East Tennessee State University Digital Commons @ East Tennessee State University Electronic eses and Dissertations Student Works 8-2004 Characterization of a Catechol-Type Siderophore and the Detection of a Possible Outer Membrane Receptor Protein from Rhizobium leguminosarum strain IARI 312. Brianne Lee Clark East Tennessee State University Follow this and additional works at: hps://dc.etsu.edu/etd Part of the Biology Commons is esis - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic eses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. Recommended Citation Clark, Brianne Lee, "Characterization of a Catechol-Type Siderophore and the Detection of a Possible Outer Membrane Receptor Protein from Rhizobium leguminosarum strain IARI 312." (2004). Electronic eses and Dissertations. Paper 922. hps://dc.etsu.edu/ etd/922
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East Tennessee State UniversityDigital Commons @ East
Tennessee State University
Electronic Theses and Dissertations Student Works
8-2004
Characterization of a Catechol-Type Siderophoreand the Detection of a Possible Outer MembraneReceptor Protein from Rhizobium leguminosarumstrain IARI 312.Brianne Lee ClarkEast Tennessee State University
Follow this and additional works at: https://dc.etsu.edu/etd
Part of the Biology Commons
This Thesis - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. Ithas been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee StateUniversity. For more information, please contact [email protected].
Recommended CitationClark, Brianne Lee, "Characterization of a Catechol-Type Siderophore and the Detection of a Possible Outer Membrane ReceptorProtein from Rhizobium leguminosarum strain IARI 312." (2004). Electronic Theses and Dissertations. Paper 922. https://dc.etsu.edu/etd/922
of the amino acid standards and the samples in each respective solvent system, and
their spot patterns. Table 3 lists the Rf values for the amino acid standards and all
samples in each system, as well as the abbreviation for each sample shown in Figure
21.
Figure 21 Detection of Amino Acid in Hydrolyzed Siderophore Sample Using Thin
Layer Chromatography. Amino acid and sample spots on TLC plates in A) methanol:
ammonium acetate (solvent 1) and B) acetonitrile: ammonium acetate (solvent 2)
A B
A C D E F G I L M Q HAc S T V W YA C D E F G I L HAc M N Q S T V W Y
72
Table 3 Rf Values for Amino Acids and Samples. Solvent system 1 (methanol:
ammonium acetate) and solvent system 2 (acetonitrile: ammonium acetate)
The results indicate the possible presence of tyrosine using solvent system 1.
However, the range of Rf values for the amino acids is very small, and many other
Rf Solvent 1 Rf Solvent 2
Alanine (A) .686 .647
Cysteine (C) .797 .696
Aspartic Acid (D) .763
Glutamic Acid (E) .788 .582
Phenylalanine (F) .792 .724
Glycine (G) .734 .622
Histidine (H) .618 .399
Isoleucine (I) .773 .639
Lysine (K) .628 .453
Leucine (L) .749 .701
Methionine (M) .773 .694
Asparagine (N) .725 .617
Proline (P) .657 .557
Glutamine (Q) .662 .591
Arginine (R) .638 .386
Serine (S) .768 .433
Hydrolyzed Serine (HS) .652
Threonine (T) .763 .643
Valine (V) .763 .656
Tryptophan (W) .652 .760
Tyrosine (Y) .802 .741
Acid Hydrolyzed Sample (HAc) .802 .659
Alkaline Hydrolyzed Sample (Hal)
.670
73
amino acids are also close to the sample Rf value. Solvent system 2 indicates the
possible presence of serine or valine. The relevance of the serine being present will be
discussed in the �Discussion� chapter.
Identification of Possible Outer Membrane Receptor Proteins
We wanted to identify the possible outer membrane receptor proteins involved in
ferric-siderophore transport of R. leguminosarum IARI 312. The SDS-PAGE results of
the whole cell pellet and outer membrane fractions are shown below for both the no
iron and high iron cultures (Figure 22). Two bands are visible in all no iron samples
with molecular weights of approximately 78 kDa and 80 kDa, and the bands are absent
in the high iron samples. This indicates repression of these proteins under high iron
conditions, which is consistent with the behavior of the iron-regulated genes of iron
transport systems.
