1 Unité de Biochimie et Structure des Protéines, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
2 Unité de Biochimie et Unité de Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
3 Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
Correspondence
Marie-Pierre Chapot-Chartier
Marie-Pierre.Chapot{at}jouy.inra.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The EMBL/GenBank accession number for the sequence reported in this paper is AJ414526.
Present address: AFSSA, LERPRA, Les Templiers, 105 route des Chappes, BP111, 06902 Sophia-Antipolis Cedex, France.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lactococcus lactis is a lactic acid bacterium widely used in starters for cheese making. During cheese ripening, the bacterial intracellular enzyme content contributes to the development of organoleptic properties (Chapot-Chartier, 1996; Crow et al., 1995
). The cell envelope appears as a physical barrier for the bacterial enzymes to reach the extracellular substrates. Bacterial autolysis was previously shown to enhance the contribution of enzymes to cheese flavour formation. Especially, autolysis leads to the release of the intracellular peptidases into the cheese curd, and as a result more free amino acids, which are aroma precursors, are produced and hydrophobic bitter peptides are degraded (Chapot-Chartier et al., 1994
; Wilkinson et al., 1994
). Besides this traditional use in dairy fermentation, L. lactis has been proposed as a vaccine vehicle or delivery vector for use in human medicine (Drouault et al., 1999
; Wells et al., 1996
). For these applications, L. lactis autolysis in the gastro-intestinal tract is also a critical parameter to obtain an optimal response. In order to get insight into the autolysis mechanism and to control it, it is necessary to identify and characterize the peptidoglycan hydrolases involved.
Up to now, only the major autolysin, named AcmA, has been characterized at the molecular level in L. lactis (Buist et al., 1995). Like most bacterial peptidoglycan hydrolases, AcmA has a modular structure with two domains. The N-terminal domain exhibits sequence similarity with the catalytic domain of Enterococcus hirae muramidase Mur-2, and the C-terminal one contains three amino acid repeats involved in cell wall binding (Buist, 1997
). As regards its enzymic specificity, AcmA was recently shown to be an N-acetylglucosaminidase (Steen et al., 2001
), rather than an N-acetylmuramidase as predicted by sequence similarity. AcmA is required for proper separation of cells after cell division and is involved in cellular autolysis in stationary phase in synthetic culture medium (Buist et al., 1997
). However, other factors than AcmA contribute to determining the autolysis rate when cells are in the cheese matrix environment (Pillidge et al., 1998
). This prompted us to search for new peptidoglycan hydrolases and evaluate their contribution to cellular autolysis in L. lactis.
In the genome sequence of L. lactis subsp. lactis IL1403 (Bolotin et al., 2001), we could identify four putative peptidoglycan hydrolases, in addition to AcmA, on the basis of a sequence similarity search. In this study, we have investigated the functionality, hydrolytic bond specificity and the role of one of these peptidoglycan hydrolases, named AcmB.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Cloning of the acmB gene from L. lactis MG1363.
With the primers AU4 (5'-GATTATCTTTATCTCGTACAC-3' and AU11 (5'-CAACTGCAGCACAAATTCC-3') selected from the IL1403 sequence genome (Bolotin et al., 2001), a 1·6 kb DNA fragment was amplified by PCR from L. lactis subsp. cremoris MG1363 total DNA. This fragment contained only part of the MG1363 acmB gene. PCR cloning of the 5'-end failed, although several pairs of primers were tested. The sequence of the 1077 bp region upstream of the 1674 bp amplified region was obtained from a long-range 10 kb DNA fragment amplified by Multiplex Long accurate PCR from MG1363 total DNA (Bolotin et al., 2002
). This 10 kb fragment was amplified with two primers derived from the IL1403 sequence: mc91.pri (5'-ATCCGTACGGCCGAAGTGCCT-3') and mb95.pri (5'-GGTATCGGTAACATCCACGGTC-3'), corresponding to metK and fbaA MG1363 sequencing tags. Finally, we obtained the sequence of a 2751 bp DNA fragment encompassing the acmB gene, which was located in the central region of the fragment.
Inactivation of acmB by double crossing-over integration.
