Abstract Listeria monocytogenes is an ubiquitous gram-positive, opportunistic food-borne human and animal pathogen. To date, five L. monocytogenes au- tolysins have been characterized: p60, p45, Ami, MurA and Auto and the preliminary results of our studies show that FlaA, a flagellar protein of L. monocytoge- nes, also has murein-degrading activity. In this study, a gene coding a 144 kDa protein (Lmo0327) with murein hydrolase activity was identified from a lambda Zap expression library of L. monocytogenes EGD genomic DNA, using a direct screening protocol involving the plating of infected Escherichia coli XL1-blue MRF¢ cells onto medium containing Bacillus subtilis murein, a substrate for autolytic proteins. Protein Lmo0327 has a signal sequence, a N-terminal LRR domain and a C-terminal wall-anchoring LPXTG motif. In order to examine the roles of this enzyme and the putative transcription regulator coded by gene lmo0326 located upstream of lmo0327, both structural genes were in- sertionally inactivated by site-specific integration of a temperature-sensitive plasmid. We show that Lmo0327 is a surface protein covalently linked to murein and that the putative transcription regulator Lmo0326 can be assumed to positively regulate the expression of gene lmo0327. The enzyme, which we have shown to have murein-hydrolysing activity, plays a role in cell separation and murein turnover. Keywords Listeria monocytogenes Murein Muramidases Autolysin Abbreviations DEPC Diethylpyrocarbonate EDTA Ethylenediaminetetraacetic acid HPLC High pressure liquid chromatography IPTG Isopropyl-beta-D- thiogalactopyranoside PMSF Phenylmethylsulfonylfluoride SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Introduction Bacterial murein hydrolases, also referred to as auto- lysins, are present in all bacteria synthesizing cell wall murein (Ghuysen et al. 1969). The possibility that au- tolysins are involved in selective hydrolysis of bonds in murein has led to suggest that they are involved in numerous cellular processes including cell growth, cell- wall turnover, murein maturation, cell division, sepa- ration, differentiation and pathogenicity (Berry et al. 1992, Shockman and Ho ¨ ltje 1994; Blackman et al. 1998). The development of renaturing SDS-PAGE and the construction of mutants inactivated in specific struc- tural genes have allowed the description of the auto- lytic complement of several bacteria. Analysis of the Bacillus subtilis genome has revealed the presence of more than 30 potential murein hydrolases (Smith et al. M. Popowska (&) Z. Markiewicz Department of General Microbiology, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland e-mail: [email protected]Arch Microbiol (2006) 186:69–86 DOI 10.1007/s00203-006-0122-8 123 ORIGINAL PAPER Characterization of Listeria monocytogenes protein Lmo0327 with murein hydrolase activity Magdalena Popowska Zdzislaw Markiewicz Received: 1 February 2006 / Revised: 24 April 2006 / Accepted: 11 May 2006 / Published online: 9 June 2006 ȑ Springer-Verlag 2006
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Abstract Listeria monocytogenes is an ubiquitous
gram-positive, opportunistic food-borne human and
animal pathogen. To date, five L. monocytogenes au-
tolysins have been characterized: p60, p45, Ami, MurA
and Auto and the preliminary results of our studies
show that FlaA, a flagellar protein of L. monocytoge-
nes, also has murein-degrading activity. In this study, a
gene coding a 144 kDa protein (Lmo0327) with murein
hydrolase activity was identified from a lambda Zap
expression library of L. monocytogenes EGD genomic
DNA, using a direct screening protocol involving the
plating of infected Escherichia coli XL1-blue MRF¢cells onto medium containing Bacillus subtilis murein,
a substrate for autolytic proteins. Protein Lmo0327 has
a signal sequence, a N-terminal LRR domain and a
C-terminal wall-anchoring LPXTG motif. In order to
examine the roles of this enzyme and the putative
transcription regulator coded by gene lmo0326 located
upstream of lmo0327, both structural genes were in-
sertionally inactivated by site-specific integration of a
temperature-sensitive plasmid. We show that Lmo0327
is a surface protein covalently linked to murein and
that the putative transcription regulator Lmo0326 can
be assumed to positively regulate the expression of
gene lmo0327. The enzyme, which we have shown to
have murein-hydrolysing activity, plays a role in cell
ration, differentiation and pathogenicity (Berry et al.
1992, Shockman and Holtje 1994; Blackman et al.
1998).
The development of renaturing SDS-PAGE and the
construction of mutants inactivated in specific struc-
tural genes have allowed the description of the auto-
lytic complement of several bacteria. Analysis of the
Bacillus subtilis genome has revealed the presence of
more than 30 potential murein hydrolases (Smith et al.
M. Popowska (&) Æ Z. MarkiewiczDepartment of General Microbiology,Institute of Microbiology, Warsaw University,Miecznikowa 1, 02-096 Warsaw, Polande-mail: [email protected]
Arch Microbiol (2006) 186:69–86
DOI 10.1007/s00203-006-0122-8
123
ORIGINAL PAPER
Characterization of Listeria monocytogenes protein Lmo0327with murein hydrolase activity
Magdalena Popowska Æ Zdzislaw Markiewicz
Received: 1 February 2006 / Revised: 24 April 2006 / Accepted: 11 May 2006 / Published online: 9 June 2006� Springer-Verlag 2006
2000) whereas zymographic analysis for Staphylococ-
cus aureus has revealed at least 20 lytic bands, although
>97% of the profile is due to processed forms of the
major bifunctional autolysin, Atl (Foster 1995; Oshida
et al. 1995).
Listeria monocytogenes is an ubiquitous, opportu-
nistic pathogen, which causes relatively infrequent but
often very serious food-borne infections in humans and
animals (Southwick and Purich 1996; Farber 1991;
Schlech 2000; Hof 2004). The infections are particularly
severe for newborns and immunocompromised indi-
viduals. L. monocytogenes has received much attention
and an impressive amount of data accumulated in recent
years has made this bacterium one of the best charac-
terized intracellular pathogens. L. monocytogenes in-
duces its own uptake into non-phagocytic mammalian
cells and then moves within the cells and spreads from
one cell to another by virtue of actin-based motility
(Portnoy et al. 1992; Cossart 1998, 2001; Braun and
Cossart 2000). Each step of the infection process is
dependent upon the production of virulence factors,
including InlA and InlB for entry, listeriolysin O (LLO)
and phospholipase (PI-PLC and Pc-PLC) for escape
from the primary and secondary vacuoles and ActA for
intra and intercellular movements. Expression of these
virulence factors is controlled by the pleiotropic tran-
scriptional activator PrfA (Vazquez-Boland et al. 2001).
The nature of the molecular association of some of these
proteins with the cell surface of L. monocytogenes has
also been elucidated in some detail (Lebrun et al. 1996).
The complete genome sequence of L. monocytogenes
strain EGD-e and of the non-pathogenic species L. in-
nocua (Glaser et al. 2001; Chakraborty et al. 2000) re-
vealed a large number of surface proteins (Cabanes
et al. 2002), including surface-bound autolytic enzymes.
