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis. Genome Biol 2:RESEARCH0019.
52. Hullo MF, Auger S, Dassa E, Danchin A, Martin-Verstraete I. 2004. The metNPQ operon of Bacillus subtilis encodes an ABC permease transporting methionine sulfoxide, D- and L-methionine. Res Microbiol 155:80-86.
53. Charney J, Fisher WP, Hegarty CP. 1951. Managanese as an essential element for sporulation in the genus Bacillus. J Bacteriol 62:145-148.
54. Swerdlow BM, Setlow B, Setlow P. 1981. Levels of H+ and other monovalent cations in dormant and germinating spore of Bacillus megaterium. J Bacteriol 148:20-29.
55. Gerhardt P, Marquis RE. 1989. Spore thermoresistance mechanisms, p. 43-63. In Smith I, Slepecky RA, Setlow P (ed.), Regulation of Prokaryotic Development. American Society for Microbiology, Washington, D.C.
56. Paidhungat M, Setlow B, Driks A, Setlow P. 2000. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512.
57. Granger AC, Gaidamakova EK, Matrosova VY, Daly MJ, Setlow P. 2011. Effects of Mn and Fe levels on Bacillus subtilis spore resistance and effects of Mn2+, other divalent cations, orthophosphate, and dipicolinic acid on protein resistance to ionizing radiation. Appl Environ Microbiol 77:32-40.
58. Raeymaekers L, Wuytack E, Willems I, Michiels CW, Wuytack F. 2002. Expression of a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation. Cell Calcium 32:93.
59. Daniel RA, Errington J. 1993. Cloning, DNA sequence, functional analysis and transcriptional regulation of the genes encoding dipicolinic acid synthetase required for sporulation in Bacillus subtilis. J Mol Biol 232:468-483.
61
60. Ramirez-Guadiana FH, Meeske AJ, Rodrigues CDA, Barajas-Ornelas RDC, Kruse AC, Rudner DZ. 2017. A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis. PLoS Genet 13:e1007015.
61. Arrieta-Ortiz ML, Hafemeister C, Bate AR, Chu T, Greenfield A, Shuster B, Barry SN, Gallitto M, Liu B, Kacmarczyk T, Santoriello F, Chen J, Rodrigues CD, Sato T, Rudner DZ, Driks A, Bonneau R, Eichenberger P. 2015. An experimentally supported model of the Bacillus subtilis global transcriptional regulatory network. Mol Syst Biol 11:839.
62. Kim HU, Goepfert JM. 1974. A sporulation medium for Bacillus anthracis. J Appl Bacteriol 37:265-267.
63. Leighton TJ, Doi RH. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 254:3189-3195.
64. Nicholson WL, Setlow P. 1990. Sporulation, germination, and outgrowth., p. 391-450. In Harwood CR, Cutting SM (ed.), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
65. González-Castro MJ, López-Hernández J, Simal-Lozano J, Oruña-Concha MJ. 1997. Determination of amino acids in green beans by derivitization with phenylisothiocyanate and high-performance liquid chromatography with ultraviolet detection. J Chrom Sci 35:181-185.
66. Vizcaino JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Rios D, Dianes JA, Sun Z, Farrah T, Bandeira N, Binz PA, Xenarios I, Eisenacher M, Mayer G, Gatto L, Campos A, Chalkley RJ, Kraus HJ, Albar JP, Martinez-Bartolome S, Apweiler R, Omenn GS, Martens L, Jones AR, Hermjakob H. 2014. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol 32:223-226.
68. Koo BM, Kritikos G, Farelli JD, Todor H, Tong K, Kimsey H, Wapinski I, Galardini M, Cabal A, Peters JM, Hachmann AB, Rudner DZ, Allen KN, Typas A, Gross CA. 2017. Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Syst 4:291-305 e297.
69. Mascher T, Margulis NG, Wang T, Ye RW, Helmann JD. 2003. Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon. Mol Microbiol 50:1591-1604.
70. Wach A. 1996. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12:259-265.
71. Hageman JH, Shankweiler GW, Wall PR, Franich K, McCowan GW, Cauble SM, Grajeda J, Quinones C. 1984. Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production. J Bacteriol 160:438-441.
72. Wyrick PB, Rogers HJ. 1973. Isolation and characterization of cell wall-defective variants of Bacillus subtilis and Bacillus licheniformis. J Bacteriol 116:456-465.
73. Meador-Parton J, Popham DL. 2000. Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation. J Bacteriol 182:4491-4499.
74. Soil and Plant Analysis Council I. 1999. Methods of Instrumental Analysis, p. 207-224. In Jones Jr. J (ed.), Soil Analysis Handbook of Reference Methods. CRC Press.
62
Figure 2.1. Predicted membrane-spanning domains of B. anthracis and B. subtilis spore
membrane proteins. Predictions of membrane association mechanisms were made for proteins
identified in membrane fractions as described in Materials and Methods. Proteins were further
classified based upon their predicted number of membrane spanning helices.
63
Figure 2.2. Mutant strains lacking yloB produce many phase-dark spores. B. subtilis strains
were grown and sporulated in 2xSG medium, and spores were purified by water washing. Phase
contrast microscopy revealed that approximately 50% of the spores produced by all strains
1 Sequences were aligned using BlastP (67). 2 The percent of the B. subtilis protein sequence that was aligned with the B. anthracis sequence. 3 A more similar protein than that detected in the spore proteome was present in the full B. subtilis genome: (B. anthracis gene:Most similar B. subtilis gene) (Q81XB0:P94421) (Q81V85:O31567) (Q81MT9:P24011) (Q81U40:P55339) (Q81TH8:O32151)
71
Table 2.3. Validation of membrane protein quantification.
B. subtilis
protein
Dormant/Germinated
spore ratio by
spectral counting
Dormant/Germinated
spore ratio by MRMa
GerAC 1.7 1.9
GerBC 0.8 1.5
GerKC 1.5 2.4
GerD 3.0 3.5
PrkC 1.9 3.3
SpoVAC 3.3 0.8
SpoVAD 0.5 0.6
YpeB 4.2 6.8
a MRM data are from reference (43).
72
Table 2.4. Changes in Bacillus spore membrane protein detection following germination.
Gene B. subtilis
Uniprot number
B. anthracis Uniprot number
Membrane prediction
Fold change in unique
spectra (D/G)
fruA P71012 Integral 5.4
qcrB P46912 Integral 4.5
ypeB P38490 Integral 4.2e
yheB O07543 Integral 3.9
znuA O34966 Lipoprotein 3.6
ylaJ O07634 Lipoprotein 3.3
oppC P24139 Integral 3.3
atpF P37814 Integral 3.3
spoVAC P40868 Integral 3.3a
fhuD P37580 Lipoprotein 3.1
yugS O05241 Q81V91 Integral 3.0 / 3.3
gerD P16450 Lipoprotein 3.0e
ythA C0SP90 Integral 2.9
secDF O32047 Integral 2.9
ypmQ P54178 Lipoprotein 2.8
yitG Q796Q1 Integral 2.7
yqfX P54481 Q81T77 Integral 2.6 / INFb, c
oppA P24141 Q81V45 Lipoprotein 2.6 / 3.8
qoxA P34957 Integral 2.2
rbsA P36947 Peripheral 2.2
yfmC O34348 Lipoprotein 2.1
yhcN P54598 Lipoprotein 2.0
yugP O05248 Integral 2.0
spoVAD P40869 Q81X67 Peripheral 0.5e / 1.4
atpG P37810 Q81JZ4 Peripheral 0.5 / 3.9
BAS4323 Q6HSW8 Lipoprotein 8.9b
GBAA_2961 Q81P56 Integral 7.0
prsA1 Q81U45 Lipoprotein 6.3
GBAA_0855 Q81UL3 Lipoprotein 5.9c
GBAA_0615 Q81V85 Lipoprotein 4.8
GBAA_3048 Q81NX4 Peripheral 4.1
GBAA_5684 Q81JM0 Integral 3.9
GBAA_422 Q81ML8 Lipoprotein 3.7d
cccA Q81LU6 Integral 3.3
psd Q81LP7 Peripheral 3.3
GBAA_1195 Q81TS0 Peripheral 3.0
GBAA_1523 Q81SX1 Integral 2.2
GBAA_3927 Q81WP4 Lipoprotein 13
GBAA_0418 Q81Z55 Peripheral 0.5d a Result is not consistent that of (43).
73
b Result is consistent with that of (21). c This protein was detected in B. anthracis dormant spores but not in germinated spores. d Result is not consistent with that of (21). e Result is consistent that of (43).
74
Table 2.5: Production of heat resistant spores by B. subtilis strains lacking putative ion transporters.
Strain Genotype Total cfu/ml
Heated cfu/ml
Heated/Total
Heated/ WT
Heated
PS832 Wild type 1.2x109 0.9x109 0.8 1
DPVB689 ΔznuA::mls 1.4x109 1.3x109 0.9 1.4
DPVB690 Δycnl::mls 1.3x109 1.4x109 1.1 1.6
DPVB691 ΔyflS::mls 1.2x109 1.0x109 0.8 1.1
DPVB693 ΔyloB::mls 4.3x109 3.4x108 0.8 0.4
DPVB706 a ΔznuA ΔyflS Δycnl 1.1x109 1.0x109 0.9 1.1
Values are averages of three independent experiments a The genotype for DPVB706 is abbreviated to “Δ3” throughout the text. b The genotype for DPVB722 is abbreviated to “Δ5ΔyloB” throughout the text.
75
Table 2.6: Production of heat resistant spores by B. subtilis ion transporter mutants with different Ca2+ concentrations.
Added CaCl2 Strain
Total cfu/ml
HeatR
cfu/ml
1 mM
PS832 3.9x107 4.5x107
DPVB693 5.8x107 4.0x107
DPVB706 4.2x107 5.0x107
DPVB722 2.9x107 6.7x107
0.2 mM
PS832 3.6x107 3.1x107
DPVB693 2.8x108 3.5x107
DPVB706 2.7x108 3.2x107
DPVB722 6.3x107 3.2x107
0.04 mM
PS832 3.9x107 4.2x106
DPVB693 1.4x108 5.7x106
DPVB706 6.8x107 4.5x106
DPVB722 1.4x108 3.5x106
0 mM
PS832 3.4x107 2.5x106
DPVB693 3.9x107 9.3x105
DPVB706 2.9x107 2.6x106
DPVB722 8.1x107 3.7x105
Values are averages of two independent experiments
76
Chapter 3
Identification of L-valine-Initiated-Germination-Active Genes
in Bacillus subtilis using Tn-seq
Cameron V. Sayer^, Bidisha Barat^, and David L. Popham*. Identification of L-valine-
initiated-germination-active genes in Bacillus subtilis using Tn-seq. PLOS One. 2019;14(6).