A 2·4 kb fragment encompassing the acmB gene was amplified by PCR with the two primers: AU57 (5'-TCCCCCCGGGAATGGAAACCCAAAGTATAGG-3'; SmaI restriction site underlined) and AU58 (5'-GGGGTACCTTTTTCATCCGAAGTTTCTG; KpnI restriction site underlined) with MG1363 DNA as template. The PCR product was cloned into the pGEMT-easy vector. The insert was recovered with SmaI and KpnI digestion and was then ligated in the corresponding sites of pG+host9 plasmid. The ligation mixture was used to transform E. coli TG1repA+competent cells. In the resulting recombinant plasmid, an internal 414 bp fragment of the acmB gene was deleted by digestion with EcoRI and PstI. A chloramphenicol-resistance cassette with a size of 0·9 kb (Trieu-Cuot et al., 1992) was then ligated into the PstI and EcoRI sites. The final plasmid pTIL328 was electroporated into L. lactis MG1363 and MG1363acmA
1. Integration of pTIL328 into the chromosome and subsequent excision was achieved according to the previously developed protocol (Biswas et al., 1993
). Mutant strains were screened first on their resistance to chloramphenicol and second on the size of the fragment amplified by PCR with primers AU57 and AU58. The presence of a correct insertional event was further verified by Southern blotting. When the properties of the acmB mutants were analysed, chloramphenicol was omitted from the culture.
Northern blotting.
Total RNA fractions were extracted from L. lactis MG1363 grown at 30 °C as previously described (Anba et al., 1995), at different stages of growth, corresponding to OD650 0·1, 0·5, 1·0, 1·5, 2·0, 2·5 and 3·0. Northern blot hybridization was performed according to a standard protocol (Sambrook et al., 1989
). Twenty micrograms of total RNA were electrophoresed in a 1 % (w/v) agarose gel (Seakem GTG Agarose, BMA Bioproducts), transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) and fixed by heat treatment (80 °C, 2 h). The membrane was probed with a 1·5 kb DNA fragment corresponding to the entire acmB gene, and labelled with [
-32P]dCTP with the Random Primed DNA Labelling Kit (Roche Molecular Biochemicals). Hybridization was performed under high-stringency conditions (50 % formamide). The membrane was then dehybridized by immersion in boiling 1x times; SSC/0·2 % SDS and subsequent incubation for 1 h at room temperature. It was rehybridized with a 16S rRNA specific probe obtained by PCR amplification as described by Cibik et al. (2000)
. Radioactivity was quantified with a PhosphorImager (Molecular Dynamics) with the ImageQuaNT program. The relative amounts of acmB transcript were standardized by hybridization with a L. lactis 16S rRNA specific probe.
Expression and purification of His-tagged proteins in E. coli.
The proteins were overexpressed in E. coli M15(pREP4) as C-terminal hexa-His-tagged proteins, with the expression vector pQE60 (Qiagen). Two truncated derivatives of the acmB gene encoding polypeptides corresponding to the AcmB catalytic domain alone, and to the catalytic domain plus the C-terminal domain of AcmB, were amplified by PCR from L. lactis MG1363 total DNA. The DNA fragments were amplified respectively with the primers AU65 (5'-CATGCCATGGTATATATGGGGCCTGTATT-3'; NcoI restriction site underlined) and AU68 (5'-GAAGATCTACTGCGACATTATCATAAAAA-3'; BglII restriction site underlined), and AU65 and AU70 (5'-GAAGATCTTTTAGGTTGGATATAAGTTGC-3'; BglII restriction site underlined). PCR fragments were digested by NcoI and BglII introduced with the primers and cloned in-frame upstream of the hexa-His box sequence in the pQE60 vector, precut by the same enzymes. E. coli M15(pREP4) competent cells were transformed with the resulting plasmids. IPTG was added at a final concentration of 1 mM to the culture at an OD650 of 0·5 to induce the expression of the hexa-His-tagged proteins. Bacteria were grown at 37 °C until IPTG addition and were then transferred at 28 °C during the expression time (4 h) to avoid the formation of inclusion bodies. The cells were harvested by centrifugation and broken by one passage at a pressure of 1600 bar with a Constant Cell Disruption System (Constant System, Warwickshire, UK). The soluble fraction containing the recombinant protein was collected by centrifugation at 15 000 g for 15 min at 4 °C. The hexa-His-tagged proteins were purified by affinity chromatography on Ni2+-nitrilotriacetic acid (Ni-NTA) spin columns (Qiagen), according to the manufacturer's instructions.
Preparation of cell wall peptidoglycan.