To date, five L. monocytogenes autolysins have been
identified: p60 (syn. CwhA), p45, Ami, MurA and Auto
(Kuhn and Goebel 1989; Bubert et al. 1992; McLaugh-
lan and Foster 1997, 1998; Schubert et al. 2000; Park
et al. 2000; Milohanic et al. 2001; Lenz et al. 2003; Car-
roll et al. 2003; Pilgrim et al. 2003a, b; Cabanes et al.
2004). We have recently identified a putative sixth lis-
terial murein-degrading enzyme, which has been iden-
tified as FlaA, a flagellar protein of L. monocytogenes
(Popowska and Markiewicz 2004).
Analysis of the L. monocytogenes genome reveals
the presence of 11 proteins with murein hydrolysis
domain, thus possibly several autolysins are still
unidentified. In this study, we have used a direct
screening method for the isolation of autolysin-coding
genes. We describe the cloning and sequencing of a
gene coding a surface protein of L. monocytogenes that
functions as an autolysin. Sequence comparison with
the complete genome sequence of L. monocytogenes
strain EGD revealed 100% homology to the internalin-
like protein Lmo0327. This protein represents a new
class of Gram-positive protein with murein lytic
activity that has two characteristic features: an N-ter-
minal LRR domain and C-terminal LPXTG motif.
Materials and methods
Bacterial strains, plasmids, primers, and growth
conditions
Listeria monocytogenes strains were grown in Tryptic
Soy Broth (TSB; Oxoid) at 37�C with constant shaking
(150 r.p.m.) unless otherwise stated, or on TSB plates
(1%, w/v agar). E. coli XLOLR and XL-1 Blue MRF’
were grown in Luria-Bertani broth [LBL or LB agar
(1%, w/v) at 37�C]. Ampicillin or kanamycin (100 lg/
ml) and erythromycin (300 lg/ml for E. coli and
1–5 lg/ml for L. monocytogenes) were added to broth
Collection of the Institute ofMicrobiology, Warsaw University
Bacillus subtilis Wild type Collection of the Institute ofMicrobiology, Warsaw University
Micrococcus luteus Wild type Collection of the Institute ofMicrobiology, Warsaw University
Table 2 Plasmids used and constructed in this study
Plasmids Marker Genotype, description Reference or source
pBK-CMV Kmr 4.5 kb; ori ColE1, MCS, LacZa StratagenepBGS18 Kmr 3.7 kb; ori ColE1, MCS, LacZa Spratt et al. (1986)pGem Apr 3.0 kb; ori ColE1, MCS, LacZa PromegaZAP Express wektor Dcl, Datt, Dint, Dxis, DKH54, Dnin5, red–gam+, bio StratagenepAUL-A Eryr 9.2 kb; ori z pMB9, MCS, LacZa thermosensitive replicon
from pE194Chakraborty et al. (1992)
pAUL-A::lmo0327 Eryr 9.7 kb; 0.5 kb fragment of gene lmo0327 from pMPZ6,cloned into the BamHI/EcoRI (MCS) site in pAULA
This work
pAUL-A::lmo0326 Eryr 9.7 kb; 0.5 kb, amplified by PCR, fragment of genelmo0326 cloned into the EcoRI (MCS) site in pAULA
This work
pMPZ6 Kmr 9.0 kb; 4.5 kb of Sau3A-Sau3A fragment of L. monocyt-ogenes DNA cloned into the BamHI (MCS) site of pBK-CMV
This work
pMPZ6/A Kmr 9.0 kb; 4.5 kb insert cloned in transverse orientation withregard to Plac
This work
pMPZ6/1 Kmr PMPZ6 with deletion of XhoI restriction site This workpMPZ6/2 Kmr PMPZ6 with deletion of ClaI restriction site This workpMPZ6/3 Kmr PMPZ6 with deletion of terminal 0.7 kb ClaI/ClaI site This workpMPZ6/4 Kmr PMPZ6 (3.2 kb) with deletion of initial 1,258 kb SmaI/
AccI siteThis work
MCS multiple cloning site
Arch Microbiol (2006) 186:69–86 71
123
Identification of lytic enzyme structural gene
A k ZAP Express library of 2–10 kb L. monocytogenes
EGD chromosomal DNA, partially digested with
Sau3AI, was constructed according to the manufac-
turer’s instructions (Stratagene), in BamHI-cut and
dephosphorylated vector. A total of 1·105 independent
insert-containing clones were produced and the library
amplified once. The library was screened for lytic
enzyme-producing clones by the plate assay, described
previously, using bacterial cell walls as the substrate
(Foster 1992). Lytic-enzyme-producing clones were
identified by a zone of clearing in the opaque wall
background and were picked and purified by further
rounds of plate screening. Purified clones were treated
to excise the phagemid (pBK-CMV) containing the
L. monocytogenes DNA inserts, according to the
manufacturer’s protocol (Stratagene), and clones were
checked for recombinant lytic activity by a plate test.
DNA was isolated from these clones and characterized
by restriction analysis.
Molecular analysis of the lytic enzyme clones
Several clones were prepared using restriction enzymes
based on the restriction map of the DNA insert and
used for sequencing. Synthetic oligonucleotide frag-
ments based on DNA sequence generated during this
work, or vector sequences, were used as primers.
Putative open reading frames were examined using the
Clone (Sci Ed Sofware) program and deduced amino
acid sequences were compared to the NCBI Entrez
protein databases by BLAST searches (http://
www.ncbi.nlm.nih.gov/BLAST/). Possible transcription
promoters were found using BDGP search tool (http://
www.fruitfly.org/seq_tools/promoter).
Mutational analysis of pMPZ6
To carry out mutational analysis, several derivatives of
plasmid pMPZ6 were constructed, pMPZ6/1-2 (Ta-
ble 2), in each of which a single gene was inactivated.
Mutations were introduced by digesting the plasmid (at
a unique site) with a restriction enzyme, removing the
single-stranded ends formed and ligating the obtained
linear fragments. Since the change (introduced in the
beginning region of each gene) embraced an 8 bp
segment, mutants with altered reading frame and
consequently not producing a given protein, were ob-
tained. Also, a deletion mutant lacking the terminal
700 bp fragment of ORF1–pMPZ6/3 and a second one
lacking the initial 1,258 bp fragment of pMPZ6 insert–
pMPZ6/4, were constructed (Table 2).