Figure 22 SDS-PAGE of Possible Outer Membrane Receptor Proteins. A 7% SDS-
PAGE of cell pellets and outer membranes of R. leguminosarum IARI 312 grown in no
and high iron optimized Fiss minimal medium. A) mol. wt. marker B) no iron cell pellet
C) high iron cell pellet D) no iron OM- 5 µl E) high iron OM- 5 µl F) no iron OM- 10 µl
G) high iron OM- 10 µl H) no iron OM- 15 µl I) high iron OM- 15µl
A B C D E F G 200k
116k
97.4k
66k
45k
74
The SDS-PAGE results of the whole cell pellet and outer membrane fractions
examining similarity to the FepA protein are shown for both R. leguminosarum Strain
IARI 312 and E. coli BL21(DE3) (Figure 23). Two bands are visible in all no iron
samples with molecular weights of approximately 78 kDa and 80 kDa, and the bands
are absent in the high iron samples. This indicates repression of these proteins under
high iron conditions, which is consistent with the behavior of the iron-regulated genes
of iron transport systems. The results indicate that the 80 kDa band observed in R.
leguminosarum Strain IARI 312 coincides with the 80 kDa FepA band, an outer
membrane receptor for ferric enterobactin expressed in E. coli BL21(DE3) with the
FepA gene in pET17b. The significance of the FepA band will be discussed in the
�Discussion� chapter.
75
Figure 23 SDS-PAGE of Possible Outer Membrane Receptor Proteins Compared to a
Known Outer Membrane Receptor Protein. A 7% SDS-PAGE of cell pellets and outer
membranes of R. leguminosarum Strain IARI 312, grown in low and high iron optimized
Fiss minimal medium, and E. coli BL21(DE3) with FepA in pET17b. (A) Mol. wt. marker
(B) IARI 312 low iron cell pellet (C) E. coli BL21(DE3) cell pellet (D) IARI 312 low iron
OM fraction (E) E. coli BL21(DE3) OM fraction (F) IARI 312 high iron OM fraction (G)
IARI 312 low iron OM fraction (H) E. coli BL21(DE3) OM fraction
A B C D E F G H 200k
116k 97.4k
66k
45k
31k
76
CHAPTER 4
DISCUSSION
Iron is a growth-limiting factor for the majority of microorganisms. It is present in
abundance but is unavailable due to its presence as insoluble iron oxyhydroxide
polymers under aerobic conditions at biological pH. Many gram-negative bacteria
express high affinity iron transport systems to overcome iron deficiency, including
members of rhizobia. Rhizobia induce N2-fixing nodules on the roots of leguminous
plants. The plant produces leghaemoglobin and components of respiration, both of
which contain iron. Because of the great demand for iron as a result of competition
with its host plant, and largely due to the iron content of the bacterial nitrogenase
complex, bacteria synthesize and secrete siderophores to overcome iron deficiency.
Poor nodulation caused by iron-deficiency affects many common agricultural
crops, such as beans and peas. Effective nodulation relies upon persistence of root
nodule bacteria in the soil. Root nodule bacteria vary widely in siderophore production
and type of siderophore produced. Much is known about E. coli and Pseudomonas iron
transport, while the components of iron transport systems of much of the genus
Rhizobium lack the same detail.
The hydroxamate-type siderophores vicibactin and rhizobactin 1021 are the
most characterized of siderophores produced by rhizobia, and also the most common
(Carson et al. 2000). However, other types of siderophores produced by rhizobia
include the carboxylate rhizobactin, vicibactin 7101, citrate, anthranilate, and other
unidentified catechol and hydroxamate-type siderophores (Carson et al. 2000). They
77
are also capable of utilizing haem, haemoglobin, and leghaemoglobin as sources of
iron (Noya et al. 1997, Nienaber et al. 2001). Many rhizobial strains have not even
been evaluated for siderophore production, are identified as CAS positive or negative,
or the siderophore is only identified as being catechol or hydroxamate (Carson et al.
2000). The biosynthesis and uptake systems of rhizobactin 1021 and vicibactin have
also been studied in greater detail, but both are hydroxamate-type siderophores, and
the same attention has not been given to other siderophores produced by rhizobia.
The agricultural importance of rhizobia demonstrates the need for understanding
its iron transport systems. Select species have been studied in greater detail (e.g., S.
meliloti), but because of the variety of species and their associated hosts within
Rhizobium, we decided to further investigate the iron transport systems of rhizobia.
The focus of this thesis is R. leguminosarum Strain IARI 312, obtained from the
Indian Agricultural Research Institute in New Dehli, India. Being a previously unstudied
strain of Rhizobium, our initial goal was to investigate the siderophore-producing
capabilities of the R. leguminosarum Strain IARI 312. Initial detection of siderophore
production was confirmed using the CAS assay, which demonstrated the production of
siderophore under iron-deficient conditions, with repression under high iron conditions.