Peptidoglycan from Bacillus subtilis HR vegetative cells was prepared as described previously (Atrih et al., 1999). Peptidoglycan from L. lactis MG1363 was prepared from an exponentially growing culture (OD650 0·7) according to the protocol of Mainardi et al. (2000)
. Briefly, cells were boiled in 4 % (w/v) SDS for 30 min. Cell walls were recovered by centrifugation for 90 min at 140 000 g and washed three times with water to eliminate SDS. To remove proteins, the cell wall pellet was treated with Pronase (200 µg ml-1) for 16 h at 37 °C, then by trypsin (200 µg ml-1) for 16 h at 37 °C. The pellet containing peptidoglycan was washed twice with water and stored at -20 °C.
SDS-PAGE and renaturing SDS-PAGE.
SDS-PAGE was performed as described by Laemmli (1970) with 15 % (w/v) polyacrylamide separating gels. Renaturing SDS-PAGE was performed as previously described (Lepeuple et al., 1998
). The polyacrylamide gels contained 0·2 % (w/v) Micrococcus luteus ATCC 4698 (Sigma) or 0·4 % (w/v) L. lactis autoclaved cells, or 0·16 % (w/v) MG1363 peptidoglycan as enzyme substrate. Gels were washed for 1 h in deionized H2O at room temperature and then incubated in buffer containing 1 % (v/v) Triton X-100, overnight at 37 °C. The following incubation buffers were used: 25 mM sodium citrate/50 mM sodium phosphate at pH 3·0, 4·0 or 5·0; 50 mM MES at pH 6·0; and 50 mM Tris/HCl at pH 7·0 and 8·0. The gels were subsequently washed for 1 h in deionized H2O and when required, with 10 mM Tris/HCl, pH 7·0, containing 0·1 % SDS to remove precipitated proteins from the gels. The gels were then stained with 0·1 % methylene blue in 0·01 % (w/v) KOH for 2 h at room temperature and destained in deionized H2O. Gel images were generated with a Duoscan T1200 scanner (Agfa-Gevaert) customized for proper gel handling and controlled by the Agfa Photolook 3.0 software.
Determination of hydrolytic bond specificity.
The hydrolytic bond specificity of AcmB was analysed using peptidoglycan from B. subtilis HR vegetative cells as substrate. Peptidoglycan (5 mg) was incubated overnight at 37 °C, with 500 µg purified hexa-His-tagged recombinant AcmB[C+Z]-His in a final volume of 500 µl buffer (25 mM sodium citrate/50 mM sodium phosphate, pH 4·0). Samples were boiled for 3 min to stop the reaction. Insoluble material was removed by centrifugation at 14 000 g. Half of the soluble muropeptide fraction was further digested with Cellosyl (250 µg ml-1). The soluble muropeptides obtained after digestion with AcmB[C+Z]-His or AcmB[C+Z]-His plus Cellosyl were reduced with sodium borohydride (8 mg ml-1) as described previously (Atrih et al., 1999). The reduced muropeptides were separated by RP-HPLC with a Biocad Sprint system (Perkin Elmer) equipped with a variable-wavelength double detector and an automatic collector (Advantec) using a Hypersyl PEP100 C18 column (250x4·6 mm, 5 µm, 100 Å) (Thermo Finnigan). Elution was carried out at a flow rate of 1 ml min-1 for 10 min with 100 % solvent A (TFA/H2O; 1·15 : 1000, v/v) and subsequently with a 120 min linear gradient (0 to 20 %) of solvent B (TFA/CH3CN; 1 : 1000, v/v). Absorbance was monitored at both 220 and 280 nm and the eluate collected in 1 ml volume fractions. Muropeptide-containing fractions were dried and then resuspended in 10 µl 0·15 % TFA in water. They were subsequently analysed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using a Voyager DE STR mass spectrometer (Perseptive Biosystem, Framingham, USA). The precision in the mass determination was 0·01 %. One microlitre of the sample was mixed with 1 µl
-cyano-4-hydroxycinnamic acid. A 1·2 µl drop of the mixture was deposited on the steel plate and was allowed to air dry. The muropeptides were desorbed and ionized by a N2 laser in the positive- and/or negative-ion mode. Each mass spectrum was a mean of 200 scans. For all experiments, the accelerating potential was held at 20 kV and laser power was set to the minimum level necessary to generate a reasonable signal. An external mass calibration standard containing bradikinin fragment 15, angiotensin I, neurotensin and melittin was employed for all the analyses.