Preparation of fractions containing recombinant
enzyme
For biochemical analysis, 4 l cultures of E. coli con-
taining pMPZ6 were grown in the presence of IPTG
(1 mM), to OD600 1.5 after inoculation with 0.01·vol.
overnight culture. The cells were harvested by centri-
fugation (11,000g, 4�C, 10 min) and broken by soni-
cation in 40 ml 50 mM Tris/HCl (pH 7.5), 200 mM
NaCl, 0.5 mM PMSF at 4�C. After removal of debris
by centrifugation (100,000g, 4�C, 2 h), the supernatant
was dialyzed overnight against 100 volumes of 20 mM
potassium phosphate buffer (pH 7.4) at 4�C. The
dialysate was then centrifuged (100,000g; 4�C, 2 h) and
the pellet was resuspended in 20 mM Tris/HCl (pH
7.5) containing 200 mM NaCl, stored at –20�C and
used directly as the enzyme source (McLaughlan and
Foster 1998).
Assay for autolytic activity
Lytic activity of the enzyme extract was determined
spectrophotometrically as the ability of the extract to
decrease the optical density of a cell wall suspension.
Unless stated otherwise, the reaction contained 1 ml
20 mM Tris/HCl (pH 7.5), 200 mM NaCl and purified
cell walls to a final OD450 of 0.3 (Foster 1991).
Insertional inactivation of the autolysin-encoding
gene
Either (a) 530 bp fragment of gene lmo0326, or (b)
557 bp fragment of gene lmo0327, was cloned into
vector pAUL-A (Emr) (Schaferkordt and Chakraborty
1995). The fragments were amplified from chro-
mosomal DNA of strain EGD by PCR using the
Table 3 Primers used and constructed in this study
fied B. subtilis cell walls (McLaughlan and Foster 1998)
or lyophilized Micrococcus luteus ATCC 4,698 cells
(Sigma). To allow for protein renaturation after elec-
trophoresis, the gels were gently shaken at room tem-
perature for 48 h, with one to three changes of 300 ml
of 25 mM Tris–HCl (pH 7.5) containing 1% (v/v)
Triton X-100. Bands of murolytic activity were visual-
ized by staining with 1% (w/v) methylene blue (Sigma)
in 0.01% (w/v) KOH and subsequent (1 to 4 h)
destaining with distilled water. Murein hydrolase
activity was detected as zones of clearing in the blue-
stained cell wall background.
Purification of murein
The cell pellet was suspended in ice-cold water and
added dropwise to the same amount of boiling 8% SDS
with vigorous stirring throughout. The samples were
kept boiling for 30 min and then allowed to stand at
room temperature overnight. Sacculi were collected by
centrifugation (30 min, 150,000g at 22�C) and the pel-
let was washed with room temperature water five
times. After each wash, the pellet was resuspended
homogeneously and centrifuged in the same condi-
tions. The SDS-free pellet was suspended in 49%
hydrofluoric acid and incubated for 40 h with stirring in
an ice bath to remove teichoic acid. The murein was
recovered by centrifugation as above and washed
Arch Microbiol (2006) 186:69–86 73
123
repeatedly to remove all hydrofluoric acid, suspended
in 10 mM Tris-HCl buffer, pH 7.0 and treated with
a-amylase (100 lg/ml) for 2 h at 37�C, then pre-
digested pronase E (200 lg/ml) was added and the
incubation was continued for 90 min at 60�C (Glauner
1988). Finally, the sample was mixed with 8% SDS and
incubated for 15 min at 100�C. SDS was removed by
washing and centrifugations as described above.
N-acetylation of murein was performed with acetic
anhydride in the presence of NaHCO3 according to
Hayashi et al. (1973). Murein and muropeptide
concentrations were calculated from their diamino acid
content. Samples were hydrolyzed in 6 N HCl (12 h,
105�C), vacuum dried and resuspended in an appro-
priate volume of distilled water.
High performance liquid chromatography (HPLC)
analysis of murein
N-acetylated murein prepared from the parental strain
and mutants was analyzed after digestion with the
muramidase Cellosyl (Hoechst AG) by HPLC using
the conditions described in detail by Glauner (1988) on
Hypersil RP18 column (250 mm·4 mm, particle size
3 lm diameter; Teknochroma). The elution buffers
were 50 mM sodium phosphate, pH 4.35 (A) and 15%
methanol in 75 mM sodium phosphate, pH 4.95 (B).
Elution conditions were 7 min isocratic elution in
buffer A, 115 of linear gradient to 100% buffer B and
28 min of isocratic elution in buffer B. The flow rate
was 0.5 ml/ml and the column temperature was 55�C.
The separated muropeptides were detected by UV-
absorption at 205 nm.
Autolysis of cell wall of L. monocytogenes
After sonication as above, the walls were sedimented
by centrifugation, washed in 10 or 50 mM Tris-hydro-
chloride buffer and resuspended in the same buffer
pre-warmed to 30 or 37�C. The suspension was incu-
bated with shaking at 30 or 37�C and changes in
absorbance were followed at 600 nm.
Cell autolysis
Culture of mutants and wild-type L. monocytogenes
strains were grown to early exponential phase (OD600
0.20) in TSB broth. To determine the effect of antibi-
otic-induced lysis, 1.20 lg penicillin G/ml (10·MIC),
1.20 lg imipenem/ml (10·MIC) or 9.6 lg nisin/ml
(4·MIC) was added, and the lysis of the culture was
followed spectrophotometrically (Novaspec II spec-
trophotometer LKB-13 Pharmacia) while continuing
incubation at 37�C with shaking. To determine the
effect of Triton X-100, the cells were grown to
OD600~0.6, harvested and resuspended in 50 mM Tris/
HCL (pH 7.5), 0.1% (v/v) Triton X-100. The lysis of
the suspension at 37�C was followed spectrophoto-
metrically.
Haemolytic activities
Bacteria were cultured on sheep blood agar at 37�C for
36 h. L. innocua and L. ivanovii were used as a nega-
tive and positive control, respectively. After incuba-
tion, the narrow ring of b-hemolysis produced by the
mutants was compared with that of L. monocytogenes
EGD.
Turnover of murein
Overnight cultures of L. monocytogenes strains were
inoculated 1:20 into warm (37�C) TSB medium sup-
plemented with [3H]N-acetyloglucosamine to 10 lCi/
ml (specific activity). When the culture reached the
value of OD A600–0.6 the culture fluid was removed
using a 0.22 lm Millipore nitrocellulose filter. The cells
deposited on the filter were washed with fresh, warm
(37�C) medium containing non-radiolabeled N-acety-
loglucosoamine (100 lg/ml). The bacteria were then
washed off the filter with fresh pre-warmed portion of
TSB and incubated with shaking. At 20 min intervals,
samples of the culture were removed and added to an
equal volume of boiling SDS solution. The samples
were boiled for 30 min, after which they were passed
through a 0.22 lm Millipore filter and the crude sacculi
remaining on the filters were washed with saline. The
filters were dried and the radioactivity remaining on
them was determined in Beckman type LS 355 scin-
tillation counter. Radioactivity at time ‘‘0’’ was taken
as 100%.