A second phenotypic group is composed of mutant strains with significantly delayed
germination via a GerA-mediated response but germinate normally through GerB and GerK
sensing. Strains lacking yybT, ygaC, yqhL, yqeF, or sipT exhibit this phenotype, and all except the
yybT mutant have significantly reduced spore GerA content. The roles of these genes in
germination are unclear, as most are relatively uncharacterized. YybT (GdpP) acts as a c-di-AMP
phosphodiesterase and exerts pleotropic effects on physiology and gene expression [71-74].
SipT, acting as a signal peptidase [75], could certainly exert effects on assembly of membrane
proteins important for germination, including GerA.
The third phenotypic group includes strains with significantly slower germination rates in
response to L-valine but either have a decreased response to AGFK or 2xYT but not both. Mutants
lacking ytpA, phoP, phoR, pcrB or ytxG feature these phenotypes. It is not clear how or why a
mutant would be deficient in GerA mediated response, have a normal GerB and GerK response,
but still be deficient for germination in rich media. The PhoPR mutants have poor vegetative
growth and pleotropic effects on gene expression [76, 77], which might exert quite variable
effects into the sporulation process. These mutants seemed to exhibit significant variability
between multiple spore preparations. Three mutants in this group may exert effects on
membrane structure. YtpA is a phospholipase [57, 78], PcrB is a heptaprenylglyceryl phosphate
synthase [79], and a ytxG mutant exhibits defects in membrane morphology [80]. Alterations in
the spore inner membrane might affect assembly or function of the germination initiation
apparatus. None of these three genes are specifically expressed in sporulating cells, and thus
their activity levels and effects on germination might be more varied among spore preparations
and possibly with regard to different germinants. Interestingly, the ytxG mutant was the only
strain in which overexpression of gerA did not correct the L-Val germination defect. This
95
overexpression in the ytxG mutant did decrease AGFK germination, as in other strains, suggesting
that the gerA overexpression was successful. Perhaps a membrane defect in this mutant renders
Ger protein complexes nonfunctional regardless of their expression level.
Four of the mutants identified here exhibit decreased gerA transcription. The predicted
functions of these gene’s products provide no simple explanation for how such an effect on
transcription could come about, and the mechanisms may therefore be indirect. The expression
of two other 𝜎G-dependent genes, pbpF and sspB, was not decreased in the mutant strains,
indicating that this was not an effect on the entire regulon. Altered activity of a transcription
factor involved in gerA transcription, SpoVT or YlyA [17, 55, 69, 81, 82], could be an expected
pathway for such an effect. Future work should examine the effects of these mutations on other
genes within forespore-specific regulons to resolve this.
Among the germination mutants identified in our Tn-seq screen, strains that could
complete Stage I of germination but were blocked in Stage II were not present. This may be due
to the mutant screening process utilized. Mutants with Tn insertions in cwlD, which should
exhibit this phenotype [83, 84], were slightly enriched in our non-germinating spore population,
but not above the significance cutoff value used. Spores blocked at stage II were expected to
pellet with dormant spores in the density gradient utilized [31]. One possibility is that spores
blocked at Stage II were unstable through the time of incubation with germinant, density gradient
separation, and subsequent washing, and thus were not efficiently recovered. Utilization of an
alternative isolation method might allow identification of mutants with this phenotype.
96
ACKNOWLEDGEMENTS
We thank Alan Grossman for providing the Tn insertion library, Peter Setlow and George
Korza for strains and antibodies, and Jennifer Meador-Parton and Isabelle Wal for technical
assistance.
97
REFERENCES
1. Setlow P. 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101:514-25.
2. Moir A. 2006. How do spores germinate? J Appl Microbiol 101:526-530. 3. Setlow P. 2003. Spore germination. Curr Opin Microbiol 6:550-556. 4. Mallozzi M, Viswanathan VK, Vedantam G. 2010. Spore-forming Bacilli and Clostridia
in human disease. Future Microbiology 5:1109-1123. 5. Setlow P, Johnson EA. 2007. Spores and Their Significance. Food Microbiology:
Fundamentals and Frontiers, Third Edition:35-67. 6. Cowan AE, Olivastro EM, Koppel DE, Loshon CA, Setlow B, Setlow P. 2004. Lipids
in the inner membrane of dormant spores of Bacillus species are largely immobile. Proc Natl Acad Sci USA 101:7733-7738.
7. Koshikawa T, Beaman TC, Pankratz HS, Nakashio S, Corner TR, Gerhardt P. 1984. Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers. J Bacteriol 159:624-32.
8. Ross C, Abel-Santos E. 2010. The Ger receptor family from sporulating bacteria. Curr Issues Mol Biol 12:147-58.
9. Vepachedu VR, Setlow P. 2007. Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores. FEMS Microbiol Lett 274:42-7.
10. Li Y, Jin K, Ghosh S, Devarakonda P, Carlson K, Davis A, Stewart KA, Cammett E, Pelczar Rossi P, Setlow B, Lu M, Setlow P, Hao B. 2014. Structural and functional analysis of the GerD spore germination protein of Bacillus species. J Mol Biol 426:1995-2008.
11. Christie G, Lowe CR. 2007. Role of chromosomal and plasmid-borne receptor homologues in the response of Bacillus megaterium QM B1551 spores to germinants. J Bacteriol 189:4375-83.
12. Igarashi T, Setlow P. 2005. Interaction between individual protein components of the GerA and GerB nutrient receptors that trigger germination of Bacillus subtilis spores. J Bacteriol 187:2513-2518.
13. Popham DL, Bernhards CB. 2015. Spore Peptidoglycan, p In Press. In Driks A, Eichenberger P (ed), The Bacterial Spore: From Molecules to Systems. ASM Press, Washington, D.C.
14. Van Opijnen T, Bodi KL, Camilli A. 2009. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767-72.
15. Johnson CM, Grossman AD. 2014. Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis. Mol Microbiol 93:1284-301.
16. Koo BM, Kritikos G, Farelli JD, Todor H, Tong K, Kimsey H, Wapinski I, Galardini M, Cabal A, Peters JM, Hachmann AB, Rudner DZ, Allen KN, Typas A, Gross CA. 2017. Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Syst 4:291-305 e7.
17. Feavers IM, Foulkes J, Setlow B, Sun D, Nicholson W, Setlow P, Moir A. 1990. The regulation of transcription of the gerA spore germination operon of Bacillus subtilis. Molec Microbiol 4:275-282.
18. Zuberi AR, Moir A, Feavers IM. 1987. The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168. Gene 51:1-11.
19. Cabrera-Martinez RM, Tovar-Rojo F, Vepachedu VR, Setlow P. 2003. Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. J Bacteriol 185:2457-64.
20. Leighton TJ, Doi RH. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 246:3189-95.
21. Stewart KA, Setlow P. 2013. Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis. J Bacteriol 195:3575-82.
98
22. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647-9.
23. Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550.
24. Nicholson WL, Setlow P. 1990. Sporulation, germination, and outgrowth., p 391-450. In Harwood CR, Cutting SM (ed), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
25. Dowd MM, Orsburn B, Popham DL. 2008. Cortex peptidoglycan lytic activity in germinating Bacillus anthracis spores. J Bacteriol 190:4541-8.
26. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676-82.
27. Hall M, Frank E, Holmes G, Pfahringer B, Reutemann P, Witten IH. 2009. The WEKA data mining software: an update. ACM SIGKDD Explorations Newsletter 11:10-18.
28. Ghosh S, Scotland M, Setlow P. 2012. Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis. J Bacteriol 194:2221-7.
29. Ramirez-Peralta A, Stewart KA, Thomas SK, Setlow B, Chen Z, Li YQ, Setlow P. 2012. Effects of the SpoVT regulatory protein on the germination and germination protein levels of spores of Bacillus subtilis. J Bacteriol 194:3417-25.
30. Ramirez-Peralta A, Zhang P, Li YQ, Setlow P. 2012. Effects of sporulation conditions on the germination and germination protein levels of Bacillus subtilis spores. Appl Environ Microbiol 78:2689-97.
31. Setlow B, Melly E, Setlow P. 2001. Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J Bacteriol 183:4894-4899.
32. Robinson DG, Chen W, Storey JD, Gresham D. 2014. Design and analysis of Bar-seq experiments. G3 (Bethesda) 4:11-8.
33. Naclerio G, Baccigalupi L, Zilhao R, De Felice M, Ricca E. 1996. Bacillus subtilis spore coat assembly requires cotH gene expression. J Bacteriol 178:4375-80.
34. Moir A, Lafferty E, Smith DA. 1979. Genetics analysis of spore germination mutants of Bacillus subtilis 168: the correlation of phenotype with map location. J Gen Microbiol 111:165-80.
35. Zheng L, Donovan WP, Fitz-James PC, Losick R. 1988. Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore. Genes & Dev 2:1047-1054.
36. Behravan J, Chirakkal H, Masson A, Moir A. 2000. Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores. J Bacteriol 182:1987-94.
37. Takamatsu H, Kodama T, Nakayama T, Watabe K. 1999. Characterization of the yrbA gene of Bacillus subtilis, involved in resistance and germination of spores. J Bacteriol 181:4986-94.
38. Fukushima T, Ishikawa S, Yamamoto H, Ogasawara N, Sekiguchi J. 2003. Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis. J Biochem 133:475-83.
39. Zhang J, Fitz-James PC, Aronson AI. 1993. Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis. J Bacteriol 175:3757-66.
40. Serrano M, Zilhao R, Ricca E, Ozin AJ, Moran CP, Jr., Henriques AO. 1999. A Bacillus subtilis secreted protein with a role in endospore coat assembly and function. J Bacteriol 181:3632-43.
99
41. Kuwana R, Okuda N, Takamatsu H, Watabe K. 2006. Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores. J Biochem 139:887-901.
42. Beall B, Driks A, Losick R, Moran CP, Jr. 1993. Cloning and characterization of a gene required for assembly of the Bacillus subtilis spore coat. J Bacteriol 175:1705-16.
43. Perez-Valdespino A, Li Y, Setlow B, Ghosh S, Pan D, Korza G, Feeherry FE, Doona CJ, Li YQ, Hao B, Setlow P. 2014. Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores. J Bacteriol 196:2077-88.
44. Chen Y, Barat B, Ray WK, Helm RF, Melville SB, Popham DL. 2019. Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores. J Bacteriol 201(6).
45. Zheng L, Abhyankar W, Ouwerling N, Dekker HL, van Veen H, van der Wel NN, Roseboom W, de Koning LJ, Brul S, de Koster CG. 2016. Bacillus subtilis Spore Inner Membrane Proteome. J Proteome Res 15:585-94.
