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PubMed2004

Enterococcal aggregation substance and binding substance are not major contributors to urinary tract colonization by Enterococcus faecalis in a mouse model of ascending unobstructed urinary tract infection.

Isogenic Enterococcus faecalis strains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS(+), constitutive EBS(+)) failed to outcompete isogenic derivative INY3000 (AS(-) EBS(-)) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS(+), EBS(+)) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by E. faecalis and that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.

Animals↗
PubMed2004

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).

Bacterial Adhesion↗
PubMed2004

Effects of the Enterococcus faecalis hypR gene encoding a new transcriptional regulator on oxidative stress response and intracellular survival within macrophages.

In order to identify regulators of the oxidative stress response in Enterococcus faecalis, an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydroperoxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis.

Animals↗
PubMed2004

Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium.

Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.

Animals↗
PubMed2005

Enterococcus faecalis mammalian virulence-related factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model.

Some pathogenic bacteria belong to a large, diverse group of species capable of infecting plants, animals, and humans. Enterococcus faecalis is an opportunistic human pathogen capable of infecting patients with a deficient immune system. Here we report that three E. faecalis strains (FA-2-2, V583, and OG1RF) are capable of infecting the leaves and roots of the model plant species Arabidopsis thaliana, causing plant mortality 7 days postinoculation. We found that E. faecalis pathogenesis in A. thaliana leaves is determined by the following series of events: attachment to leaf surface, entry through stomata or wounds, and colonization in intercellular spaces, leading to rotting and to the disruption of plant cell wall and membrane structures. The three E. faecalis strains colonize the roots of A. thaliana by forming a mosaic of large clusters of live bacteria on the root surface, as observed by scanning electron microscopy, phase-contrast microscopy, and fluorescence microscopy. To dissect the involvement of mammalian virulence-related factors in plant pathogenicity, we tested E. faecalis mutant strains DeltafsrA (TX5240), DeltafsrB (TX5266), DeltafsrC (TX5242), DeltagelE (TX5264), and DeltasprE (TX5243), which correspond to virulence factors involved in pathogenesis in different animal models. Two E. faecalis virulence-related factors that play an important role in mammalian and nematode models of infection, a putative quorum-sensing system (DeltafsrB) and serine protease (DeltasprE), were also found to be important for plant pathogenesis. The development of an E. faecalis-A. thaliana model system could potentially be used to circumvent certain inherent limitations that an animal model imposes on the identification and study of virulence factors. Furthermore, our study suggests an evolutionary crossover of virulence factors in plant, animal, and nematode pathogenesis.

Arabidopsis↗
PubMed2005

Gelatinase is important for translocation of Enterococcus faecalis across polarized human enterocyte-like T84 cells.

Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE, as well as disruption of its regulatory system (fsrA, fsrB, and fsrC) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE- SprE+) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA, fsrB, and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB, are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 (gelE) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.

Bacterial Proteins↗
PubMed2005

Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice.

Enterococcus faecalis is a gram-positive bacterium that can cause a variety of nosocomial infections of which urinary tract infections are the most common. These infections can be exceptionally difficult to treat because of drug resistance of many E. faecalis isolates. Despite their troublesome nature, little is known about the host or bacterial factors necessary for E. faecalis to cause disease in the urinary tract. Using a mouse model of urinary tract infection, we have shown that E. faecalis is capable of persisting in the kidneys of mice for at least 2 weeks. In contrast, bacterial titers from the bladders of the same mice were inconsistent and tended to be much lower than those recovered from the kidney. This preference for the kidney over the bladder is also observed in other clinical E. faecalis strains. Histologic examination of bladder and kidney tissues demonstrated that E. faecalis induced an inflammatory response in the kidney but not in the bladder. This inflammatory response was TLR2 independent and did not induce inflammatory markers typically associated with uropathogenic Escherichia coli. Using a competition assay, we demonstrated that a pyelonephritis clinical isolate had a growth advantage over a laboratory strain of E. faecalis in the kidneys but not in the bladders of mice. Taken together, these results demonstrate that E. faecalis has tropism for the kidneys in the urinary tracts of mice and that this system can be used to study factors involved in the pathogenesis of urinary tract infections.