Autolysis in buffer solution.
L. lactis strains were grown in M17 medium to mid-exponential phase (OD650 1·0). Cells were harvested by centrifugation at 5000 g for 15 min at 4 °C and washed once with sterile deionized water. They were resuspended in 50 mM potassium phosphate buffer pH 7·0 at OD650 0·8 and incubated at 30 °C. Autolysis was monitored by measuring the OD650 of the cell suspension. The extent of autolysis was expressed as the percentage decrease in OD650. The intracellular X-prolyldipeptidyl aminopeptidase (PepX) was chosen as a marker for cell autolysis (Lepeuple et al., 1998). Its release into the culture supernatant was monitored by measuring enzymic activity with Ala-Pro-p-nitroanilide (Bachem) chromogenic substrate. Enzymic activity was expressed in katals (1 katal is the quantity of enzyme releasing 1 mol p-nitroaniline s-1) per ml of culture.
Determination of the proportion of cells with a damaged membrane.
The membrane integrity of the cells incubated in buffer solution as described above was investigated by the fluorescence labelling procedure described by Niven & Mulholland (1998). The fraction of permeable cells in the whole L. lactis cell population was estimated by labelling with propidium iodide (PI), a fluorescent dye, which stained only bacteria with a damaged membrane. The whole population of cells was estimated by PI-induced fluorescence after treatment with cetyltrimethylammonium bromide (CTAB) (Sigma), a cationic surfactant used to permeabilize bacterial cytoplasmic membranes. Bacterial suspension (1 ml) was incubated with 30 µM PI (Molecular Probes) with or without 200 µM CTAB for 30 min at 25 °C in the dark. Labelled cell suspensions were kept on ice until fluorescence measurement (less than 15 min). Fluorescence was measured in a 1 cm cuvette with an excitation wavelength of 500 nm and emission wavelength of 600 nm with a Kontron Instrument SFM25 spectrofluorimeter. The fraction of permeable cells in the cell population was calculated with the following formula (cellsPI-cellsalone-bufferPI)/(cellsPI+CTAB-cellsalone-bufferPI+CTAB) as described by Walker & Klaenhammer (2001)
.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AcmB, a peptidoglycan hydrolase with an uncommon three-domain modular structure
The acmB gene encodes a 538-residue protein with calculated molecular mass of 57·1 kDa and pI of 4·72. The first 33 N-terminal residues of AcmB possess the characteristics of a signal peptide (Nielsen et al., 1997). They could also constitute an N-terminal signal anchor sequence since a transmembrane helix is predicted in positions 1433 (Tusnady & Simon, 2001
). According to protein sequence similarities, the AcmB protein has a three-domain modular organization (Fig. 1
A).
|
|
The N-terminal domain (residues 34211) has a remarkably high content of the amino acids Ser (29 %), Thr (15 %), Asn (10 %) and Pro (8 %). This characteristic is encountered in protein domains associated with the cell wall in Gram-positive bacteria (Fischetti et al., 1991). A very similar Ser-, Thr-, Gly-, Pro-rich domain was previously described in the cell-bound fructosyltransferase of Streptococcus salivarius (Rathsam et al., 1993
) and this domain was shown to play a role in cell surface attachment of the protein (Rathsam & Jacques, 1998
).
A putative protein similar to AcmB and composed of three homologous domains with 39 % sequence similarity was found only in the Staph. aureus genome sequence (Kuroda et al., 2001).
acmB expression is regulated during cellular growth
Transcriptional analysis of acmB during cellular growth was studied by Northern blot analysis. Total RNA was isolated from cells harvested at different stages of growth at 30 °C in M17 medium. Using an acmB probe, we detected a single 1·6 kb transcript (Fig. 3A). This size corresponds to a monocistronic organization of acmB. Quantitative mRNA analysis revealed that the relative abundance of the transcript varied during growth (Fig. 3B
). Early-exponential-phase cells (OD650 0·1) contained a substantial amount of acmB mRNA, which rapidly declined to become 3·5 times less abundant at an OD650 of 0·5. Then, the amount of acmB mRNA increased slightly during the exponential growth phase, followed by a slow decline until stationary phase. These results indicate that acmB is transcribed in L. lactis MG1363 and its transcription is regulated during cell growth.