In order to chemically characterize the siderophore being produced, we used Arnow�s
and Atkin�s methods for detecting catechol-type and hydroxamate-type siderophores,
respectively. The results of the assays indicated that both a catechol-type and a
hydroxamate- type siderophore were being produced under iron-deficient conditions by
R. leguminosarum Strain IARI 312.
78
The detection of both a hydroxamate-type siderophore and a catechol-type
siderophore produced by a strain of R. leguminosarum was interesting because
catechol-type siderophores are much more uncommon in rhizobial species than
hydroxamate-type siderophores (Carson et al. 2000). For this reason, our primary
focus was to purify and characterize the catechol-type siderophore produced by R.
leguminosarum Strain IARI 312. Because purification procedures lead to loss of a
significant amount of sample, the growth conditions of the strain were first explored to
provide conditions for optimum siderophore production.
The modified Fiss minimal medium composition, temperature, and incubation
time were tested to obtain optimum catechol-type siderophore production. The
resulting optimized growth conditions included an increase in the concentrations of
some media components, no iron added to the media, and incubation at 37°C for 24
hours, resulting in a nearly four-fold increase of catechol-type siderophore production
relative to the original growth conditions. The most surprising change in growth
conditions was the increase in the incubation temperature from 27°C to 37°C, because
the organism is a soil bacterium. However, only siderophore production is increased at
37°C, growth of the strain is still best at 27°C.
The optimized growth conditions were utilized to grow R. leguminosarum Strain
IARI 312 in large volume batch cultures. Supernatant collected was acidified and
purified using a XAD-2 column, which binds cyclic compounds. Because siderophores
are cyclic compounds by nature, the column should bind any siderophore produced.
Siderophore content of the collected fractions were confirmed using TLC, as described
earlier, and Arnow�s assay. The sample was then further purified using a hydrophobic
Sephadex LH20 column, which separates compounds based on their hydrophobicity,
with methanol as an eluting solvent. Catechol-type siderophores are highly
79
hydrophobic, should bind with a higher affinity than compounds with low
hydrophobicity, and so were eluted in the latter fractions. Hydroxamate-type
siderophores are fairly hydrophilic, binding with less affinity, and were eluted in earlier
fractions. This purification method allowed the separation of the catechol-type
siderophore and the hydroxamate-type siderophore produced by R. leguminosarum
Strain IARI 312, as well as other cyclic impurities that might have present. The
collected fractions were tested for siderophore content using TLC.
In order to minimize contaminating cyclic compounds, the collected fractions
testing positive for catechol-type siderophore were dried by rotary evaporation,
redissolved in methanol, and again purified using the LH20 column. The fractions
testing positive for catechol-type siderophore were again dried and stored at -20°C until
chemically characterized. The siderophore was stored in a dry state because catechol-
type siderophores are highly unstable and are easily oxidized.
The purified catechol-type siderophore was chemically characterized using a
number of methods. UV spectroscopy compared the UV absorbance spectra of the
sample to those of 2,3-DHBA, 2,4-DHBA, 2,5-DHBA, and 3,4-DHBA standards, NMR
spectroscopy showed the functional groups of the sample, analytical HPLC gave
retention times of the sample compared with the standards, and cyclic voltammetry was
performed to analyze the oxidation and reduction potential of the sample compared to
the standards. All analyses detected 2,3-DHBA as a component of the siderophore
produced by R. leguminosarum Strain IARI 312. Amino acid analysis of the hydrolyzed
sample yielded a number of possible amino acids as conjugates of the siderophore,
including serine.
The catechol-type siderophore produced by R. leguminosarum Strain IARI 312
seemed similar to enterobactin, a trimer of 2,3-dihydroxybenzoylserine, composed of
both 2,3-DHBA and serine. To investigate the mass of the catechol-type siderophore,
80
ESMS spectroscopy was performed. The ESMS instrument was equipped with
analytical HPLC (LC/MS), which separated the sample into pure compounds, and each
peak was analyzed separately using ESMS. The data were analyzed using the
structural program MS Interpreter, distributed by the National Institute of Standard and
Technology (NIST), which yielded the structures drawn by ChemSketch, distributed by
Advanced Chemistry Development (ACD).