Animals↗
PubMed2005

Effects of a probiotic strain of Enterococcus faecium on the rate of natural chlamydia infection in swine.

Chlamydiae are obligately intracellular pathogens which cause infections associated with a broad range of diseases in both livestock and humans. In addition, a large proportion of animals may become persistently infected asymptomatic carriers and serve as reservoirs for other animals which also shed these potential zoonotic pathogens. Reducing the chlamydial load of animals is therefore of major importance, and since large-scale antibiotic treatment is neither desired nor feasible, alternative means of prevention are needed. Here we performed a study comparing the efficacy of a probiotic strain of Enterococcus faecium on the reduction of both the rate of natural infection and the shedding of chlamydiae in swine. The presence of Chlamydiaceae was detected by species-specific PCR of fecal samples of sows taken at three times prior to the birth of piglets. Piglets delivered from chlamydia-positive sows in either the control or the probiotic group were also examined for the frequency of chlamydiae at various ages. Eighty-five percent of the piglets from the control group were found to be chlamydia positive, whereas chlamydiae were found in only 60% of piglets from the probiotic group, results confirmed by fluorescence in situ hybridization and immunohistology, which showed higher rates of infection in the control group. In addition to the reduced frequency of chlamydia-positive piglets in the probiotic group, the time of appearance of positive samples was delayed. To our knowledge, these data show for the first time that a probiotic strain of E. faecium can reduce the rate of carryover infections of piglets by obligate intracellular pathogens.

Animals↗
PubMed2005

Fsr-independent production of protease(s) may explain the lack of attenuation of an Enterococcus faecalis fsr mutant versus a gelE-sprE mutant in induction of endocarditis.

An Enterococcus faecalis gelE insertion disruption mutant (TX5128), which produces neither gelatinase (GelE) nor the cotranscribed (in the wild type) serine protease (SprE), was found to be attenuated in a rat endocarditis model with a significant decrease in the endocarditis induction rate versus wild-type E. faecalis OG1RF (GelE(+), SprE(+)). TX5266, which has a nonpolar deletion in fsrB and, like TX5128, is phenotypically GelE(-) under usual conditions, was also studied; fsrB is a homologue of agrB of staphylococci and participates in regulation of gelE-sprE expression. Unexpectedly, TX5266 approximated wild-type OG1RF in the endocarditis model and was significantly less attenuated than TX5128. This is in contrast to other models which have found fsr mutants to be as or more attenuated than TX5128. Further study found that the fsrB mutant produced very low levels of gelatinase activity after prolonged incubation in vitro versus no gelatinase activity with TX5128 and did not show the extensive chaining characteristic of TX5128. Reverse transcription-PCR confirmed that gelE was expressed in TX5266 at a very low level versus wild-type OG1RF and was not expressed at all in TX5128. Possible explanations for the increased induction of endocarditis by TX5266 versus TX5128 include the production of low levels of protease(s) or some other effect(s) of the inactivation of the E. faecalis fsr regulator. The equivalent ability of OG1RF and its fsr mutant to initiate endocarditis may explain why we did not find naturally occurring fsr mutants, which account for ca. 35% of E. faecalis isolates, unrepresented in endocarditis versus fecal isolates.

Animals↗
PubMed2006

PBP5 complementation of a PBP3 deficiency in Enterococcus hirae.

The low susceptibility of enterococci to beta-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed in Enterococcus hirae SL2, a mutant whose pbp5 gene was interrupted by the nisRK genes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-borne pbp5 genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184 region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209 segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.

Enterococcus↗
PubMed2006

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Bacillus subtilis↗
PubMed2006

Comparative genomic hybridization analysis of Enterococcus faecalis: identification of genes absent from food strains.