|
The His-tagged derivatives of AcmB were purified by nickel affinity chromatography under native conditions (Fig. 4A). Their peptidoglycan-hydrolysing activity was examined by renaturing SDS-PAGE with M. luteus autoclaved cells as substrate (Fig. 4B
). The two proteins gave a clear hydrolysis band with incubation buffer at pH 4·0. The detection of AcmB[C]-His activity indicates that the [C] domain retains peptidoglycan hydrolase activity and confirms its assignment as the catalytic domain. In addition, these results indicate that cell wall binding is not an absolute requirement for enzyme activity. It is worth noting that a rather high amount of the purified His-tagged proteins was loaded on the gel for activity to be detected. Thus, the absence of detection of AcmB activity in L. lactis cellular extracts results most probably from too low an amount of protein expressed in the cells.
|
The influence of the incubation buffer pH on activity detection was tested on M. luteus cell substrate. AcmB[C+Z]-His activity was detected at pH 4·0 and 5·0 but not at pH 6·0 and higher, nor at pH 3·0. AcmB[C+Z]-His has a low pI (5·03) and interestingly, its activity was detected only at acidic pH in the renaturing electrophoresis test. This result suggests that interaction with the negatively charged cell walls is favoured by positive or neutral charge of the protein.
AcmB is an N-acetylglucosaminidase
The purified recombinant N-terminal-domain-truncated AcmB (AcmB[C+Z]-His) was found by renaturing SDS-PAGE to hydrolyse B. subtilis vegetative cell walls (data not shown). Therefore, the hydrolytic bond specificity of AcmB was investigated on B. subtilis vegetative peptidoglycan, whose fine structure has been previously studied in detail (Atrih et al., 1999).
Peptidoglycan extracted from B. subtilis vegetative cells was incubated with purified recombinant AcmB[C+Z]-His overnight at 37 °C in phosphate/citrate buffer at pH 4·0. A slight reduction of OD450 was observed (less than 5 %). The soluble fraction was recovered and analysed by RP-HPLC (Fig. 5A). Peaks 1 and 2 were products of AcmB digestion since they were absent from the control consisting of cell walls incubated without enzyme (data not shown). When analysed by MALDI-TOF mass spectrometry, they gave molecular ions with m/z values of 892·36 and 1814·70 respectively. According to the data of Atrih et al. (1999)
, these m/z values correspond to peak 1 containing the disaccharide tripeptide muropeptide, and peak 2 containing the disaccharide tripeptide disaccharide tetrapeptide muropeptide (Table 2
, Fig. 6
). The AcmB-soluble muropeptides were subsequently incubated with Cellosyl, which is an N-acetylmuramidase. New major peaks appeared on the RP-HPLC profile (Fig. 5B
), whereas the peaks 1 and 2 present in the AcmB digest became minor peaks. By MALDI-TOF analysis, peaks 3, 4 and 5 gave molecular ions with m/z values of 689·11, 1386·66 and 1611·96 respectively. These values correspond to peak 3 containing disaccharide tripeptide missing one N-acetylglucosamine, and peaks 4 and 5 containing disaccharide tripeptide disaccharide tetrapeptide missing two and one N-acetylglucosamine respectively (Table 2
, Fig. 6
). These results indicate that the muropeptides generated by AcmB hydrolysis can be cleaved by Cellosyl and thus that N-acetylglucosamine is present on the reducing end of the disaccharide of these muropeptides. AcmB hydrolyses the peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid and thus AcmB is an N-acetylglucosaminidase.
|
|
|
The acmB mutant was not impaired in cellular growth nor in cell separation compared to the wild-type strain. No change in the cellular morphology of the mutants was observed by phase-contrast microscopy. The double acmA acmB mutant was not affected in its growth rate and formed long chains of cells compared to MG1363, as previously described for the acmA single mutant (Buist et al., 1995).
Autolysis of the acmB mutant was measured in 50 mM potassium phosphate buffer solution at pH 7·0, and compared to that of the wild-type MG1363. As shown in Fig. 7, the acmB mutant autolysed at a slightly but significantly lower rate than the wild-type strain. Also the final extent of autolysis was reduced in the mutant strain (61 %) compared with the wild-type strain (78 %). The OD650 decrease was accompanied by a concomitant release of intracellular peptidase PepX into the culture supernatant (Fig. 7
), which confirms that the OD650 decrease reflected cellular autolysis and release of intracellular content. The acmB mutant released a lower amount of PepX activity than the wild-type MG1363, confirming the differences revealed by optical density measurement.