Modeling of the structure of Lmo0327
The potential tertiary structure of the LRR domain of
Lmo0327 was created based on ORFeus search server
available to the academic community via Structure
Prediction Meta Server (http://www.BioInfo.PL/Meta/)
(Ginalski et al. 2003) and BLAST and FFAS Software
(Rychlewski et al. 2000) (http://www.ncbi.nlm.nih.gov/
BLAST/). For molecular graphics visualization, Ra-
sMol program (version 2.7.2.1) from RCSB PDB
Software (http://www.rcsb.org/pdb/software-list) was
used. The atomic coordinates of the modeled LRR
structures are available at http://www.cmm.info.-
nih.gov/kajava.
74 Arch Microbiol (2006) 186:69–86
123
Results
Identification of lytic enzyme structural genes
A k Zap library of L. monocytogenes genomic DNA
was screened for lytic-enzyme-producing clones by a
direct screening protocol. Lytic enzyme clones are
characterized by a zone of clearing in the opaque wall
which overlays the plaque. After an initial screening of
approximately 2·103 insert-containing clones, a total of
10 lytic-enzyme-producing clones were isolated using
B. subtilis cell walls as substrate. The putative phage
clones were purified by re-screening prior to excision
as phagemids.
The ability of the clones to hydrolyse the cell walls
was not IPTG-dependent. E. coli expressing pBK-
CMV (vector without insert) showed no activity on
wall overlays. Restriction analysis and Southern blot-
ting (Kit DIG-High Prime, Boehringer Mannheim,
Germany) revealed the isolation of three distinct lytic-
enzyme-encoding genes. One of these genes was
present in three of the clones originally isolated by
plating on B. subtilis walls. For subsequent experi-
ments plasmid pMPZ6 containing an insert of 4,472 kb
was used for sequencing and further characterization.
Renaturing gel analysis
Cells of E. coli XLOLR strains expressing the re-
combinant enzymes were harvested at mid-exponential
phase (OD600 1.2) and cell extracts were prepared by
sonication and SDS method. The protein samples were
analyzed by renaturing SDS-PAGE. Lytic bands with
different molecular mass were seen in extracts from E.
coli XLOLR (pMPZ6). The E. coli XLOLR (pBK-
CMV) extract (plasmid without insert) showed no lytic
enzyme activity (results not shown).
Mutational analysis of pMPZ6
Cell suspensions of E. coli carrying derivatives of
plasmid pMPZ6 constructed by us, pMPZ6/1–4 (Ta-
ble 2) were disrupted with ultrasonic waves and the
soluble fractions were taken for determination of
murein-hydrolyzing activity. Electrophoresis in 12%
polyacrylamide gel was performed, with protein-free
cell wall of B. subtilis incorporated in the gel matrix
(zymogram). No murein-hydrolysing activity was ob-
served for pMPZ6/1–3, even when the incubation time
was prolonged to ten days, otherwise than for pMPZ6/
4 and parental pMPZ6 plasmids. Experiments in which
the orientation of the cloned DNA was reversed with
respect to the promotor of gene lacZ, pMPZ6/A (Ta-
ble 2) demonstrated that the gene coding for a protein
with murein hydrolase properties is expressed from its
own promoter. The change in the orientation of the
insert did not affect the production of active protein.
Construct pMPZ6/4 (3217 kb), shortened by a 1.2 kb
fragment, which also showed zymographic activity, was
used for further analysis.
Sequencing of the lytic enzyme clones
and identification of a new L. monocytogenes
protein with autolytic activity
Sequence analysis
The DNA sequence of the pMPZ6/4 insert was deter-
mined and three putative ORFs were identified
(Fig. 1). A comparison of all obtained sequences re-
vealed 100% identity and enabled the identification of
the cloned genes. It is worth mentioning that in the
case of all identified L. monocytogenes genes, coun-
terparts in the L. innocua genome were identified.
Fig. 1 Genetic map of fragment of L. monocytogenes EGDgenome cloned into pMPZ6 (4,472 bp) and pMPZ6/4 (3,217 bp).Open reading frames and direction of transcription are indicated
with arrows. The broken line indicates the sequence of listerialgenome embracing regions lmo0323 and lmo0327, which werenot present in the sequenced fragment. T putative terminators
Arch Microbiol (2006) 186:69–86 75
123
The 3.217 kb pMPZ6 insert contains three genes,
from lmo0325 to lmo0327 (Fig. 1). The two genes
lmo0325 and lmo0326 encode putative positive tran-
scriptional regulators of the Rgg type with typical
conserved HTH XRE domain. A comparison of the
sequences of proteins Lmo0325 and Lmo0326 revealed
high homology (32% identity, 58% similarity). The
greatest similarity is in the N-terminal part of both
proteins, in which the HTH XRE domains are located.
Gene lmo0327 is incomplete in the cloned insert,
1,036 out of 4,101 bp, and codes only the N-terminal
segment of the protein that is 310 amino acids long.
The presence of a proline/glycine-rich region
(LPXTG) (Schubert et al. 2001) in protein Lmo0327
indicates covalent binding to murein. Further searches
in the Listeria genome database (Glaser et al. 2001;
Cabanes et al. 2002) indicated the presence of other
served domain LRR (leucine reach repeat) character-
istic for internalin, which consists of tandem repeats of
20–22 amino acids with conserved leucine or isoleucine
residues. The potential LRR domain of protein
Lmo0327 is composed of 151 amino acids (from amino-
acid 25 to 176) (Fig. 2b, c). LRRs are known to be
involved in protein–protein interactions and in a vari-
ety of functions, such as adhesion, ligand-receptor
interactions and signalling (Kajava 1998; 2002). The
central part of the protein contains regions of amino
acid repeats with varied length (Fig. 2c), and in this
part of the protein, no known domains have been
identified. A comparison of the amino acid sequence of
the protein (BLAST), both of entire protein and only
its N-terminal end, revealed a similarity to murein-
bound surface proteins, such as the internalins and
other leucine-rich proteins. Protein database searches
showed that the Lmo0327 protein of L. monocytogenes
EGD and L. innocua (NP469697) are highly con-
served, with 95% identities at the amino acid level. At
this stage of our studies (taking into account the results
of sequence analysis of all the genes cloned into
pMPZ6), we formulated the hypothesis that the pro-
tein responsible for the studied murolytic activity is
Lmo0327 (N-terminal part) and that its expression may
Fig. 2 a Schematicrepresentation of domainstructure of protein Lmo0327.b Structure of LRR domain ofLmo0327. Secondarystructure is indicated by bars(a-helices) and arrows(b strands). c Amino acidsequence of the Lmo0327.The signal peptide is shown inbold, and the LRR domain(LRR 1–5) is shaded in darkgrey. Regions of amino acidrepeats are shaded in grey andLPXTG motif is shown inbold and underlined
76 Arch Microbiol (2006) 186:69–86
123
depend on the presence of potential transcription
activators coded upstream of lmo0327.