46. Wetzstein M, Volker U, Dedio J, Lobau S, Zuber U, Schiesswohl M, Herget C, Hecker M, Schumann W. 1992. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bacteriol 174:3300-10.
47. Dambach M, Irnov I, Winkler WC. 2013. Association of RNAs with Bacillus subtilis Hfq. PLoS One 8:e55156.
48. Au N, Kuester-Schoeck E, Mandava V, Bothwell LE, Canny SP, Chachu K, Colavito SA, Fuller SN, Groban ES, Hensley LA, O'Brien TC, Shah A, Tierney JT, Tomm LL, O'Gara TM, Goranov AI, Grossman AD, Lovett CM. 2005. Genetic composition of the Bacillus subtilis SOS system. J Bacteriol 187:7655-66.
49. Puri-Taneja A, Paul S, Chen Y, Hulett FM. 2006. CcpA causes repression of the phoPR promoter through a novel transcription start site, P(A6). J Bacteriol 188:1266-78.
50. Kaushal B, Paul S, Hulett FM. 2010. Direct regulation of Bacillus subtilis phoPR transcription by transition state regulator ScoC. J Bacteriol 192:3103-13.
51. Tjalsma H, Bolhuis A, van Roosmalen ML, Wiegert T, Schumann W, Broekhuizen CP, Quax WJ, Venema G, Bron S, van Dijl JM. 1998. Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases. Genes Dev 12:2318-31.
52. Allenby NE, Watts CA, Homuth G, Pragai Z, Wipat A, Ward AC, Harwood CR. 2006. Phosphate starvation induces the sporulation killing factor of Bacillus subtilis. J Bacteriol 188:5299-303.
53. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R. 2003. The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50:1683-701.
54. Strauch MA, Bobay BG, Cavanagh J, Yao F, Wilson A, Le Breton Y. 2007. Abh and AbrB control of Bacillus subtilis antimicrobial gene expression. J Bacteriol 189:7720-32.
55. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, Eichenberger P. 2006. The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358:16-37.
56. DeLoughery A, Lalanne JB, Losick R, Li GW. 2018. Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis. Proc Natl Acad Sci U S A 115:E5585-E5594.
57. Eiamphungporn W, Helmann JD. 2008. The Bacillus subtilis sigma(M) regulon and its contribution to cell envelope stress responses. Mol Microbiol 67:830-48.
58. Petersohn A, Brigulla M, Haas S, Hoheisel JD, Volker U, Hecker M. 2001. Global analysis of the general stress response of Bacillus subtilis. J Bacteriol 183:5617-31.
59. Rao F, See RY, Zhang D, Toh DC, Ji Q, Liang ZX. 2010. YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity. J Biol Chem 285:473-82.
100
60. Luo Y, Helmann JD. 2012. A sigmaD-dependent antisense transcript modulates expression of the cyclic-di-AMP hydrolase GdpP in Bacillus subtilis. Microbiology 158:2732-41.
61. Atluri S, Ragkousi K, Cortezzo DE, Setlow P. 2006. Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor. J Bacteriol 188:28-36.
62. Yi X, Liu J, Faeder JR, Setlow P. 2011. Synergism between different germinant receptors in the germination of Bacillus subtilis spores. J Bacteriol 193:4664-71.
63. Setlow B, Cowan AE, Setlow P. 2003. Germination of spores of Bacillus subtilis with dodecylamine. J Appl Microbiol 95:637-48.
64. Straus D, Walter W, Gross CA. 1990. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of s32. Genes & Dev 4:2202-2209.
65. Kavita K, de Mets F, Gottesman S. 2018. New aspects of RNA-based regulation by Hfq and its partner sRNAs. Curr Opin Microbiol 42:53-61.
66. Gonzalez-Pastor JE, Hobbs EC, Losick R. 2003. Cannibalism by sporulating bacteria. Science 301:510-3.
67. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH. 2017. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200-D203.
68. Zhou R, Cusumano C, Sui D, Garavito RM, Kroos L. 2009. Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP. Proc Natl Acad Sci U S A 106:16174-9.
69. Steil L, Serrano M, Henriques AO, Volker U. 2005. Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiol 151:399-420.
70. Korza G, Camilleri E, Green J, Robinson J, Nagler K, Moeller R, Caimano MJ, Setlow P. 2019. Analysis of the Messenger RNAs in Spores of Bacillus subtilis. J Bacteriol 201(9):e00007-19.
71. Gundlach J, Mehne FM, Herzberg C, Kampf J, Valerius O, Kaever V, Stulke J. 2015. An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis. J Bacteriol 197:3265-74.
72. Gundlach J, Rath H, Herzberg C, Mader U, Stulke J. 2016. Second Messenger Signaling in Bacillus subtilis: Accumulation of Cyclic di-AMP Inhibits Biofilm Formation. Front Microbiol 7:804.
73. Luo Y, Helmann JD. 2012. Analysis of the role of Bacillus subtilis sigma(M) in beta-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis. Mol Microbiol 83:623-39.
74. Gandara C, Alonso JC. 2015. DisA and c-di-AMP act at the intersection between DNA-damage response and stress homeostasis in exponentially growing Bacillus subtilis cells. DNA Repair (Amst) 27:1-8.
75. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, van Dijl JM. 2000. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515-47.
76. Allenby NE, O'Connor N, Pragai Z, Ward AC, Wipat A, Harwood CR. 2005. Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis. J Bacteriol 187:8063-80.
77. Antelmann H, Scharf C, Hecker M. 2000. Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J Bacteriol 182:4478-90.
101
78. Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, Ochi K. 2002. Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrob Agents Chemother 46:315-20.
79. Guldan H, Matysik FM, Bocola M, Sterner R, Babinger P. 2011. Functional assignment of an enzyme that catalyzes the synthesis of an archaea-type ether lipid in bacteria. Angew Chem Int Ed Engl 50:8188-91.
80. Meeske AJ, Rodrigues CD, Brady J, Lim HC, Bernhardt TG, Rudner DZ. 2016. High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis. PLoS Biol 14:e1002341.
81. Bagyan I, Hobot J, Cutting S. 1996. A compartmentalized regulator of developmental gene expression in Bacillus subtilis. J Bacteriol 178:4500-7.
82. Traag BA, Ramirez-Peralta A, Wang Erickson AF, Setlow P, Losick R. 2013. A novel RNA polymerase-binding protein controlling genes involved in spore germination in Bacillus subtilis. Mol Microbiol 89:113-22.
83. Popham DL, Meador-Parton J, Costello CE, Setlow P. 1999. Spore peptidoglycan structure in a cwlD dacB double mutant of Bacillus subtilis. J Bacteriol 181:6205-6209.
84. Sekiguchi J, Akeo K, Yamamoto H, Khasanov FK, Alonso JC, Kuroda A. 1995. Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which effects germination in Bacillus subtilis. J Bacteriol 177:5582-5589.
102
Fig 3.1. Germination rates of B. subtilis strains. Purified spores were heat activated,
stimulated to germinate by addition of 10 mM L-Val, and shaken at 37°C, during which
the OD600 was monitored. Values are averages of three assays and error bars are standard
deviations. Each assay was performed on three replicate spore preparations. For the ylbC
(■) and phoP (▲) mutants, all points after 10 min are significantly different from those of
the wild type (●); P≤0.05.
103
Fig 3.2. Phase-contrast microscopy of germinating B. subtilis spore populations.
Purified spores of B. subtilis wild type and mutant strains were heat-activated and
stimulated to germinate by addition of 10 mM L-Val followed by incubation at 37°C for
60 mins. A) PS832 B) ytpA mutant strain C) ylbC mutant strain All panels are the same
magnification; the bar in panel C is 5 µm.
104
Fig 3.3: Expression of a gerA-lacZ transcriptional fusion. Purified spores carrying a gerA-
lacZ transcriptional fusion were decoated and lysed, and extracts were assayed for β-
galactosidase. Values are expressed as a percentage of that detected in DPVB761, the
wild type strain containing the gerA-lacZ fusion. Values are averages of triplicate assays
and error bars are standard deviations. * indicates a significant difference from the wild
type (p ≤ 0.05).
105
Fig 3.4: GerAC is reduced in the spores of several B. subtilis mutant strains. Equal
quantities of spore suspensions were decoated and broken, and proteins were extracted,
serially diluted, run on SDS-PAGE, and transferred to PVDF membrane as described
previously [21]. The membrane was probed with anti-GerAC antibodies [30] (Panel A and
Fig S4). Strain genotype (All strains were also gerB.) and sample dilution is indicated
above each lane. Protein load and transfer to membrane in each lane was normalized as
described in Materials and Methods, and the amount of GerAC detected in each strain
was compared to that found in the wild type (Panel B). Error bars indicate standard
deviations. * indicates a significant difference from the wild type (p ≤ 0.05).
106
Table 3.1: Genes in which Tn insertions altered germination
Gene p-valuea Fold-change Sample 1b
Fold-change Sample 2b
Reference for Ger defect
cotH 2.2E-35 15.0 10.9 [33]
gerAA 4.4E-31 150.1 43.7 [18, 34]
gerAC 2.0E-29 279.9 73.9 [18, 34]
cotE 3.8E-24 17.0 12.0 [35]
ygaC 2.3E-18 4.6 4.2
yqfT 3.0E-16 15.4 8.8
ypzK 6.3E-16 10.1 9.7
yqeF 2.0E-15 3.3 5.5
ymzD-ymcCc 5.7E-13 2.5 2.7
gerPF 5.8E-13 7.4 4.1 [36]
safA 1.1E-11 7.3 4.1 [37]
pcrB 2.3E-11 3.0 2.6
ylbC 3.9E-11 4.1 2.9
gidA 1.3E-10 4.2 2.8
gerPB 1.0E-09 5.9 3.6 [36]
gerPC 3.1E-09 11.7 3.9 [36]
nocA 4.2E-09 2.0 2.2
veg 1.1E-08 4.8 3.2 [38]
gerE 1.8E-08 Infinite 32.0 [34]
ytoA 2.2E-08 5.1 4.1
ytpA 1.0E-07 5.3 4.6
ytpB 1.1E-07 3.9 3.5
rsbW 1.2E-07 2.2 2.5
yfhD 2.8E-07 4.2 4.6
cotZ 7.7E-07 2.1 3.1 [39]
yqhL 1.1E-06 4.3 2.5
kinB 1.8E-06 1.6 1.9
skfC 1.9E-06 7.9 3.7
skfE 3.8E-06 8.4 3.7
gerAB 5.8E-06 181.7 73.6 [18, 34]
ymaB 6.6E-06 1.9 2.9
sipT 7.5E-06 2.5 2.8
skfG 9.7E-06 3.5 2.3
phoR 1.0E-05 2.5 3.0
cotN (tasA) 1.5E-05 2.1 2.2 [40]
yhbJ 2.8E-05 1.8 1.7
yhaM 3.3E-05 4.6 2.5
ymaF 3.7E-05 8.1 2.8
107
yabG 1.7E-04 2.5 2.3 [41]
spoVID 1.8E-04 2.7 2.1 [42]
yqhR 3.6E-04 2.3 2.0
yonF 4.7E-04 1.8 3.0
spoVAF 4.8E-04 2.2 1.8 [43]
hfq 6.7E-04 3.2 2.5
yosK 9.2E-04 4.4 6.4
yozE 9.4E-04 3.0 4.8
yopI 1.1E-03 2.3 2.8
flgN 1.2E-03 14.2 3.6
gerD 1.2E-03 2.6 1.9 [34]
fliW 2.0E-03 1.9 3.4
yfbJ 2.0E-03 3.1 2.3
ytmO 2.1E-03 4.0 3.5
gerPE 2.1E-03 6.5 1.7 [36]
phoP 2.3E-03 3.2 3.4
gerPD 4.7E-03 8.2 3.2 [36]
tufA 5.2E-03 5.7 3.1
ispA 5.7E-03 2.9 2.8
yoqL 1.7E-02 6.1 3.7
dnaJ 4.7E-02 2.5 2.9
yaaB (remB) 5.0E-02 7.4 2.3
ytxG 2.1E-01 2.7 2.9
yybT (gdpP) 2.4E-01 7.5 2.1 a p-value determined using DESeq2 [23] comparing Dormant and Germinated sample read counts. b Fold change in read counts of Dormant/Germinated samples
c Intergenic region
108
Table 3.2: Genes without previously known germination role identified by Tn-seq and in
spore membrane proteome.