|
For the double MG1363 acmA acmB mutant, no difference in autolytic properties in buffer solution was observed compared with the single MG1363 acmA mutant. Both of them exhibited a lower autolysis rate and lower autolysis extent compared with the wild-type strain MG1363 (Fig. 7). Thus, AcmB does not appear to be responsible for the residual autolytic activity detected in the MG1363 acmA mutant. The fact that acmB inactivation leads to a reduction of the autolysis rate and extent in wild-type MG1363, but not in the acmA mutant, suggests that AcmB could act only in concert with AcmA and that AcmA could potentiate the hydrolytic action of AcmB.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AcmB exhibits sequence similarity with the Ent. hirae muramidase-2 catalytic domain (Joris et al., 1992). However, like the major L. lactis autolysin AcmA (Steen et al., 2001
) and B subtilis peptidoglycan hydrolase LytG (Horsburgh, 2001
), which are also homologous to the Ent. hirae muramidase-2, AcmB exhibits N-acetylglucosaminidase hydrolytic specificity. Thus, it appears that the enterococcal muramidase family defined by Smith et al. (2000)
on the basis of sequence similarity contains glycosidases with the two types of specificities: muramidase or glucosaminidase. Hitherto, one muramidase, Mur-2 of Ent. hirae, and three glucosaminidases, LytG, AcmA and AcmB, had been identified, whereas the hydrolytic specificity of the other homologous enzymes was not determined experimentally.
B. subtilis LytG was proposed to have exoglucosaminidase specificity (Horsburgh, 2001). The fact that AcmB leads to only limited hydrolysis of B. subtilis peptidoglycan (less than 5 % optical density decrease), and that in these conditions only small muropeptides were released, suggests that AcmB could also be an exoglucosaminidase.
AcmB is devoid of sequence repeats termed LysM domains (Bateman & Bycroft, 2000) previously described as cell-wall-targeting and peptidoglycan-binding sequences. In contrast, three such LysM repeats are found in L. lactis AcmA major autolysin. Also, no LPXTG motif, enabling covalent binding to peptidoglycan (Fischetti et al., 1990
), could be found close to the C-terminus. However, AcmB contains a domain with a high content of Ser, Thr, Asn and Pro residues, which could be involved in cell wall interaction. Such a domain was previously described in Strep. salivarius fructosyltransferase, and was shown to be required in combination with a C-terminal transmembrane anchor, for cell wall attachment of the enzyme (Rathsam et al., 1993
). By analogy, in AcmB, this domain plus the N-terminal putative transmembrane sequence could be involved in cell wall attachment of the protein. Like other Pro-, Ser-, Thr-rich domains (Fischetti et al., 1991
), the AcmB N-terminal domain is likely to have an extended conformation, and could serve as a cell wall spacer allowing exposure of the catalytic domain of AcmB at the cellular surface. Another remarkable feature of this domain is the presence of several consensus sequences (Asn-X-Ser/Thr) for N-glycosylation and the high amount of Ser and Thr residues, which could be sites for O-glycosylation. Glycosylation of the protein could protect it from proteolytic degradation in the cell wall (Moens & Vanderleyden, 1997
).
Recent studies provided evidence that actively metabolizing B. subtilis cells secrete protons, which bind to anionic sites in the cell wall (Calamita et al., 2001). It was proposed that acidification of the cell wall during growth may be one means for autolysin inhibition (Kemper et al., 1993
). We can speculate that in contrast AcmB, which has a low pI (4·65) and is active at acidic pH, may be active during cellular growth.
In this study, we have shown that AcmB contributes to cellular autolysis but to a lesser extent than AcmA. In addition, AcmB action appears to be potentiated by that of AcmA. This feature is consistent with AcmB being an exoenzyme. It has been demonstrated previously that the amino acid repeats present in Staph. aureus Atl autolysin direct the enzyme to cell division sites (Baba & Schneewind, 1998). AcmB is devoid of such repeats and thus could have a uniform localization around the cell surface. In contrast to AcmA, which plays a role in cell separation, AcmB could be more likely involved in other cellular processes such as cell enlargement and insertion of new peptidoglycan units. The fact that inactivation of AcmB did not impair cellular growth does not exclude such a role for this protein. Indeed, functional redundancy was previously observed among B. subtilis autolysins (Smith et al., 2000
). Thus, other L. lactis peptidoglycan hydrolases could compensate for its absence. Besides AcmA and AcmB, at least three other putative peptidoglycan hydrolases are present in the L. lactis IL1403 genome sequence.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atrih, A., Bacher, G., Allmaier, G., Williamson, M. P. & Foster, S. J. (1999). Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation. J Bacteriol 181, 39563966.