In silico modeling of the structure of Lmo0327 and
analysis of its potential tertiary structure
Available data bases contain crystallographic models
elaborated for the internalin proteins of L. mono-
cytogenes InlA, InlB and InlH (Kajava and Kobe
2002). Computer analysis of the amino acid sequence
of the LRR domain of Lmo0327 and of the
remaining part of the protein, enabled determination
of the probable tertiary structures of both parts. The
N-terminal domain—LRR (151 aa) forms a right-
handed beta-helix with a turn after each repeat of
the amino acid sequence. This model structurally
corresponds to an analogous domain identified in
proteins belonging to the very large internalin family
(Fig. 2b). Nucleotide sequence analysis of the
L. monocytogenes, genome identified 20 proteins of
this type in this bacterium (Cabanes 2002). The
spatial arrangement of the N-terminal domain prob-
ably enables more effective binding to other proteins,
as in the binding of InlA by means of the LRR
domain to E-cadherin—a surface receptor on
eukaryotic cells (Bergmann 2002).
A model of the tertiary structure of the remaining
part of protein Lmo0327 was also elaborated. The re-
peats in the central part of the protein form an a-helix
with a turn after repeat of the amino acid sequence and
a probable b-helix was also identified (data not shown).
Characterization of recombinant lytic enzyme
Partial purification by selective precipitation of re-
combinant lytic enzyme has been previously reported
(McLaughlan and Foster 1998). The enzyme from
pMPZ6 was not able to hydrolyze the cell wall of
L. monocytogenes and B. subtilis when the lytic
activity was determined by the ability of the extract
to decrease the optical density of cell wall suspen-
sion. The same sample was analyzed on 12% SDS-
polyacrylamide renaturing gel containing cell walls of
B. subtilis as substrate and clear zones in the opaque
gel indicating murolytic activity (data not shown)
were observed. The reason for this observation is
most probably that in the former case the formation
of protein aggregates takes place, which prevents the
degradation of murein. A similar situation was ob-
served when attempting the purification of protein
p60, which precluded determination of the hydrolytic
bond specificity the enzyme (Wuenscher et. al. 1993).
Construction of insertional mutations in the
chromosome of L. monocytogenes EGD
To determine the physiological role of Lmo0327 and to
determine the effect of a potential positive transcrip-
tion regulator on the studied murolytic activity, two
mutants of L. monocytogenes EGD insertionally inac-
tivated in genes lmo0326, lmo0327 were constructed.
Internal fragments of 530 bp from the lmo0326 gene
and 557 bp from the lmo0327 gene were amplified by
PCR and cloned into pAUL-A in E. coli to create two
clones, pAUL-A::lmo0326 and pAULA::lmo0327,
respectively (Table 2). The obtained plasmids were
introduced into L. monocytogenes EGD cells by elec-
troporation and maintained at a permissive tempera-
ture (30�C). Plasmid pAUL-A contains a temperature
sensitive replicon and when the cells were successively
subcultured at the non-permissive temperature (42�C),
the plasmid recombined at the region of homology
with the host chromosome, thus inactivating the
appropriate gene. The obtained insertion mutants
(Emr) EGD/pAUL-A::lmo0326 were designated: MP2
and EGD/pAUL-A::lmo0327 MP1. These mutants
were used in an analysis of the role of proteins
Lmo0326 and Lmo0327 in cell physiology.
Transcriptional analysis
The non-polar effects of insertion into lmo0327 (mu-
tant MP1) and into lmo0326 (mutant MP2) were con-
firmed by an RT-PCR reaction using MP1 or MP2
generated cDNA and specific primer pairs (Table 3)
for downstream/upstream gene. As a positive control
analogous PCR reactions were performed using cDNA
generated from EGD. The achieved results (the
insertions had no-polar effect) are in accordance with
the sequence prediction, which reveals the presence of
transcriptional terminators downstream of lmo0326
and lmo0327 (Fig. 1). All PCR amplifications were
performed according to the scheme presented in
Fig. 3a. The reactions resulted in products of equal
intensity, confirming the formation of transcripts for
lmo0326 and lmo0328 in the case of mutant MP1 and
also for lmo0325 and lmo0327 in the case of mutant
MP2 (Fig. 3b).
Autolytic activity of Lmo0327
Cell surface proteins (SDS and LiCl extractions) of
EGD and mutants MP1 and MP2 were isolated and
analyzed on SDS-polyacrylamide gels. As expected, a
band at ~144 kDa in the surface extracts of EGD was
Arch Microbiol (2006) 186:69–86 77
123
absent in the lmo0327 mutant (MP1) extracts. In
addition, we observed the absence of one other band at
~80 kDa in the mutant LiCl-extracts and different
pattern of bands at ~69 and 80 kDa from SDS-extracts
of the mutant strains. Significant quantitative changes
regarding other bands in the studied fractions were
observed (Fig. 3a). In the case of mutant MP2, the LiCl
fraction lacked a ~75 kDa band and there were
considerable quantitative changes in the bands in the
85–45 kDa range. Significant differences were also
observed in the protein profiles in the SDS fraction,
including the absence of ~144 kDa and ~80 kDa bands.
In the supernatant fraction, significant quantitative and
qualitative differences were observed, especially in the
case of mutant MP2 (Fig. 4c).
Cell surface proteins of both strains were also ana-
lyzed in renaturing SDS-polyacrylamide gels (12%)
containing 0.2% purified cell walls of Bacillus subtilis.
Multiple lytic bands, which represented cell wall
hydrolase activity, were detected in renaturing gels
(Fig. 4b, d). A ~144 kDa lytic band present in bacterial
surface LiCl-extracts and total protein fraction from
the EGD strain was absent in these fractions isolated
from the MP1 and MP2 mutants. We also observed
very weak murolytic activity of bands at ~80, ~75 and
~67 kDa in LiCl-extracts from mutant MP1 (Fig. 4b).
Similarly, in the case of LiCl-extracts and total protein
from mutant MP2 we observed the loss of p144 cell
wall hydrolase activity and in addition, in the LiCl
fraction, a protein with mass ~67 kDa (Fig. 4d). In the
culture supernatant from MP1 slight quantitative dif-
ferences were visible, whereas in the case of mutant
MP2 no zones of hydrolysis in this fraction were ob-
served (Fig. 4b, d). Weaker activity of an enzymatic
protein with mass ~60 kDa was also noted. No differ-
ence was observed in the bacterial surface SDS-ex-
tracts from all strains.
Taken together, the obtained results indicate that
Lmo0327 with hydrolase activity is a cell surface pro-
tein covalently linked to the murein of L. monocytog-
enes and that the transcription regulator Lmo0326
presumably positively affects the expression of gene
lmo0327.