Gene Function Locus structure Regulation of expression
dnaJ Protein quality control
hrcA-grpE-dnaK-dnaJ-yqeTUV
σA, HrcA [46]
hfq RNA chaperone hfq Increased protein during transition to stationary phase [47]
pcrB Heptaprenylglyceryl phosphate synthase
pcrB-pcrA-ligA-yerH
LexA regulon [48]
phoP Response regulator, phosphate metabolism
phoPR σA, σB, σE, CcpA, ScoC [49, 50]
phoR Sensor kinase, phosphate metabolism
phoPR σA, σB, σE, CcpA, ScoC [49, 50]
sipT Signal peptidase I sipT DegU [51]
skfE Export of spore killing factor (SkfA)
skfABCEFGH Spo0A, AbrB, PhoP [52-54]
ygaC Unknown ygaCD
ylbC Unknown ylbBC σF [55]
yqeF Unknown yqeF
yqhL Unknown yqhL mRNA processed by RNase Y [56]
ytpA Phospholipase, Bacilysocin synthesis
ytpAB σM [57]
ytxG General stress ytxGHJ σB, σH [58]
yybT (gdpP)
c-di-AMP phosphodiesterase. Functions in DNA damage and acid resistance [59]
yybS-gdpP-rplI σA, σD-induced antisense RNA [60]
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Table 3.3: Phenotypic properties of B. subtilis strains
Genotype Doubling timea (min)
Sporulation efficiencyb (%)
DPA releasec (µg/ml/OD)
NAM releasec (nmole/ml/OD)
% phase-dark sporesd
Wild type 20 66 5.3 ± 0.1 61.0 ± 14.2 95
dnaJ 31 89 1.1 ± 0.4* 25.9 ± 12.7 14
hfq 40 63 2.2 ± .03* 30.1 ± 6.2 48
pcrB 31 68 3.0 ± 0 30.3 ± 8.0 41
phoP 44 83 2.7 ± 0.4* 46.6 ± 5.8 63
phoR 35 54 3.9 ± 0.5 39.2 ± 9.5 86
sipT 34 54 2.3 ± 1.0* 23.5 ± 10.2* 22
skfE 27 59 1.8 ± 0* 40.6 ± 10.7 38
ygaC 21 71 3.1 ± 0.4* 36.6 ± 9.9 60
ylbC 29 48 1.1 ± 0.3* 14.3 ± 3.4* 19
yqeF 23 84 1.9 ± 0* 37.2 ± 9.4 50
yqhL 20 53 2.6 ± 0.4* 36.1 ± 8.1 69
ytpA 22 95 1.63 ± 0* 17.7 ± 2.7* 55
ytxG 31 18 4.8 ± 0.3 50.9 ± 4.8 90
yybT 21 69 4.7 ± 0.4 56.6 ± 14.1 92 a Growth in 2xSG medium at 37°C b Heat-resistant count/total viable count after 24 hr incubation on 2xSG medium at 37°C. c Release of DPA and NAM 30 or 45 min, respectively, after exposure to 10 mM L-Val at 37°C. *
indicates a significant difference from the wild type (T-test, p<0.05). Values are indicative of averages and standard deviations of three biological replicates.
d Spores pixel intensities quantified and classified as described in Materials and Methods after 60 min exposure to 10 mM L-Val at 37°C.
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Table 3.4: Response of B. subtilis strains to varied germinants.
Genotype
% OD600 lossa
% DPA released by dodecylamineb
L-Val (60 mins)
AGFK (60 mins)
2xYT (40 mins)
Wild type 60 ± 1 41 ± 2 60 ± 2 75 ± 1
dnaJ 6 ± 0** 28 ± 10* 40 ± 3* 68 ± 4
hfq 26 ± 5* 30 ± 1* 52 ± 2* 75 ± 1
pcrB 47 ± 10* 27 ± 3* 58 ± 4 87 ± 2
phoP 28 ± 1* 36 ± 10 53 ± 1* 59 ± 6
phoR 44 ± 2* 22 ± 10* 56 ± 1 52 ± 6
sipT 7 ± 0** 33 ± 10 57 ± 7 69 ± 5
skfE 35 ± 10* 28 ± 6* 50 ± 3* 82 ± 5
ygaC 22 ± 3* 37 ± 10 55 ± 2 67 ± 5
ylbC 8 ± 1** 13 ± 2** 23 ± 8** 66 ± 3
yqeF 38 ± 3* 33 ± 7 58 ± 1 84 ± 1
yqhL 33 ± 6* 37 ± 10 54 ± 3 71 ± 2
ytpA 32 ± 3* 41 ± 3 47 ± 3* 73 ± 4
ytxG 35 ± 0* 28 ± 8 53 ± 1* 72 ± 4
yybT 47 ± 2* 44 ± 7 60 ± 0 76 ± 4 a Values are averages and standard deviations of assays on three replicate spore preparations.
OD600 of purified spore suspension monitored at the indicated time after addition of 10 mM L-valine, 1X AGFK, or 2xYT while shaking at 37°C. * indicates a significant difference (T-test, p<0.05) or ** indicates a significant difference (T-test, p<0.01) from the wild type.
b Values are averages and standard deviations of assays on three replicate spore preparations. DPA release by purified spore suspension monitored 100 min after addition of 1 mM dodecylamine while shaking at 37°C.
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Table 3.5. Overexpression of gerA suppresses germination defect of multiple mutants.
Genotype
% OD Lossa
without sspDp-gerA
with sspDp-gerA
Wild type 35 ± 4 38 ± 2
𝛥skfE 23 ± 5* 38 ± 3
𝛥pcrB 34 ± 7 37 ± 1
𝛥ygaC 26 ± 1* 36 ± 1
𝛥sipT 9 ± 5* 41 ± 1
𝛥ylbC 7 ± 0* 37 ± 0
𝛥hfq 27 ± 3* 38 ± 3
𝛥yqhL 29 ± 3* 37 ± 0
𝛥dnaJ 12 ± 2* 36 ± 2
𝛥yqeF 28 ± 2* 31 ± 2
𝛥phoR 37 ± 1 42 ± 9
𝛥phoP 32 ± 1 36 ± 0
𝛥ytxG 25 ± 6* 15 ± 4*
𝛥ytpA 25 ± 0* 38 ± 2
𝛥yybT 37 ± 2 37 ± 2 a Values are averages and standard deviations of assays on three replicate spore preparations. OD600 of purified spore suspension monitored 45 min after addition of 10 mM L-valine while shaking at 37°C. * indicates a significant difference from the wild type (T-test, p<0.05)
112
Chapter 4
Role of YlbC and YlbB in GerA-mediated spore germination in
Bacillus subtilis
Bidisha Barat and David L. Popham
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ATTRIBUTIONS
Bidisha Barat performed the research, experimentation, and data analysis. David L. Popham is
the principal investigator.
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ABSTRACT
Germination of dormant B. subtilis spores with specific nutrient germinants starts at the
inner membrane with the interaction of a germinant with Ger receptor proteins and progresses
through core rehydration and cortex breakdown. Deficiencies in Ger receptors such as GerA,
GerB, GerK, receptor-associated proteins such as GerD, Ca2+-dipicolinic acid channels, and lytic
enzymes can potentially inhibit the germination process. Using transposon sequencing, genes
newly implicated in germination were identified in a previous study. One such gene was ylbC,
encoding a protein of unknown function. The ylbC mutant strain showed a significant reduction
in germination efficiency with L-valine, about 80% of the population failed to initiate germination,
suggesting a defect in the GerA-mediated response. YlbC-deficient spores demonstrated a 30%
reduction in gerA transcription and 50% reduced GerA abundance relative to those of wild-type
spores. The ylbC gene is expressed in an operon with another gene ylbB under the control of a
forespore-specific sigma factor, σF. In an effort to better understand the underlying mechanism,
genetic studies were performed to determine if ylbC works with other known genes in altering
the production of germinant receptors. YlbC seemed to have an overall positive effect while YlbB
seemed to have a negative effect on GerA-dependent germination. Genetic characterization of
YlbC and YlbB demonstrated that they seem to act independently of each other in affecting GerA-
mediated germination despite being expressed in the same operon.
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INTRODUCTION
Bacillus subtilis spores are formed under adverse conditions and are resilient to a range
of environmental agents including ultraviolet radiation, heat, desiccation and toxic chemicals [1].
These spores remain metabolically dormant for years, until nutrients are available in the
environment and they can revert to the vegetative state by spore germination followed by
outgrowth [2, 3]. Spore germination is an irreversible process that is generally initiated by the
addition of certain nutrients called germinants that are sensed by specific germinant receptors
present within the inner membrane of the spore. In B. subtilis , spore germination may be induced
by nutrients such as L-alanine, L-valine, a mixture of L-asparagine, D-glucose, D-fructose and
potassium ions (AGFK) or rich media as well as with non-nutrient germinants such as
dodecylamine (a cationic surfactant) Ca2+-DPA, or high pressure [4, 5]. B. subtilis germinant
receptors are expressed during late sporulation in the forespore, encoded by the homologous
tricistronic gerA, gerB, and gerK operons [4, 5]. The individual germinant receptors are composed
of A, B and C subunits, which work synergistically to function. The GerA receptor senses either L-
valine or L-alanine, whereas the GerB and GerK receptors respond to a combination of AGFK [6].