The molecular masses of fragments obtained in ESMS were consistent with the
previously reported ESMS analysis of enterobactin, a catechol-type siderophore
produced by E. coli (Berner et al. 1991).The structural results of the sample are shown
in Figure 24, which shows the structures of intact enterobactin, with a molecular weight
of 669, hydrolyzed enterobactin, with a molecular weight of 687, and methylated
enterobactin, with a molecular weight of 701, consistent with the production of
enterobactin by R. leguminosarum Strain IARI 312. Enterobactin production is also
supported by the identification of serine as one of the possible amino acid conjugates
using TLC because enterobactin in composed of 2,3-DHBA and serine (tris-(N-(2,3-
dihydroxaybenzoyl)serine)) (Ehmann et al. 1999). The ESMS spectra shown in figure
20 illustrates that methylated enterobactin is the predominant product in the sample.
This is not surprising given the purification of the sample using methanol.
The detection of enterobactin production by a strain of rhizobia is not as
surprising as one might expect. Rhizobia are already known to express an outer
membrane receptor FhuA homologue (Yeoman et al. 2000). FhuA is the outer
membrane receptor protein for ferrichrome, a hydroxamate-type siderophore produced
by fungi, yet is commonly found in E. coli and other Enterobacteriaceae (Crosa and
Walsh 2002). In addition, enterobactin has been shown to be produced by plant-
associated bacteria, including Enterobacter cloacae (Loper & Henkels1999) and other
nitrogen-fixing bacteria including Klebsiella pneumoniae (Höfte 1993).
81
Figure 24 Proposed Catechol-type Siderophore Structures. A) intact enterobactin B)
hydrolyzed enterobactin C) methylated enterobactin
Because ferric siderophores are transported across the outer membrane with the
help of outer membrane receptor proteins, it was of interest to possibly identify outer
membrane receptor proteins produced by R. leguminosarum Strain IARI 312. SDS-
PAGE analysis of the outer membrane fractions from the organism grown under iron-
deficient conditions clearly indicates the expression of two distinct outer membrane
proteins of approximately 78 kDa and 80 kDa, which were completely repressed under
A
C
B
82
high iron conditions. It is very likely that these proteins are involved in the siderophore
transport because the molecular weights of these proteins are similar to the outer
membrane ferric siderophore receptor proteins reported so far and are repressed under
high iron conditions. The outer membrane fraction of R. leguminosarum Strain IARI
312 was also compared with the outer membrane fraction of E. coli BL21(DE3) with the
FepA gene in pET17b using SDS-PAGE analysis to confirm the possibility of a FepA
homologue. The results indicate that the 80 kDa band observed in R. leguminosarum
Strain IARI 312 coincides with the FepA band, an outer membrane receptor for ferric
enterobactin expressed in E. coli BL21(DE3). Thus, R. leguminosarum Strain IARI 312
may be expressing a FepA homologue, although this should be confirmed with western
blot analysis using a monoclonal antibody against FepA.
Future studies on R. leguminosarum Strain IARI 312 could include purification
and identification of the outer membrane receptor proteins, as well as characterization
of the hydroxamate-type siderophore. Because this is the first report of enterobactin
production in rhizobia, it would be of interest to know what type of hydroxamate
siderophore is produced by this strain. Also, it is important to confirm the identity of this
organism as Rhizobium. In addition, iron uptake studies using radiolabeled
siderophore will be useful to characterize the kinetics of tranport.
83
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VITA
BRIANNE L. CLARK
Personal Data: Date of Birth: August 14, 1979 Place of Birth: Columbus, Georgia Marital Status: Married Education: Bearden High School, Knoxville, Tennessee East Tennessee State University, Johnson City, Tennessee;
Health Sciences, B.S., 2001 East Tennessee State University, Johnson City, Tennessee;
Biology, M.S., 2003 Professional Experience: Graduate Assistant, East Tennessee State University, College of
Public and Allied Health, 2001 � 2004 Publications: Moretz, S.E., Clark, B.L., and Lampson, B. C. (2002) Siderophore
production by selected species within the genus Rhodococcus. Abstract, 213. 18th Annual Student Research Forum, East Tennessee State University, Johnson City, TN.
Clark, B.E., Storey, E.P., Mohseni, R., and Chakracborty, R.N. (2003) Characterization of a catechol-type siderphore and the detection of an outer membrane receptor protein from Rhizobium leguminosarum RL 312. Abstract, 103th Annual General Meeting for the American Society for Microbiology.
Clark, B.E., Storey, E.P., Mohseni, R., Little, J., and Chakraborty,
R.N. (2004) Characterization of hydroxamate and enterobactin-like siderophore production by Rhizobium leguminosarim RL 312. Abstract, 104th Annual General Meeting for the American Society for Microbiology.
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Honors and Awards: ASM member 2001-2003 Dean�s Graduate Student Research Award. (2004) College of
Public and Allied Health, East Tennessee State University, Johnson City, Tennessee.