The role of the lmo0327 gene was examined by a
phenotypic comparison of MP1 (lmo0327) and EGD
(wild-type). A similar experiment was carried out for
mutant MP2. No differences were detected with re-
spect to hemolytic activity on blood agar plates, growth
in TSB medium at 37�C, motility on swarm plates and
colony formation (data not shown). The cells of EGD,
Fig. 3 a Scheme of RT-PCRs. Arrows indicate theprimer, broken lines indicatecDNA, short lines indicatePCR product and rectanglesindicate insertionalinactivation. b RT-PCRexamination. 1 DNA ladderband (Fermentas), 2 10;Orf2L/Orf2R PCR products(MP1) and 3 (EGD), 4 RTC/RTB PCR products (MP1)and 5 (EGD) 6 Orf3L/Orf3RPCR products (MP2) and 7(EGD), 8 RT3/RT2 PCRproducts (MP2) and 9 (EGD)
78 Arch Microbiol (2006) 186:69–86
123
MP1 and MP2 were also examined by light and elec-
tron microscopy when the cultures were in logarithmic
phase of growth (OD600 of 0.6). Mutants MP1 and MP2
formed long chains, composed of cells that did not
separate following division, with fully formed septum
(Fig. 5). Sixty-eight percent of MP1 cells were
arranged in chains of 5 to 11 bacteria compared to no
chains formed by the wild-type strain. The data suggest
the involvement of Lmo0327 in cell separation. How-
ever, during stationary-phase growth, the MP1 cells
formed shorter chains than in the log phase and more
than 50% of MP1 cells grew as separate cells. These
Fig. 5 Scanning electron micrographs of L. monocytogenes EGD (a, d), mutant MP1 (b, e), mutant MP2 (c). Bar represents 2 lm (a, b,c) and 1 lm (d, e)
Fig. 4 Surface expression andautolytic activity of Lm00327.Samples were prepared asdescribed in Materials andmethods. a, c Surface proteinsof L. monocytogenes EGD,MP1 and MP2 were preparedby SDS-extraction and LiCl-extraction, separated on 12%SDS-PAGE gel and stainedwith Coomassie blue. b, d Theautolytic enzyme profile of L.monocytogenes EGD, MP1and MP2 was analyzed on a12% SDS-PAGE gelcontaining purified cell wallsof B. subtilis as substrate.Bands with autolytic activitywere observed as clear zonesin the opaque gel. Arrowsindicate the position ofLmo0327 at 144 kDa, lines,those of the other proteins
Arch Microbiol (2006) 186:69–86 79
123
observations suggest that other cell wall hydrolases
may compensate for the loss of p144 in MP1. Ultra-
structural analysis by electron microscopy did not re-
veal any significant differences in the morphology of
the cell wall. Analysis by HPLC of purified cell walls of
wild type EGD and mutants (MP1 and MP2) showed
no alteration in muropeptide profile (data not pre-
sented), except for small quantitative differences in the
case of some of the individual muropeptides. The
growth of MP1 and MP2 was compared to that of the
wild type at 25, 30, 37, and 42�C. No significant dif-
ference in the growth rate of the all strains was de-
tected at 30 and 37�C temperature (generation time at
both temperatures was 45 min). At 25 and 42�C the
growth of the strains differed—mutants MP1 and MP2
grew slower than the wild type (generation time for
EGD at 25�C was 90 min versus 180 and 240 min for
MP1 and MP2, respectively). At 42�C the generation
times were 55 min, 90 min and 240 min, respectively,
indicating that these mutations confer temperature
sensitivity but that protein Lmo0327 is not essential for
cell growth.
Penicillin- or imipenem-induced cell lysis was
determined by the addition of b-lactam to growing
cultures. After 5 h the treated EGD, MP1 and MP2
cultures all had final OD600 of 0.3–0.35, and after 24 h,
there still was no difference between the strains (data
not shown). From these results it appears that
Lmo0327 does not have a role in b-lactam-induced
lysis. During nisin-induced lysis a decrease of OD600
from 0.35 to 0.2 after 3 h treatment of MP1 cultures
and from 0.32 to 0.2 after 3 h treatment of MP2 cul-
tures compared to EGD culture (OD600 of 0.35 was at
the same level throughout the experiment) was ob-
served. The obtained results indicate the greater sus-
ceptibility of the cells of mutants MP1 and MP2 to the
action of nisin (Fig. 6). Lmo0327 is probably not in-
volved in Triton-X-induced lysis, as both the mutant
and the wild-type lysed at a similar rate (Fig. 7). Sim-
ilarly, the rate of autolysis of murein, isolated from
both EGD and MP1 strain, was similar. After 4 h
autolysis, the absorbance dropped by about 50% for
MP1, compared to about 60% for EGD (Fig. 8a). The
autolysis of mutant MP2 cells following induction with
Triton X-100 was significantly slower than for the wild-
type control. The drop in absorbance at the end of the
experiment was 35%, compared to 60% for the control
(Fig. 7). The second difference concerns the autolysis
of murein, including murein labeled with [3H]N-acet-
ylglucosamine, in which case MP2 was clearly distin-
guished by slower rate of murein degradation (Fig. 8b).
These differences are probably related to the possi-
bility of the regulation of other listerial proteins by the
regulator Lmo0326.
After labeling the bacterial cell wall with tritiated
N-acetyloglucosamine, it is possible to follow the re-
lease of old murein into the medium as new murein is
incorporated into it. By measuring the amount of
radioactivity either released to the medium, or
remaining in the cell wall the turnover of the macro-
molecule in time can be identified. In the case of mu-
tants MP1 and MP2, the rate of murein turnover was
much slower than for the wild type strain. After
150 min of the experiment, the radioactivity remaining
in the cell wall (murein) of the wild type strain EGD
was only 8% of the initial value, compared to 44% for
mutant MP1 and 46% for MP2 (Fig. 9). These results
show that Lmo0327 is involved in the process of mur-
ein turnover.
Discussion
Murein hydrolases are ubiquitous in murein-containing
bacteria but the participation of these enzymes in cell
elongation and division remains to be fully elucidated.
Fig. 6 Nisin-induced cell lysis of L. monocytogenes EGD, MP1 and MP2. Lysis was measured as decline in optical density. Filled circleEGD, symbol, MP1, filled triangle MP2, symbol nisin (4·MIC). The results are means from at least three independent experiments
80 Arch Microbiol (2006) 186:69–86
123
It is obvious that enzymes involved in such important
processes as cell growth and division or turnover of
murein should be under topological and temporal
control. The role of bacterial murein hydrolases in
cellular processes has been demonstrated in but a
limited number of cases, and more often their roles are
inferred indirectly. They are involved in cell growth
but on the other hand they can participate in autolytic
events resulting in cell death (Holtje 1998).
Analysis of the L. monocytogenes genome reveals
the presence of at least 11 proteins with possible
murein hydrolysis domain and so far, as mentioned in
the Introduction, six such enzymes have been identi-
fied. The rather unusual phenomenon among bacteria
of the death of L. monocytogenes cells in the presence
of penicillin, without accompanying autolysis (Chen
et al. 1996), indicates that the autolytic enzymes of this
bacterium are likely tightly regulated and their activity
is to some extent inhibited even after the death of the
cells. This fact makes the study of the autolysins of
L. monocytogenes particularly interesting.
In this study, we have identified a novel cell wall
hydrolase of L. monocytogenes and have characterized
the gene, lmo0327, encoding the enzyme. In addition,
we have characterized a putative positive transcription
regulator, coded by gene lmo0326, which may posi-
tively affect the expression of protein Lmo0327.