Although the process of germination in B. subtilis has been studied extensively, there are still
many undetermined aspects in this area. The exact mechanism of commitment to germinate and
the activation of germinant receptors by binding of nutrients is still not clear.
Recently, the YlbC protein was reported to be important in B. subtilis spore germination
with L-valine [7]. YlbC deficient spores demonstrated a >50-fold reduction in germination rate, a
30% reduction in gerA transcription, and 50% reduced GerA abundance relative to those of wild-
type spores [7]. YlbC is an uncharacterized membrane protein in Bacillus subtilis. ylbC is
expressed as a part of a two-gene operon under the control of σF, an early sporulation forespore-
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specific sigma factor [8]. YlbC contains two conserved domains, including a CAP domain which is
a cysteine-rich secretory domain and a YkwD domain that is conserved in spore-forming bacteria
but with unknown function [9]. The protein contains a likely transmembrane domain near the N-
terminus [9] which together with indications from previous work could indicate that YlbC is
anchored to the inner spore membrane.
The upstream gene in the operon, ylbB, is annotated as a putative oxidoreductase. It has
a paralogous gene yhcV, which is expressed in the forespore later in sporulation under σG control
[8, 10]. YlbB contains conserved domains of cystathionine-beta-synthase (CBS) pairs [9], which
while largely uncharacterized, are predicted to have a role in ligand binding, most likely adenosyl
groups such as AMP or ATP [11]. YlbB shares a 38% sequence identity to a signaling protein from
Burkholderia which was shown to bind both NAD and AMP [12]. It could be theorized that YlbB
in a partially redundant manner with YhcV, acts as a sensor for the function of YlbC whose role
has yet to be determined. The ylbB-ylbC operon is transcribed under σF control [8], and thus
mutations to ylbC may have implications for spore assembly, thus affecting germination. YlyA is
produced during the late stage of sporulation and fine tunes forespore-specific transcription by
regulating the expression of σG-dependent genes involved in spore germination by binding to
RNA polymerase [13].
In the quest to provide more insight to what the function of YlbC is within spore formation
and germination, we have examined the roles of YlbB and YhcV as well as YlyA, which is involved
in modulation of genes that encode germinant receptors (GR), and their potential interaction
with YlbC.
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RESULTS
Germination rates of deletion strains. Single mutants of ylbC, ylbB, yhcV, ylyA were
received from the Bacillus Genetic Stock Center. The wildtype strain PS832 was naturally
transformed with each of these mutations. Multiple mutants of the various genes were
generated (Table S5) and the impact of these mutations on spore germination was analyzed.
Purified spores were heat activated and germinated with 10 mM L-valine and changes in OD600
was monitored. All mutant strains with a ylbC deletion show a significantly reduced rate of
germination in comparison to the wildtype as seen in our previous studies for ylbC, which was
attributed to a defect in GerA receptor function. Single mutants ylbB::kan, ylbB, and
yhcV::kan have a germination rate significantly better than the wildtype, while the double
mutant ylbB::mls yhcV::kan has a germination rate comparable to the wildtype (Figure 4.1,
Table 4.1). Thus, YlbB and YhcV may have a negative effect or act as negative regulators of GerA,
therefore a ylbB and/or yhcV results in increased germination rates. The double mutants,
ylbB::kan ylbC::mls and ylbC::mls yhcV::kan show a significantly greater germination defect
than the wildtype and ylbC::mls. Since ylbB::kan ylbC::mls, ylbC::mls yhcV::kan, and
ylbB::kan ylbC::mls yhcV strains show germination rates similar to the ylbC strain (Figure
4.1, Table 4.1), YlbB and YhcV seem to be acting upstream of YlbC. Spores from ylyA show a
moderately reduced germination rate in comparison to the wildtype, though not as drastic as the
ylbC mutant strains. From previous studies on ylyA, it has been seen that a ylyA deletion leads to
an increase in SpoVT, which in turn may repress genes under σG control such as gerA [13].
However, germinant receptor levels of GerAA, GerAC, GerKA and GerD were found to be normal
in ylyA mutant spores [13]. Hence some additional factor may be responsible for this germination
defect.
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Microscopic analysis of germination. In mutant strains that show a significant decrease
in germination rate, this phenotype could be attributed to the majority of the spore culture
germinating poorly or to a subpopulation of spores germinating normally and the rest remaining
dormant. To compare and quantify the number of germinated and dormant spores in the various
mutant strains, purified spores were induced to germinate with 10 mM L-valine and 25mM HEPES
buffer (pH 7.4), imaged at 1 hour pre and post germination by phase-contrast microscopy, and
categorized as phase-bright and phase-dark spores based on pixel intensity of the spores (Figure
S8). Prior to germination, all spore samples from the various strains had 95-99% phase- bright
spores. Post germination, PS832 (wildtype) and strains with a ylbB or yhcV deletion had ~90%
phase-dark spores and strains with a ylyA deletion had ~60% phase-dark spores while all the
mutant strains with a ylbC deletion had only ~20% phase- dark spores (Figure 4.2, S8). The ylbC
phenotype is conserved in the double and triple mutants with a ylbC deletion. The failure to
change from phase-bright to phase-dark indicates that these mutant spores failed to initiate
germination.
Polar effects of ylbB mutations. Since ylbB and ylbC are expressed in the same operon,
creating a single ylbB::kan or ylbB::mls mutant could lead to polar effects on expression of
ylbC. In order to check for polarity, a transcriptional fusion of ylbC to lacZ was generated to allow
quantification of ylbC transcription. The ylbC-lacZ expression in the wildtype, ylbB::kan, and
ylbB was compared by measuring the β-galactosidase activity. The ylbB::kan ylbC-lacZ strain
had a high ylbC expression in dormant spores in comparison to the wildtype, while a ylbB ylbC-
lacZ strain had a significantly lower ylbC expression in comparison to the wildtype (Figure 4.3).
However, a germination assay of ylbB spores in response to L-valine shows a similar rate of
germination in comparison to ylbB::kan, both faster than the wildtype.
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The ylbC expression during sporulation was also measured for the various strains. Samples
were collected at timepoints throughout sporulation for assay of β-galactosidase. During sporulation,
ylbC expression was similar between mutant strains and wildtype. However, at T20 (20 hours after
initiation of sporulation and dormant spore formation), the ylbB::kan ylbC-lacZ strain had an
exceptionally high level of ylbC expression in comparison to the wildtype, while a ylbB ylbC::lacZ
strain had a significantly lower ylbC expression in comparison to the wildtype (Table 4.2). This
suggests that although ylbB::kan and ylbB have significantly different levels of ylbC expression,
these levels of YlbC production are sufficient to support rapid germination, and the effect on ylbB on
germination is through another YlbB function.
Expression of the GerA receptor. To determine if there was any difference in the gerA
transcription level in the single mutant strains ylbB::kan, ylbB, yhcV::kan, ylbC::kan, ylyA::kan
in comparison to the wildtype, gerA expression was determined using a gerA-lacZ transcriptional
fusion and measurement of β-galactosidase activity. The single mutants, ylbB::kan, ylbB,
yhcV::kan and ylyA::kan had comparable gerA transcription to the wildtype, while the ylbC::kan
mutant had a significant decrease in gerA transcription (Figure 4.4).
Previously, through the germination assays targeting different germinant receptors, it was
observed that a GerA germinant receptor mediated response (L-valine) may have led to the reduction
in germination initiation in ylbC::mls. [7] . This was supported by the observation that ylbC::mls
strain showed a ~50% decrease in the abundance of the GerA receptor in comparison to the wildtype.
Since the ylbB::kan,ylbB and yhcV::kan strains showed a better rate of germination than the
wildtype, the GerA abundance in these spores was measured using quantitative GerAC western blots
in comparison to the wildtype. All three mutant strains had similar GerAC abundance in comparison
to the wildtype (Figure 4.6).
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Overexpression of ylbB. As mutants with a ylbB or yhcV deletion have a better rate of
germination than the wildtype, YlbB/YhcV could be having a negative effect on function of YlbC
or GerA. Overexpression of YlbB was obtained by cloning ylbB downstream of a sigma F-
dependent promoter (dacFp) and then insertion to the B. subtilis chromosome [14]. Purified
spores of PS832 (Wildtype), PS832 with ylbB overexpression, ∆ylbC, and ∆ylbC with ylbB
overexpression were stimulated to germinate with 10 mM L-valine and changes in optical density
were monitored. Overexpression of ylbB did not affect the germination rate in the wildtype
however, it significantly reduced the rate of germination in ∆ylbC with ylbB overexpression in
comparison to ∆ylbC (Figure 4.6, Table 4.3). This suggests that in the ∆ylbC strain which already
has reduced GerA abundance, overexpression of ylbB may inhibit the low amounts of GerA.
While, in the wildtype strain that has normal amounts of GerA, overexpression of ylbB may not
affect the higher levels of GerA.
MATERIALS AND METHODS
Strain constructions. Single mutants lacking four genes (ylbC, ylbB, yhcV, and ylyA) were
received from the Bacillus subtilis Genetic Stock Center. Each mutation was a deletion with the
replacement of the gene of interest by an erythromycin resistance gene flanked by two loxP sites
[21]. B. subtilis strain PS832 was naturally transformed with the mutations with selection for
erythromycin (2.5μg/ml) and lincomycin (12.5μg/ml) (MLS) resistance. To promote deletion of
the resistance gene, the Cre recombinase was expressed from plasmid pDR244 [21], which left
an unmarked in-frame deletion mutation. Multiple mutants were produced by repeating this
process. All strains are listed in Table S5.
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To construct a ylbC-lacZ transcriptional fusion, the first 400 bases of ylbC was inserted into
plasmid pDPC87 [15], which contains a promoterless lacZ. The 5’-region of ylbC was amplified and
restriction sites were added onto the 5’ and 3’ ends. This 400 bp PCR product and vector pDPC87
[15] were digested with EcoRI and BamHI and ligated. Escherichia coli cells (DPVE3) were transformed
with the resulting plasmid (pDPV500), and transformants were screened by restriction mapping
followed by sequencing. Rec+ E. coli cells (DPVE2) were transformed with the plasmid with the correct
insert. The resulting plasmid DNA was transformed into B. subtilis strains PS832, ylbB::kan, and
ylbB with selection for chloramphenicol resistance when recombined into the chromosome via a
single crossover at the ylbC locus.