Baba, T. & Schneewind, O. (1998). Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus. EMBO J 17, 46394646.
Bateman, A. & Bycroft, M. (2000). The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J Mol Biol 299, 11131119.[CrossRef][Medline]
Biswas, I., Gruss, A., Ehrlich, S. D. & Maguin, E. (1993). High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175, 36283635.[Abstract]
Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S. D. & Sorokin, A. (2001). The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11, 731753.
Bolotin, A., Ehrlich, S. D. & Sorokin, A. (2002). Studies of genomes of dairy bacteria Lactococcus lactis. Sci Aliment 22, 4553.
Buist, G. (1997). AcmA of Lactococcus lactis, a cell-binding major autolysin. PhD thesis, University of Groningen, Netherlands.
Buist, G., Kok, J., Leenhouts, K. J., Dabrowska, M., Venema, G. & Haandrikman, A. J. (1995). Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. J Bacteriol 177, 15541563.[Abstract]
Buist, G., Karsens, H., Nauta, A., van Sinderen, D., Venema, G. & Kok, J. (1997). Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA. Appl Environ Microbiol 63, 27222728.[Abstract]
Calamita, H. G., Ehringer, W. D., Koch, A. L. & Doyle, R. J. (2001). Evidence that the cell wall of Bacillus subtilis is protonated during respiration. Proc Natl Acad Sci U S A 98, 1526015263.
Chapot-Chartier, M. P. (1996). Les autolysines des bactéries lactiques. Lait 76, 91109.
Chapot-Chartier, M. P., Deniel, C., Rousseau, M. & Gripon, J. C. (1994). Autolysis of two strains of Lactococcus lactis during cheese ripening. Int Dairy J 4, 251269.
Chopin, A., Chopin, M.-C., Moillo-Batt, A. & Langella, P. (1984). Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid 11, 260263.[Medline]
Chopin, A., Bolotin, A., Sorokin, A., Ehrlich, S. D. & Chopin, M. (2001). Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations. Nucleic Acids Res 29, 644651.
Chu, C. P., Kariyama, R., Daneo-Moore, L. & Shockman, G. D. (1992). Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. J Bacteriol 174, 16191625.[Abstract]
Cibik, R., Lepage, E. & Talliez, P. (2000). Molecular diversity of Leuconostoc mesenteroides and Leuconostoc citreum isolated from traditional French cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification. Syst Appl Microbiol 23, 267278.[Medline]
Cibik, R., Talliez, P., Langella, P. & Chapot-Chartier, M. P. (2001). Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum. Appl Environ Microbiol 67, 858864.
Crow, V. L., Coolbear, T., Gopal, F. G., Martley, F. G., McKay, L. L. & Riepe, H. (1995). The role of autolysis of lactic acid bacteria in the ripening of cheese. Int Dairy J 5, 855875.
Drouault, S., Corthier, G., Ehrlich, S. D. & Renault, P. (1999). Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Appl Environ Microbiol 65, 48814886.
Fischetti, V. A., Pancholi, V. & Schneewind, O. (1990). Conservation of a hexapeptide sequence in the anchor region of surface proteins from Gram-positive cocci. Mol Microbiol 4, 16031605.[Medline]
Fischetti, V. A., Pancholi, V. & Schneewind, O. (1991). Common characteristics of the surface proteins from gram-positive cocci. In Genetics and Molecular Biology of Streptococci, Lactococci and Enterococci, pp. 290294. Edited by G. M. Dunny, P. P. Cleary & L. L McKay. Washington, DC: American Society for Microbiology.
Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154, 19.[Medline]
Gibson, T. J. (1984). Studies on the Epstein-Barr genome. PhD thesis, University of Cambridge, UK.
Holo, H. & Nes, I. F. (1995). Transformation of Lactococcus by electroporation. Methods Mol Biol 47, 195199.[Medline]
Horsburgh, G. J. (2001). The role of autolysins of Bacillus subtilis 168 during cell growth and differentiation. PhD thesis, University of Sheffield, UK.