Comparative genome analysis of the region surround-
ing the gene lmo0327 shows that it is highly conserved
in the genus Listeria. For each of the genes cloned into
plasmid pMPZ6 (lmo0325–lmo0327) a counterpart
exists in the genome of the non-pathogenic L. innocua.
The similarity of the amino acid sequences of the
protein products of these genes is very high and aver-
ages 90%. Sequence and comparative analysis of the
genes identified in this study cloned into plasmid
pMPZ6 demonstrated that genes lmo0325 and lmo0326
code for positive transcription regulators of the Rgg
type. The sequence of both proteins contains a HTH
(helix-turn-helix) domain responsible for binding to
DNA and is characteristic of transcription regulators
belonging to the XRE family. Proteins of this type
function as transcription activators. This group of
regulators embraces, among others, protein PlcR of
Bacillus cereus (Slamati and Lereclus 2002), which
plays the role of a positive regulator for virulence
factors, as well as an activator of proteins participating
in germination and sporulation in B. subtilis (Foster
1992). Similarity to many other proteins being poten-
tial transcription activators in L. monocytogenes and
Fig. 7 Triton X-100-stimulated autolysis of L. monocytogenesEGD, MP1 and MP2. Autolysis was measured as decline inoptical density. The results are means from at least threeindependent experiments
Fig. 8 a Autolysis of L. monocytogenes EGD, MP1 and MP2 murein. Autolysis was measured as decline in optical density. b [3H]N-acetyloglucosamine labeled murein autolysis of L. monocytogenes EGD, MP2. Autolysis was measured as the amount of radioactivityreleased to the medium. The results are means from at least three independent experiments
Fig. 9 Turnover of murein of L. monocytogenes EGD, MP1 andMP2. The rate of turnover was measured as the amount ofradioactivity remaining in the cell wall. The results are meansfrom at least three independent experiments
Arch Microbiol (2006) 186:69–86 81
123
L. innocua has also been demonstrated. A comparison
of the amino acid sequences of both proteins showed
that they are homologues, with 58% similarity and
32% identity. The greatest similarity is in the N-ter-
minal part of the proteins, in which the domains HTH
XRE are located.
Computer analysis of the amino acid sequence of
protein Lmo0327 revealed the presence, in its N-ter-
minal part, of the domain LRR (leucine reach repeat).
In the C-terminal part, the strongly conserved motif
LPXTG has been identified, which points to the
probable sortase-mediated covalent binding of the
protein to murein. Comparative analysis of Lmo0237
revealed similarity to murein-bound surface proteins,
internalins and other leucine-rich proteins. Analysis in
silico of the sequence of domain LRR of Lmo0327 and
the remaining part of the protein allowed determining
its probable tertiary structure. The N-terminal do-
main—LRR (151 aa) forms a right-handed beta-helix
with a turn after each repetition of the amino acid se-
quence. Structurally, this model corresponds to an
analogous domain identified in proteins belonging to
the very numerous internal family. The amino acid
sequence is strongly conserved in this region in all
known internalins (Schubert et al. 2002). Twenty-two
internalin-like proteins have been identified in
L. monocytogenes, 14 of which carry both the LPXTG
motif as well as domain LRR. A model of the tertiary
structure of the remaining part of Lmo0327 was also
elaborated. The repeats in the central part of the
protein form an a-helix with a turn after each repeti-
tion of the amino acid sequence and two putative
b-helices were also identified. A comparison of the
possible tertiary structure of protein Lmo0327 with
crystallographic models in databases did not reveal any
similarity to other known proteins.
Since analysis of the DNA fragment of L. mono-
cytogenes EGD cloned into plasmid pMPZ6/4 did not
allow determining the potential protein responsible for
the observed murolytic activity, mutational analysis of
genes lmo0325, lmo0326 and lmo0327 in pMPZ6 was
carried out, accompanied by investigation of the mur-
olytic activity of the mutated clones. Zymographic
analysis of total proteins obtained after ultrasonic dis-
ruption of E. coli cells carrying the plasmid constructs
demonstrated the lack of murolytic activity in every
case, suggesting that the protein products of all the
studied genes are necessary for expression of this
activity. Computer analysis of the amino acid sequence
of the proteins suggests, however, that only one pro-
tein, Lmo0327, may be a candidate for a murein
hydrolase, especially since the other two proteins seem
to be putative transcription activators. An experiment
in which the orientation of the cloned DNA with re-
spect to the lacZ promotor was reversed indicated that
the gene coding a protein with murein-hydrolase is
expressed from its own promotor. The change of ori-
entation of the insert does not affect the production of
active protein.
In order to confirm that protein Lmo0327 is in fact
responsible for the investigated murolytic activity and
to determine the effect of the potential positive regu-
lator Lmo0326 on the enzyme, insertional inactivation
of genes lmo0326 and lmo0327 in the chromosome of
L. monocytogenes EGD was performed. Mutants were
selected under conditions being restrictive for the
autonomous replication of the vector pAUL-A (42�C)
in the presence of erythromycin (Schaferkordt and
Chakraborty 1995). The correctness of the construction
was confirmed by hybridization and the obtained mu-
tants were designated MP2 and MP1, respectively, and
subjected to physiological characterization.
Microscopic observations showed that MP1 and
MP2 cells grow in the form of chains following cell
division. No significant differences in colony mor-
phology of the mutant strains compared to strain EGD
were observed. Mutants devoid of autolysins fre-
quently do not manifest striking changes in cell mor-
phology or growth: a mutant of L. monocytogenes
lacking amidase activity (Ami–) does not demonstrate
any phenotypic changes besides reduced motility
(McLaughlan and Foster 1998) and no phenotypic
changes compared to wild type cells have been
observed in the case of the aut mutant of L. mono-
cytogenes (Cabanes et al. 2004). However, some
L. monocytogenes mutants defective in autolytic
activity show a tendency to form shorter or longer
chains, as observed for the deletion mutant DmurA or
mutant defective in p60 activity. Similarly, the only
consequence of the absence of LytA amidase activity
in a mutant of Streptococcus pneumoniae, was growth
in the form of chains composed of daughter cells with
developed septum (Tomasz et al. 1988).
The growth rate of wild-type L. monocytogenes and
the mutants (without antibiotic pressure) at several
temperatures was studied. The growth of both mutants
was found to be sensitive at the temperature extremes
(25�C and 42�C) examined. The growth of mutant MP2
carrying a mutation in the putative regulator was even
slower than that of mutant MP1. Its doubling time was
four times that of strain EGD. At 30�C and 37�C, the
growth of all the studied strains was comparable. At
25–30�C, L. monocytogenes EGD is able to move in
liquid medium by means of flagella. The role of auto-
lytic enzymes in the proper formation of flagellum has
been demonstrated, for instance for L. monocytogenes
82 Arch Microbiol (2006) 186:69–86
123
amidase Ami (McLaughlan and Foster 1998) as well as
for other specific muramidases involved in building a
functional flagellum (Holtje and Tuomanen 1991).