To reduce cross-reactivity of GerBC during quantitative western blot analysis of GerAC, strains
were converted to GerB- by transformation with chromosomal DNA from B. subtilis strain DPVB724
with an insertion of a chloramphenicol resistance gene and a gerB deletion.
B. subtilis strain with a gerA-lacZ fusion and MLS resistance gene (DPVB761) was received from the
Setlow lab [18, 19]. The gerA-lacZ fusion was naturally transformed into the ylbB::kan and yhcV::kan
mutant strains with selection for kanamycin and MLS and into the ylbB deletion strain with selection
for MLS. Since the strain with a ylbC deletion was also marked with a MLS resistance gene, it was
necessary to eliminate the MLS resistance gene for transformation of the ylbC mutant strain with
chromosomal DNA bearing the gerA-lacZ fusion. An unmarked in-frame deletion mutant was
generated by the Cre recombinase that deleted the MLS resistance gene followed by the introduction
of the gerA-lacZ fusion into the ylbC deletion strain by natural transformation with selection for MLS
resistance.
For overexpression of ylbB, the entire ylbB gene without the promoter region but
including the RBS was inserted into the plasmid pDPV115 which allows cloning of a gene
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downstream of a sigma F-dependent promoter (dacFp) and then insertion to the B. subtilis
chromosome [14]. The promoterless ylbB gene was amplified and restriction sites were added
onto the ends. This 447 bp PCR product and vector pDPV115 were digested with SpeI and SacII
and ligated together. E. coli cells (DPVE3) were transformed with the resulting plasmid
(pDPV498), and transformants were screened by restriction mapping, followed by sequencing.
Rec+ E. coli cells (DPVE2) were transformed with the plasmid with a correct insert.. The resulting
plasmid DNA was transformed into B. subtilis with selection for chloramphenicol resistance
(3.0μg/ml) with recombination into the chromosome via a double crossover at the amyE locus.
All mutations were verified by PCR and agarose gel electrophoresis.
Spore preparation. Spores of B. subtilis strain PS832 and the various mutant strains were
prepared in 2xSG broth [20]. Cultures were incubated at 37°C and harvested after 3-4 days.
Spores were washed in cold water with repeated centrifugation for several days until >98% of
spores were free and phase-bright when observed by phase-contrast microscopy. Prior to assays,
purified spores were checked for >95% phase-bright phenotype by microscopy.
Germination assays. To quantify change in OD600 as a measure the of rate of germination,
purified spores were heat activated at 70˚C for 30 minutes and quenched on ice for 5 minutes. The
spores were then germinated at 37°C at an OD600 of 0.2 with 10 mM L-valine in 25mM HEPES buffer
(pH 7.4). Changes in OD600 over time was observed using a Tecan M200 microplate reader.
Assay of gerA and ylbC transcription. B. subtilis strains with a ylbC-lacZ or gerA-lacZ
transcriptional fusion were induced to sporulate by the nutrient exhaustion method in 2xSG at 37˚C.
Purified dormant spores were chemically decoated to remove the outer membrane and spore coat
proteins, washed, and extracted, followed by assay of β-galactosidase activity using methyl-
umbelliferyl-D-galactoside (MUG) as previously described [16]. A microplate reader (Tecan M200)
123
was used to measure the MUG fluorescence at excitation and emission wavelengths of 365 nm and
450 nm, respectively. To calibrate the fluorescence readings, methylumbelliferone standard
solutions were made in the same buffer mixture. The average β-galactosidase activity of PS832
(wildtype without gerA-lacZ or ylbC-lacZ fusion) was deducted from the readings for each sample
containing the gerA-lacZ or ylbC-lacZ fusion, and the decoated spore OD600 values were used for
normalization of the readings.
Western blot analysis. In order to avoid cross-reactivity of GerB with the GerAC antibody,
strains with a gerB deletion were used for performing quantitative western blots. Purified dormant
spores of B. subtilis (~100 OD600 units) were chemically decoated followed by extraction of proteins
as previously described [7]. Samples were then serially diluted with ∆gerA ∆gerB strain extract and
2x SDS-PAGE sample loading buffer. The Bio-Rad TGX Stain-Free Fast Cast premixed acrylamide
solution was used for SDS-PAGE as described previously [7]. A trihalo compound present in the
electrophoresis gel modified the proteins after separation, which made the proteins fluorescent
directly within the gel. Following membrane transfer, the total protein amount in each lane was
measured directly from the stain-free image of the membrane. The total density for each lane was
measured from the blot. The anti-GerAC antibody was used for probing via western blot. Individual
band intensity was compared between sample lanes and normalized to the total protein amount
measured from the stain-free image of the membrane for a particular lane. Dilutions of 1.0 and 0.5
were blotted and used for quantification. Data analysis of quantitative blots was performed using
Biorad Image Lab 6.0. Quantitative GerAC western blots were performed in triplicate.
Microscopy. Purified spores were observed both 1 hour prior and post germination with
10mM L-valine and 25 mM HEPES buffer via phase-contrast microscopy. Images were collected
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and analyzed for each sample, using three visual fields each containing 70–300 spores per field,
as described previously [7].
DISCUSSION
The ylbC gene was identified in a previous Tn-Seq study of genes associated with L-valine
germination [7]. That study demonstrated a reduction in germination rate, a 30% reduction in gerA
transcription, and 50% reduced GerA abundance within dormant spores containing the ylbC null
mutation [7]. This additional study was performed to understand the specific mechanism by which
YlbC alters GerA-mediated spore germination. In the Tn-seq germination screen, Tn insertions were
significantly underrepresented in ylbB (p=0.014) however it did not qualify for the 2-fold difference
in reads cutoff used in that study [7]. ylbC and ylbB are expressed as a part of a bicistronic operon
under the control of a forespore-specific sigma factor, σF [8]. There are two conserved CBS domains
in YlbB which may be involved in binding of ligands such as ATP [11]. ylbB also has a paralogous gene
yhcV which is under the control of σG [8, 10]. Hence, ylbB may be functioning in conjunction with
ylbC or as a sensor for ylbC and potentially in a redundant manner with yhcV.
It could be theorized that YlbC is either acting positively on the transcription of gerA
and/or positively on GerA production (Figure 4.7). Alternatively, YlbC may act negatively on some
intermediate regulators (repressors) of gerA transcription such as YlyA, hence a ylbC would
result in a reduced gerA expression and thus reduced levels of GerA (Figure 4.8). The role of
YlbB/YhcV with respect to YlbC is uncertain; YlbB/YhcV could be acting upstream or downstream
of YlbC or completely independently (Figure 4.8). The potential role of YlbB/YhcV was studied
using both single and double mutants of ylbB, yhcV, and ylbC. If YlbB/YhcV is activating YlbC and
has an overall positive effect, ∆ylbB and ∆yhcV would in turn result in downregulation of gerA
and reduced GerA production. Alternatively, if YlbB/YhcV has a negative effect on YlbC or acts as
125
a negative regulator of GerA, then a ∆ylbB and ∆yhcV would result in more GerA production.
Germination with L-valine revealed that single mutants of ylbB and yhcV had a significantly
greater germination rate in comparison to the wildtype suggesting that YlbB/YhcV may have a
negative effect on either GerA or YlbC.
If YlbB/YhcV functions upstream of YlbC, then ∆ylbB ∆ylbC and ∆yhcV ∆ylbC spores should
behave the same as ∆ylbC spores. If YlbB/YhcV functions downstream of YlbC, ∆ylbB ∆ylbC and
∆yhcV ∆ylbC spores should behave the same as ∆ylbB spores. The germination assay of the
double mutants revealed that YlbB/YhcV may be acting upstream of YlbC. To rule out any polar
effect on the expression of ylbC in our ylbB::kan strain,ylbC transcription was studied using a lacZ
transcriptional fusion. Surprisingly, ∆ylbB produced a significantly lower level of ylbC expression
in comparison to ∆ylbB::kan, despite having similar germination rates suggesting that the
germination is not affected by YlbC levels in ylbB mutant strains instead could be through another
YlbB function. Apart from ∆ylbC, none of the single mutants showed any significant change in
gerA transcription and GerA production, suggesting that YlbB/YhcV may not be affecting GerA
mediated germination directly. Microscopy revealed that only strains with a ylbC deletion failed
to initiate germination.
To further investigate if YlbB has a negative effect on YlbC or GerA, ylbB was
overexpressed under the control of a sigma F-dependent promoter (dacFp). If YlbB has a negative
effect on YlbC, then overexpression of ylbB would result in poor germination in a ylbC+ strain and
would have no effect on the germination of the ylbC deletion mutant, which already has a
germination defect. Alternatively, if YlbB has a negative effect on GerA then overexpression of
ylbB would result in poor germination in a ylbC+ strain and the germination rate should be worse
in a ylbC- strain with ylbB overexpression than in the ylbC deletion mutant. Overexpression of
126
ylbB did not affect wildtype spore germination rate but significantly reduced the rate of
germination of ∆ylbC spores, indicating that ylbB overexpression may only have an inhibitory
effect in the presence of reduced amounts of GerA.
The exact mechanism by which YlbC affects transcription and protein production of GerA
still remains unclear as there is no clear connection between ylbC and other known genes in
altering the production of GerA. However, YlbC and YlbB may be acting independently of each
other despite being expressed in the same operon.
127
REFERENCES
1. Setlow P. 1994. Mechanisms which contribute to the long-term survival of spores of Bacillus species. J Appl Bacteriol Sympos Suppl 76:49S-60S.
2. Errington J. 1993. Bacillus subtilis sporulation: Regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1-33.
4. Paidhungat M, Setlow P. 2000. Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182:2513-2519.
5. Setlow P. 2003. Spore germination. Curr Opin Microbiol 6:550-556. 6. Setlow P. 2014. Germination of spores of Bacillus species: what we know and do not
know. J Bacteriol 196:1297-305. 7. Sayer CV, Barat B, Popham DL. 2019. Identification of L-Valine-initiated-germination-
active genes in Bacillus subtilis using Tn-seq. PLoS One 14:e0218220. 8. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R,
Eichenberger P. 2006. The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358:16-37.
9. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH. 2017. CDD/SPARCLE: Functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200-D203.
10. Steil L, Serrano M, Henriques AO, Volker U. 2005. Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiol 151:399-420.
11. Zhou R, Cusumano C, Sui D, Garavito RM, Kroos L. 2009. Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP. Proc Natl Acad Sci USA 106:16174-9.
12. Baugh L, Gallagher LA, Patrapuvich R, Clifton MC, Gardberg AS, Edwards TE, Armour B, Begley DW, Dieterich SH, Dranow DM, Abendroth J, Fairman JW, Fox D, 3rd, Staker BL, Phan I, Gillespie A, Choi R, Nakazawa-Hewitt S, Nguyen MT, Napuli A, Barrett L, Buchko GW, Stacy R, Myler PJ, Stewart LJ, Manoil C, Van Voorhis WC. 2013. Combining functional and structural genomics to sample the essential Burkholderia structome. PLoS One 8:e53851.