Husson-Kao, C., Mengaud, J., Benbadis, L. & Chapot-Chartier, M. P. (2000). Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain. FEMS Microbiol Lett 187, 6976.[CrossRef][Medline]
Joris, B., Englebert, S., Chu, C. P., Kariyama, R., Daneo-Moore, L., Shockman, G. D. & Ghuysen, J. M. (1992). Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin. FEMS Microbiol Lett 70, 257264.[Medline]
Kemper, M. A., Urrutia, M. M., Beveridge, T. J., Koch, A. L. & Doyle, R. J. (1993). Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis. J Bacteriol 175, 56905696.[Abstract]
Kuroda, M., Ohta, T., Uchiyama, I. & 34 other authors (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 12251240.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lepeuple, A. S., Van Gemert, E. & Chapot-Chartier, M. P. (1998). Analysis of the bacteriolytic enzymes of the autolytic Lactococcus lactis subsp. cremoris strain AM2 by renaturing polyacrylamide gel electrophoresis: identification of a prophage-encoded enzyme. Appl Environ Microbiol 64, 41424148.
Mainardi, J. L., Legrand, R., Arthur, M., Schoot, B., van Heijenoort, J. & Gutmann, L. (2000). Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium. J Biol Chem 275, 1649016496.
McIver, K. S., Subbarao, S., Kellner, E. M., Heath, A. S. & Scott, J. R. (1996). Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci. Infect Immun 64, 25482555.[Abstract]
Moens, S. & Vanderleyden, J. (1997). Glycoproteins in prokaryotes. Arch Microbiol 168, 169175.[CrossRef][Medline]
Morton, T. M., Eaton, D. M., Johnston, J. L. & Archer, G. L. (1993). DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1. J Bacteriol 175, 44364447.[Abstract]
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 16.[Abstract]
Niven, G. W. & Mulholland, F. (1998). Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures. J Appl Microbiol 84, 9096.[CrossRef]
Pillidge, C. J., Govindasamy-Lucey, S., Gopal, P. K. & Crow, V. L. (1998). The major lactococcal cell wall autolysin AcmA does not determine the rate of autolysis of Lactococcus lactis subsp. cremoris 2250 in cheddar cheese. Int Dairy J 8, 843850.[CrossRef]
Pospiech, A. & Neumann, B. (1995). A versatile quick-prep of genomic DNA from gram-positive bacteria. Trends Genet 11, 217218.[CrossRef][Medline]
Ramadurai, L., Lockwood, K. J., Nadakavukaren, M. J. & Jayaswal, R. K. (1999). Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 145, 801808.[Abstract]
Rathsam, C. & Jacques, N. A. (1998). Role of C-terminal domains in surface attachment of the fructosyltransferase of Streptococcus salivarius ATCC 25975. J Bacteriol 180, 64006403.
Rathsam, C., Giffard, P. M. & Jacques, N. A. (1993). The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system. J Bacteriol 175, 45204527.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shockman, G. D. & Höltje, J. V. (1994). Microbial peptidoglycan (murein) hydrolases. In New Comprehensive Biochemistry, vol. 27, Bacterial Cell Wall, pp.131167. Edited by J.-M. Guysen & R. Hakenbeck. Amsterdam: Elsevier Science.
Smith, T. J., Blackman, S. A. & Foster, S. J. (2000). Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146, 249262.
Steen, A., Buist, G., Horsburgh, G., Foster, S., Kuipers, O. & Kok, J. (2001). AcmA is a glucosaminidase. In Abstracts, Eurolab Conference, 36 July 2001, Cork, Irish Republic.
Trieu-Cuot, P., de Cespedes, G. & Horaud, T. (1992). Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501. Plasmid 28, 272276.[Medline]
Tusnady, G. E. & Simon, I. (2001). The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849850.
van Asseldonk, M., Rutten, G., Oteman, M., Siezen, R. J., de Vos, W. M. & Simons, G. (1990). Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95, 155160.[CrossRef][Medline]
Walker, S. A. & Klaenhammer, T. R. (2001). Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways. Appl Environ Microbiol 67, 251259.
Wells, J. M., Robinson, K., Chamberlain, L. M., Schofield, K. M. & Le Page, R. W. (1996). Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek 70, 317330.[Medline]
Wilkinson, G. M., Guinee, T. P., O'Callaghan, D. M. & Fox, P. F. (1994). Autolysis and proteolysis in different strains of starter bacteria during cheddar cheese ripening. J Dairy Res 61, 249262.
Received 12 July 2002;
revised 24 October 2002;
accepted 28 November 2002.