Since at 25�C the constructed mutants grow very
slowly, the test for motility was performed at 30�C. No
differences in motility in TSB medium between the two
mutants as well as between either of the mutants and
wild-type listeria, was observed.
Proteins containing the LPXTG motif have been
shown to play an important role in the adhesion and
entry of pathogens, including L. monocytogenes, into
non-professional phagocytic cells and colonization of
the intestine, using the mouse model (e.g. Cabanes
et al. 2005; Lalioui et al. 2005). In our studies we car-
ried out experiments aimed at determining the possible
role of Lmo0327 in adherence and entry of L. mono-
cytogenes into cells of the eukaryotic lines Int407 and
found that there is no difference in this regard between
wild-type cells and mutants carrying inactive Lmo0327
(data not presented). However, other cell lines should
be tested too.
The murein of Gram-positive bacteria undergoes
constant turnover. The process involves the highly
coordinated participation of murein hydrolases and
synthetases. The role of an amidase in the turnover of
murein in Bacillus subtilis (Hobot and Rogers 1991)
and of a muramidase in Lactobacillus acidophilus
(Coyette and Shockman 1973) has been determined. In
the case of L monocytogenes mutants MP2 and MP1,
slower metabolic turnover compared to the wild-type
was observed, which may reflect the role of protein
Lmo0327 in the process.
The phenomenon of autolysis is caused by the
degradation of murein by endogenous cellular
enzymes. L. monocytogenes has been shown to be
refractory to lysis induced by such agents as EDTA or
SDS, which may indicate the stricter regulation of
autolysis in this bacterium. As mentioned, in the
presence of b-lactam antibiotics L. monocytogenes
behaves differently compared to most other bacteria.
Death without autolysis is a very rare phenomenon
that has also been described for group A streptococci
(McDowell and Lemanski 1988). No differences in the
response of the mutants obtained by us to the presence
of penicillin or imipenem in the culture medium,
compared to the wild-type strain, were observed. The
induction of autolysis by the surfactant Triton X-100
showed significant differences for mutant MP2 com-
pared to strain EGD. The course of the process was
much slower, and a 40% drop in absorbance for the
mutant versus 80% for the wild-type strain during the
same time was observed. In the case of mutant MP1,
the rate of autolysis induced by Triton X-100 was
comparable with strain EGD, which points to the lack
of Lmo0327 involvement in induced autolysis. The
results for the mutant defective in the putative regu-
lator gene lmo0326 may suggest that Lmo0326 may
possibly also regulate the expression of other proteins,
whose absence may affect the course of autolysis.
The effect of the lantibiotic nisin on the studied
strains was examined. Nisin causes local disruption of
the cytoplasmic membrane and also binds to the mur-
ein precursor lipid II, resulting in inhibited cell wall
synthesis and violent lysis (Sahl and Bierbaum 1988).
The results indicate the greater susceptibility of the
mutant cells to nisin. This may be related to the growth
of the cells in the form of chains due to disturbed
separation of daughter cells following division but the
actual reason for this observation remains to be eluci-
dated, especially since spontaneous nisin-resistant
mutants of L. monocytogenes have been shown to have
elevated levels of PBP4, caused by an increase in his-
tidine kinase expression (Gravesen et al. 2004).
Because the autolysins of many Gram-positive bac-
teria are present in the inner layers of the cell wall, we
examined the rate of degradation of murein isolated
from the studied strains. Mutant MP2, with a mutation
in the gene coding positive transcription regulator,
showed a slower rate of murein degradation compared
to strain EGD. In the case of mutant MP1, in which the
mutation directly concerns the identified surface pro-
tein, no significant differences in the course of cell wall
autolysis compared to the wild-type strain were
observed. An analogous experiment in which [3H]N-
acetyloglucosamine was used, showed a similar
effect—slower rate of release of labeled fragments of
murein in the case of MP2, compared to strain EGD.
The use of high performance liquid chromatography
(Glauner 1988) to study the muropeptide composition
of murein from wild-type L. monocytogenes and mu-
tants MP1 and MP2 demonstrated the absence of the
role of the studied gene products on the structure of
the macromolecule. A comparison of the three chro-
matograms showed no qualitative differences in the
composition of murein.
Earlier studies have shown that proteins isolated
from various cell fractions show hydrolytic activity
against murein, as revealed by zymography
(McLaughlan and Foster 1997). Protein was isolated
from the cellular fractions of the studied strains and
both quantitative and qualitative differences in the
studied fractions between the studied mutants and wild
type strain were observed. In the case of all cellular
fractions from mutants MP1 and MP2, both SDS-PAGE
and zymography showed a protein of 144 kDa to be
missing, compared to analogous fractions from strain
Arch Microbiol (2006) 186:69–86 83
123
EGD. Worth attention is that in the case of mutant MP2,
no zones of hydrolysis were observed in a zymogram of
the fraction of proteins released to the growth medium.
Moreover, the protein profile of this fraction in SDS-
PAGE decidedly differs from that of the wild type
strain. This result may point to the role of protein
Lmo0326 in the expression or transport of proteins re-
leased to the medium. Further studies conducted by our
group will attempt to explain this observation.
The above observations taken together show that
Lmo0327 is an enzyme that is covalently linked to
murein and has murein-hydrolyzing activity. In view of
the fact that gene lmo0327 is incomplete (1,036 bp) in
the studied L. monocytogenes genome fragment
(pMPZ6) and it is the studied product (N-terminal part
of the protein—310 amino acids) that demonstrates
murein-hydrolysing activity, it can be concluded that
the active centre is in the N-terminal part of the protein.
Lmo0327 appears to be involved in cell separation and
turnover of murein and does not seem to be involved in
adhesion to and entry into eukaryotic cells, at least in
the case of the cell lines examined. A further study of
Lmo0327 may reveal other roles for this protein in
L. monocytogenes. The expression of gene lmo0327
coding for the autolysin is presumably positively regu-
lated by the transcription regulator of the Rgg type
Lmo0326, though conclusive proof, gained from further
studies, is required to justify this statement.
The characterization of mutant MP3, with a muta-
tion in a second regulatory gene lmo0325, has also
been initiated, which will allow studying the possible
effect of protein Lmo0325 on the expression of
Lmo0327 and/or other proteins with similar character.
Preliminary studies have revealed differences in pro-
tein and zymographic profiles between the mutants
characterized herein and MP3, which may suggest the
effect of transcription regulator Lmo0325 on the
expression and activity of other proteins.
Acknowledgments We are grateful to Paweł Szczesny (IBB,Polish Academy of Sciences) for modeling the potential tertiarystructure of the LRR domain of Lmo0327.
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