13. Traag BA, Ramirez-Peralta A, Wang Erickson AF, Setlow P, Losick R. 2013. A novel RNA polymerase-binding protein controlling genes involved in spore germination in Bacillus subtilis. Mol Microbiol 89:113-22.
14. Gilmore ME, Bandyopadhyay D, Dean AM, Linnstaedt SD, Popham DL. 2004. Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan. J Bacteriol 186:80-9.
15. Popham DL, Setlow P. 1994. Cloning, nucleotide sequence, mutagenesis, and mapping of the Bacillus subtilis pbpD gene, which codes for penicillin-binding protein 4. J Bacteriol 176:7197-7205.
16. Ghosh S, Scotland M, Setlow P. 2012. Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis. J Bacteriol 194:2221-7.
17. Paidhungat M, Setlow B, Driks A, Setlow P. 2000. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512.
18. Feavers IM, Foulkes J, Setlow B, Sun D, Nicholson W, Setlow P, Moir A. 1990. The regulation of transcription of the gerA spore germination operon of Bacillus subtilis. Molec Microbiol 4:275-282.
19. Zuberi AR, Moir A, Feavers IM. 1987. The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168. Gene 51:1-11.
128
20. Leighton TJ. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 246(10):3189-95.
21. Koo, B.M., Kritikos, G., Farelli, J.D., Todor, H., Tong, K., Kimsey, H., Wapinski, I., Galardini, M., Cabal, A., Peters, J.M. and Hachmann, A.B. 2017. Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. Cell Syst 4:291-305 e297.
129
50
60
70
80
90
100
0 5 10 15 20 25 30
% I
NIT
IAL
OD
TIME (MINUTES)
AWild type ylbC::mls ylbB::kan yhcV::kan ylyA::kan ylbB
50
60
70
80
90
100
0 5 1 0 15 20 25 30
% IN
ITIA
L O
D
TIME (MINUTES)
BWild type ylbc::mls ylbB::kan ylbB::kan ylbC::mls
130
Figure 4.1 (A-C). Germination rates of B. subtilis strains. Purified spores of B. subtilis wild type and
mutant strains were heat activated, stimulated to germinate with 10 mM L-valine, and shaken at
37°C, during which the OD600 values were monitored. Values are averages of three assays and error
AC, Rudner DZ. 2017. A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis. PLoS Genet 13:e1007015.
4. Tovar-Rojo F, Chander M, Setlow B, Setlow P. 2002. The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis. J Bacteriol 184:584-587.
5. Chen Y, Barat B, Ray WK, Helm RF, Melville SB, Popham DL. 2019. Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores. J Bacteriol 201(6).
6. Meeske AJ, Rodrigues CD, Brady J, Lim HC, Bernhardt TG, Rudner DZ. 2016. High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis. PLoS Biol 14:e1002341.
7. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, Eichenberger P. 2006. The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358:16-37.
8. Steil L, Serrano M, Henriques AO, Volker U. 2005. Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiol 151:399-420.
9. Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M. 2018. A proximity-tagging system to identify membrane protein–protein interactions. Nature methods 15(9):715-22.
148
APPENDIX A
Supplementary Materials for Chapter 2
149
Table S1. Oligonucleotides used in this work.
Name Sequence (5’-3’) Use
DLP668 AGTAGCCCGGCATTTTTAGC PCR verification of znuA
DLP669 TAGCGGCCTGACTGAACAAG PCR verification of znuA
DLP670 CCGAACCTTGCGCTGATGTC PCR verification of ycnL
DLP671 GCCGCCTTGCTGCTGTTCTT PCR verification of ycnL
DLP672 TCAAGCGGGTGCGGGTAAAT PCR verification of yflS
DLP673 TGTATGCTGAACGGCTAACG PCR verification of yflS
DLP676 GTTCGCTTGGATTTTCATAA PCR verification of yloB
DLP677 CACCCAATTCTTCCCTGTTC PCR verification of yloB
DLP687 GATCCGACTGCCATTCCTGC Construction of chaA::spec
DLP688 CAATAAACCCTTGCCCTCGCTACGCAGAAAGCGGAACACCGGCC Construction of chaA::spec
DLP689 CGTTACGTTATTAGCGAGCCAGTCATGTCATTATGGCGATCGGC Construction of chaA::spec
DLP690 CCAGCCTTGCAGTAAGACGG Construction of chaA::spec
DLP683 ATCGGTACGCAGCTGGCGGC Construction of yugS::mls
DLP684 CGATTATGTCTTTTGCGCAGTCGGCACCATAAATGGTAAGGGGCC Construction of yugS::mls
DLP685 GAGGGTTGCCAGAGTTAAAGGATCCCTCGACGCCGAAGATCACC Construction of yugS::mls
DLP686 GCTTCTTTTGCAGCGACGCC Construction of yugS::mls
DLP675 CTGCTTTTTCGCGTGGATGG PCR verification of chaA::spec
DLP677 CGTAGCGAGGGCAAGGGTTTATTGTTTTCTAAAATCTG PCR verification of chaA::spec
DLP679 CTACGGCTTCCTTCCACCAA PCR verification of yugS::mls
DLP565 GCCGACTGCGCAAAAGACATAATCG PCR verification of yugS::mls
150
Figure S1. Growth and sporulation of putative ion transporter mutant strains of B. subtilis. A) Strains
were grown with shaking in 2xSG medium at 370C and O.D.600 was measured. Time 0 was the estimated
151
time of initiation of sporulation (T0) for each strain. B) Samples were collected for measuring GDH activity
as previously described [1]. Values are averages of three assays and error bars are standard deviations.
C) Samples were removed starting at T3 for measurement of DPA accumulation. DPA was quantified using
a colorimetric assay as previously described [1]. Values are averages of three assays and error bars are
standard deviations.
152
APPENDIX B
Supplementary Materials for Chapter 3
153
Table S2. B. subtilis strains used in this study.
Strain Genotype Source/Construction
DPVB724 gerB CmR FB72 [2]→PS832
DPVB726 gerA SpR gerB CmR FB72 [2]→PS832
DPVB747 skfE MLSR BKE01950a→PS832
DPVB748 pcrB MLSR BKE06600a →PS832
DPVB749 ygaC MLSR BKE08680a →PS832
DPVB750 sipT MLSR BKE14410a →PS832
DPVB751 ylbC MLSR BKE14960a →PS832
DPVB752 hfq MLSR BKE17340a →PS832
DPVB753 yqhL MLSR BKE24540a →PS832
DPVB754 dnaJ MLSR BKE25460a →PS832
DPVB755 yqeF MLSR BKE25700a →PS832
DPVB756 phoR MLSR BKE29100a →PS832
DPVB757 phoP MLSR BKE29110a →PS832
DPVB758 ytxG MLSR BKE29780a →PS832
DPVB759 ytpA MLSR BKE30510a →PS832
DPVB760 yybT MLSR BKE40510a →PS832
DPVB761 gerA-lacZ MLSR PS767 [3, 4]→PS832
DPVB763 skfE MLSR gerB CmR DPVB724→DPVB747
DPVB764 pcrB MLSR gerB CmR DPVB724→DPVB748
DPVB765 ygaC MLSR gerB CmR DPVB724→DPVB749
DPVB766 sipT MLSR gerB CmR DPVB724→DPVB750
DPVB767 ylbC MLSR gerB CmR DPVB724→DPVB751
DPVB768 hfq MLSR gerB CmR DPVB724→DPVB752
DPVB769 yqhL MLSR gerB CmR DPVB724→DPVB753
DPVB770 dnaJ MLSR gerB CmR DPVB724→DPVB754
DPVB771 yqeF MLSR gerB CmR DPVB724→DPVB755
DPVB772 phoR MLSR gerB CmR DPVB724→DPVB756
DPVB773 phoP MLSR gerB CmR DPVB724→DPVB757
DPVB774 ytxG MLSR gerB CmR DPVB724→DPVB758
DPVB775 ytpA MLSR gerB CmR DPVB724→DPVB759
DPVB776 yybT MLSR gerB CmR DPVB724→DPVB760
DPVB805 skfE Cre expression for deletion of MLSR
DPVB806 pcrB Cre expression for deletion of MLSR
DPVB807 ygaC Cre expression for deletion of MLSR
DPVB808 sipT Cre expression for deletion of MLSR
DPVB809 ylbC Cre expression for deletion of MLSR
DPVB810 hfq Cre expression for deletion of MLSR
DPVB811 yqhL Cre expression for deletion of MLSR
DPVB812 dnaJ Cre expression for deletion of MLSR
DPVB813 yqeF Cre expression for deletion of MLSR
DPVB814 phoR Cre expression for deletion of MLSR
DPVB815 phoP Cre expression for deletion of MLSR
DPVB816 ytxG Cre expression for deletion of MLSR
DPVB817 ytpA Cre expression for deletion of MLSR
DPVB818 yybT Cre expression for deletion of MLSR
DPVB819 skfE gerA-lacZ MLSR DPVB761→DPVB805
DPVB820 pcrB gerA-lacZ MLSR DPVB761→DPVB806
154
DPVB821 ygaC gerA-lacZ MLSR DPVB761→DPVB807
DPVB822 sipT gerA-lacZ MLSR DPVB761→DPVB808
DPVB823 ylbC gerA-lacZ MLSR DPVB761→DPVB809
DPVB824 hfq gerA-lacZ MLSR DPVB761→DPVB810
DPVB825 yqhL gerA-lacZ MLSR DPVB761→DPVB811
DPVB826 dnaJ gerA-lacZ MLSR DPVB761→DPVB812
DPVB827 yqeF gerA-lacZ MLSR DPVB761→DPVB813
DPVB828 phoR gerA-lacZ MLSR DPVB761→DPVB814
DPVB829 phoP gerA-lacZ MLSR DPVB761→DPVB815
DPVB830 ytxG gerA-lacZ MLSR DPVB761→DPVB816
DPVB831 ytpA gerA-lacZ MLSR DPVB761→DPVB817
DPVB832 yybT gerA-lacZ MLSR DPVB761→DPVB818
DPVB833 PsspD::gerA MLSR PS3476 [5]
DPVB834 skfE PsspD::gerA MLSR DPVB833→DPVB805
DPVB835 pcrB PsspD::gerA MLSR DPVB833→DPVB806
DPVB836 ygaC PsspD::gerA MLSR DPVB833→DPVB807
DPVB837 sipT PsspD::gerA MLSR DPVB833→DPVB808
DPVB838 ylbC PsspD::gerA MLSR DPVB833→DPVB809
DPVB839 hfq PsspD::gerA MLSR DPVB833→DPVB810
DPVB840 yqhL PsspD::gerA MLSR DPVB833→DPVB811
DPVB841 dnaJ PsspD::gerA MLSR DPVB833→DPVB812
DPVB842 yqeF PsspD::gerA MLSR DPVB833→DPVB813
DPVB843 phoR PsspD::gerA MLSR DPVB833→DPVB814
DPVB844 phoP PsspD::gerA MLSR DPVB833→DPVB815
DPVB845 ytxG PsspD::gerA MLSR DPVB833→DPVB816
DPVB846 ytpA PsspD::gerA MLSR DPVB833→DPVB817
DPVB847 yybT PsspD::gerA MLSR DPVB833→DPVB818 a Strain obtained from the Bacillus Genetic Stock Center
155
Table S3. Long-term germination efficiency of B. subtilis mutant strainsa
Strain Colonies after 24 hr
(cfu/mL/OD) New colonies after 48 hr
(cfu/mL/OD)
Wild type 3.5x108 0
dnaJ 9.6x108 0
hfq 3.2x108 0
pcrB 1.7x108 6.0x106
phoP 3.7x108 0
phoR 3.5x108 0
sipT 1.4x108 4.0x106
skfE 9.2x107 1.0x106
ygaC 4.0x108 1.0x107
ylbC 1.4x108 7.0x106
yqeF 1.7x108 0
yqhL 2.5x108 0
ytpA 1.8x108 0
ytxG 1.2x108 0
yybT 2.8x108 1.0x107
a Values are from a single determination for each strain. Purified spores were serially diluted, plated on 2xSG medium, and incubated at 37°C.