Enterococcus faecalis, a member of the natural microbiota of animal and human intestinal tracts, is also present as a natural contaminant in a variety of fermented foods. Over the last decade, E. faecalis has emerged as a major cause of nosocomial infections. We investigated the genetic diversity in 30 clinical and food isolates, including strains V583 and MMH594, in order to determine whether clinical and food isolates could be distinguished. Data were obtained using comparative genomic hybridization and specific PCR with a total of 202 probes of E. faecalis, selected using the available V583 genome sequence and part of the MMH594 pathogenicity island. The cognate genes encoded mainly exported proteins. Hybridization data were analyzed by a two-component mixture model that estimates the probability of any given gene to be either present or absent in the strains. A total of 78 genes were found to be variable, as they were absent in at least one isolate. Most of the variable genes were clustered in regions that, in the published V583 sequence, related to prophages or mobile genetic elements. The variable genes were distributed in three main groups: (i) genes equally distributed between clinical and dairy food isolates, (ii) genes absent from dairy food-related isolates, and (iii) genes present in MMH594 and V583 strains only. Further analysis of the distribution of the last gene group in 70 other isolates confirmed that six of the probed genes were always absent in dairy food-related isolates, whereas they were detected in clinical and/or commensal isolates. Two of them corresponded to prophages that were not detected in the cognate isolates, thus possibly extending the number of genes absent from dairy food isolates. Genes specifically detected in clinical isolates may prove valuable for the development of new risk assessment markers for food safety studies and for identification of new factors that may contribute to host colonization or infection.

DNA, Bacterial↗
PubMed2025

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus.

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, which encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalis&#x394;ldh1 or &#x394;ldh1&#x394;ldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how E. faecalis mitigate assaults by L. crispatus.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, E. faecalis must interact with Lactobacillus crispatus, but how E. faecalis overcomes or mitigate assaults by L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.

Enterococcus faecalis↗
PubMed2006

Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis.

The Fst toxin of the Enterococcus faecalis pAD1-encoded par addiction module functions intracellularly to kill plasmid-free segregants. Previous results had shown that Fst induction results in membrane permeabilization and cessation of macromolecular synthesis, but only after 45 min. Electron micrographs of toxin-induced cells showed no obvious membrane abnormalities but did reveal defects in nucleoid segregation and cell division, begging the question of which is the primary effect of Fst. To distinguish the possibilities, division septae and nucleoids were visualized simultaneously with fluorescent vancomycin and a variety of DNA stains. Results showed that division and segregation defects occurred in some cells within 15 min after induction. At these early time points, affected cells remained resistant to membrane-impermeant DNA stains, suggesting that loss of membrane integrity is a secondary effect caused by ongoing division and/or segregation defects. Fst-resistant mutants showed greater variability in cell length and formed multiple septal rings even in the absence of Fst. Fst induction was also toxic to Bacillus subtilis. In this species, Fst induction caused only minor division abnormalities, but all cells showed a condensation of the nucleoid, suggesting that effects on the structure of the chromosomal DNA might be paramount.

Bacillus subtilis↗
PubMed2006

C-terminal WxL domain mediates cell wall binding in Enterococcus faecalis and other gram-positive bacteria.

Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.

Amino Acid Sequence↗
PubMed2006

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.

Amino Acid Sequence↗
PubMed2006

Siamycin attenuates fsr quorum sensing mediated by a gelatinase biosynthesis-activating pheromone in Enterococcus faecalis.

The expression of two Enterococcus faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the fsr gene cluster. In this system, E. faecalis secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts, fsrBDC and gelE-sprE. In the present study, we screened actinomycete metabolites for inhibitors of fsr quorum sensing. E. faecalis was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of Streptomyces sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting E. faecalis cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited E. faecalis cell growth at concentrations above micromolar concentrations. Quantitative analysis of fsrBDC and gelE-sprE transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections.

Bacterial Proteins↗
PubMed2006

Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

Acetylglucosaminidase↗