156
Table S4. Spore germination in response to diverse germinants following overexpression of gerA.
Genotype
% OD Loss at 60 mins with 1X AGFK % OD Loss at 40 mins with 2xYT
without sspDp-gerA
with sspDp-gerA without
sspDp-gerA with sspDp-gerA
Wild type 13 ± 4 4 ± 1.3* 34 ± 2 34 ± 1
dnaJ 6 ± 3* 1 ± 2* 23 ± 0* 29 ± 2
hfq 9 ± 5 5 ± 1* 33 ± 1 33 ± 2
pcrB 5 ± 1* 0 ± 1* 31 ± 0 36 ± 3
phoP 8 ± 3 1 ± 1* 33 ± 0 30 ± 1
phoR 5 ± 4* 1 ± 1* 32 ± 3 36 ± 4
sipT 6 ± 6* 0 ± 1* 30 ± 6 33 ± 1
skfE 4 ± 5* 3 ± 1* 32 ± 4 35 ± 0
ygaC 10 ± 1* 1 ± 4* 31 ± 3 32 ± 5
ylbC 5 ± 4* 0 ± 1* 16 ± 2* 31 ± 0
yqeF 5 ± 2* 4 ± 2* 33 ± 5 28 ± 3
yqhL 16 ± 5 0 ± 1* 34 ± 4 30 ± 1
ytpA 18 ± 2 13 ± 1 30 ± 1 33 ± 1
ytxG 7 ± 5 0 ± 2* 26 ± 2* 29 ± 8
yybT 17 ± 2 0 ± 0* 29 ± 1 36 ± 4 a Values are averages and standard deviations of assays on three replicate spore preparations.
OD600 of purified spore suspension monitored at the indicated time after addition of 1X AGFK or
2xYT while shaking at 37°. * indicates a significant difference from the wild type without PsspD-
gerA (p<0.05).
157
Figure S2. Germination rates of B. subtilis strains. Purified spores of B. subtilis wild type and
mutant strains were heat activated, stimulated to germinate by addition of 10 mM L-valine, and
shaken at 37°C, during which the OD600 was monitored. Values are averages of three assays and
20%
40%
60%
80%
100%
0 20 40 60 80 100 120 140 160 180
% I
nitia
l O
D
Time (min)
B
20%
40%
60%
80%
100%
0 20 40 60 100 140 180
% I
nitia
l O
D
Time (min)
C
20%
40%
60%
80%
100%
0 20 40 60 80 100 120 140 160 180
% I
nitia
l O
D
Time (min)
A
158
error bars are standard deviations. Each assay was performed on three replicate spore
preparations. A) Wild type ♦, ytxG X, yqhL ▲, ygaC +, dnaJ ■ B) Wild type ♦, phoR X, hfq +,
sipT ■, yybT ● C) Wild type ♦, sfkE X, pcrB ▲, yqeF +, ytpA ■.
159
Figure S3. Release of DPA and NAM by B. subtilis strains. Purified spores were heat activated,
stimulated to germinate by addition of 10 mM L-valine, and shaken at 37°C. Samples were taken
at designated intervals, centrifuged, and the supernatant was saved for later analysis. Values are
0
2
4
6
0 10 20 30 40 50
DP
A (
µg/m
L)
Time (min)
A
0
20
40
60
0 10 20 30 40 50
Norm
aliz
ed
Pe
ak A
rea
Time (min)
D
0
2
4
6
0 10 20 30 40 50
DP
A (
µg/m
L)
Time (min)
B
0
20
40
60
0 10 20 30 40 50
Norm
aliz
ed
Pe
ak A
rea
Time (min)
E
0
2
4
6
0 10 20 30 40 50
DP
A (
µg/m
L)
Time (min)
C
0
20
40
60
0 10 20 30 40 50
No
rmla
ize
d P
ea
k A
rea
Time (min)
F
160
averages of three assays for DPA (panels A-C) and NAM (panels D-F), and error bars are standard
deviations. Each assay was performed on three replicate spore preparations. Panels A and D:
Wild type ♦, ytxG X, yqhL ▲, ygaC +, dnaJ ■, ylbC ●. Panels B and E: Wild type ♦, phoR X,
phoP ▲, hfq +, sipT ■, yybT ●. Panels C and F: Wild type ♦, sfkE X, pcrB ▲, yqeF +, ytpA ■. For
DPA release: dnaJ, ylbC, yqeF, and ytpA strains are significantly different from the wild type at all
time points; hfq, phoP, sipT, ygaC, and yqhL strains are significantly different from 10 min
onwards; and pcrB, phoR, yybT, and ytxG, strains are not significantly different from the wild
type. For NAM release: sipT , ylbC, and ytpA strains are significantly different from the wild type
from 20 min onwards. Some other strains exhibited reduced NAM release, but this was not found
to be significant due to high variability between replicates.
were subjected to germination conditions, 10 mM L-valine at 37°C, for 1 hour. For each strain,
pixel intensities were averaged for each spore detected in three images, including a total of at
least 100 spores. Blue dots indicate spores classified as phase bright, which had similar intensities
as spores in the initial dormant population, and orange dots indicate spores classified as phase-
dark, in order to determine population percentages.
1,000
11,000
21,000
31,000
41,000
51,000
0 100 200
Sp
ore
m
ea
n p
ixe
l in
ten
sity
Spore number (ylbC ylyA )
1,000
11,000
21,000
31,000
41,000
51,000
0 100 200 300 400
Sp
ore
m
ea
n p
ixe
l in
ten
sity
Spore number (ylbC ylbB yhcV )
1,000
11,000
21,000
31,000
41,000
51,000
0 200 400
Sp
ore
m
ea
n p
ixe
l in
ten
sity
Spore number (ylbB yhcV )
171
Table S5. B. subtilis strains used in this study
Strain Genotype Source/Construction
DPVB724 gerB CmR FB72 [2]→PS832
DPVB751 ylbC MLSR BKE14960a →PS832
DPVB761 gerA-lacZ MLSR PS767 [3, 4]→PS832
DPVB877 ylbB MLSR BKE14950a →PS832
DPVB878 ylyA MLSR BKE15440a →PS832
DPVB880 yhcV KnR BKK09230a →PS832
DPVB881 ylbB KnR BKK14950a →PS832
DPVB882 ylbC KnR BKK14960a →PS832
DPVB883 ylyA KnR BKK15440a →PS832
DPVB890 ylbC MLSR yhcV KnR DPVB880→DPVB751
DPVB891 ylbC MLSR ylyA KnR DPVB883 →DPVB751
DPVB892 ylbB MLSR yhcV KnR DPVB880 →DPVB877
DPVB898 yhcV Cre expression for deletion of KnR
DPVB909 ylbC MLSR ylbB KnR DPVB881 →DPVB751
DPVB910 yhcV ylbC MLSR ylbB KnR DPVB909→DPVB898
DPVB911 ylbB Cre expression for deletion of KnR
DPVB913 ylbC-lacZ CmR pDPV500 →PS832
DPVB915 ylbB KnR gerA-lacZ MLSR DPVB761→DPVB881
DPVB916 ylbC KnR gerA-lacZ MLSR DPVB761→DPVB882
DPVB917 ylyA KnR gerA-lacZ MLSR DPVB761→DPVB883
DPVB918 yhcV KnR gerA-lacZ MLSR DPVB761→DPVB880
DPVB919 ylbB KnR ylbC-lacZ CmR pDPV500 →DPVB881
DPVB920 ylbB ylbC-lacZ CmR pDPV500→DPVB911
DPVB921 ylbB KnR gerB CmR DPVB724 →DPVB881
DPVB922 yhcV KnR gerB CmR DPVB724 →DPVB880
DPVB929 dacFp::ylbB CmR pDPV498 →PS832
DPVB930 ylbC MLSR dacFp::ylbB CmR pDPV498→DPVB751
DPVB936 ylbB gerB CmR DPVB724→DPVB911
DPVB938 ylbB yhcV KnR DPVB880→DPVB911 a Strain obtained from the Bacillus Genetic Stock Center
172
REFERENCES
1. Nicholson WL, Setlow P. 1990. Sporulation, germination, and outgrowth., p 391-450. In Harwood CR, Cutting SM (ed), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
2. Paidhungat M, Setlow B, Driks A, Setlow P. 2000. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512.
3. Feavers IM, Foulkes J, Setlow B, Sun D, Nicholson W, Setlow P, Moir A. 1990. The regulation of transcription of the gerA spore germination operon of Bacillus subtilis. Molec Microbiol 4:275-282.
4. Zuberi AR, Moir A, Feavers IM. 1987. The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168. Gene 51:1-11.
5. Cabrera-Martinez RM, Tovar-Rojo F, Vepachedu VR, Setlow P. 2003. Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. J Bacteriol 185:2457-64.
6. Stewart KA, Setlow P. 2013. Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis. J Bacteriol 195:3575-82.
7. Ramirez-Peralta A, Zhang P, Li YQ, Setlow P. 2012. Effects of sporulation conditions on the germination and germination protein levels of Bacillus subtilis spores. Appl Environ Microbiol 78:2689-97.