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PubMed1986

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin.

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.

Amidohydrolases↗

PubMed1992

Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.

Amino Acid Sequence↗

PubMed1998

The role of autolysins during vegetative growth of Bacillus subtilis 168.

A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (sigma D, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be sigma D-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.

Anti-Bacterial Agents↗

PubMed2001

Polymorphic outer-membrane proteins of Chlamydophila abortus are glycosylated.

Antigenic profiles of mono-, bi- and poly-specific monoclonal antibodies against 90 kDa polymorphic outer-membrane proteins (POMPs) and a 105 kDa POMP-related protein of Chlamydophila abortus ATCC VR 656(T), after one- and two-dimensional electrophoretic analysis, helped identify each one of the triplets POMP 90, 91A and 91B, and a POMP-related protein at 85 kDa. The lectin concanavalin A bound to the four POMPs and the POMP-related protein in a specific manner and the binding was sensitive to treatment with the amidase N-endoglycosidase F, suggesting the presence of small asparagine-linked oligosaccharide chains. The exposure of the five proteins on the chlamydial surface and the orientation of the attached oligosaccharide chains was examined by protease and endoglycosidase treatments of intact bacteria. The results were consistent with the concept that some of the oligosaccharides in the POMPs face outwards, possibly protecting the polypeptides from proteolytic enzymes, whereas the oligosaccharides in the 105 kDa POMP-related protein are oriented inwards, thereby rendering the polypeptide chain accessible to proteases. A possible role for the N-linked oligosaccharides in the POMPs might be the promotion of the proper folding and processing of these proteins.

Amidohydrolases↗

PubMed1999

Crystallization of a precursor penicillin acylase from Escherichia coli.

The crystallization of a mutant precursor penicillin acylase [penicillin amidohydrolase (amidase) E.C. 3.5.1.11] from Escherichia coli W (ATCC 11105) using the hanging-drop method is reported. The crystals are in space group P1 with unit-cell parameters a = 51.04, b = 63.58, c = 71.17 A, alpha = 103.0, beta = 110.6, gamma = 105.3 degrees, with one molecule in the asymmetric unit, and diffract to 1. 8 A using synchrotron radiation.

Bacterial Proteins↗

PubMed1994

Isolation and characterization of Staphylococcus aureus mutants which form altered cell clusters.

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-beta-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.

Acetylglucosaminidase↗

PubMed1993

Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme.

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Amino Acid Sequence↗

PubMed1995

Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes.

Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.

Amino Acid Sequence↗

PubMed2001

The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor.

Adherence of pathogenic microorganisms to the cell surface is a key event during infection. We have previously reported the characterization of Listeria monocytogenes transposon mutants defective in adhesion to eukaryotic cells. One of these mutants had lost the ability to produce Ami, a 102 kDa autolytic amidase with an N-terminal catalytic domain and a C-terminal cell wall-anchoring domain made up of repeated modules containing the dipeptide GW ('GW modules'). We generated ami null mutations by plasmid insertion into L. monocytogenes strains lacking the invasion proteins InlA (EGDDeltainlA), InlB (EGDDeltainlB) or both (EGDDeltainlAB). These mutants were 5-10 times less adherent than their parental strains in various cell types. The adhesion capacity of the mutants was restored by complementation with a DNA fragment encoding the Ami cell wall-anchoring domain fused to the Ami signal peptide. The cell-binding activity of the Ami cell wall-anchoring domain was further demonstrated using the purified polypeptide. Growth of the ami null mutants constructed in EGD and EGDDeltainlAB backgrounds was attenuated in the livers of mice inoculated intravenously, indicating a role for Ami in L. monocytogenes virulence. Adhesive properties have recently been reported in the non-catalytic domain of two other autolysins, Staphylococcus epidermidis AtlE and Staphylococcus saprophyticus Aas. Interestingly, we found that these domains were also composed of repeated GW modules. Thus, certain autolysins appear to promote bacterial attachment by means of their GW repeat domains. These molecules may contribute to the colonization of host tissues by Gram-positive bacteria.

Amino Acid Sequence↗

PubMed2006

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

Adhesins, Bacterial↗

PubMed1995

Site-specific and complete enzymic deglycosylation of the native human chorionic gonadotropin alpha-subunit.

Numerous studies have shown that glycosylation of the alpha-subunit of human chorionic gonadotropin (alpha hCG) is essential for the biological activity of this hormone. To obtain detailed insight into the function of N-glycosylation, the availability of site-specifically and fully deglycosylated alpha-subunits obtained under non-denaturing conditions is a prerequisite. NMR spectroscopy in combination with FAB-mapping demonstrates that only Asn52 of the alpha-subunit is accessible to digestion by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F under native conditions. Treatment of native alpha hCG with endo-beta-N-acetylglucosaminidase B results in full deglycosylation yielding alpha hCG with one GlcNAc residue at both Asn52 and Asn78.

Amidohydrolases↗

PubMed1997

Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from alphaD-hemocyanin of Helix pomatia.

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein alphaD-hemocyanin (alphaD-Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an alphaD-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH2, was characterized using 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of alphaD-Hc were released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH2/Lichrosphere-NH2, and/or Lichrosphere-C18. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 1H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, [structures: see text] which are all attached to O-2 of alphaMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally beta-1,2-xylosylated and alpha-1,6-fucosylated, Man(alpha1-6)[Man(alpha1-3)][+/-Xyl(beta1-2)]Man(beta1-4)GlcNAc(beta1-4) [+/-Fuc(alpha1-6)]GlcNAc.

Amidohydrolases↗

PubMed2005

Components of the peptidoglycan-recycling pathway modulate invasion and intracellular survival of Salmonella enterica serovar Typhimurium.

beta-Lactam resistance in enteric bacteria is frequently caused by mutations in ampD encoding a cytosolic N-acetylmuramyl- l-alanine amidase. Such mutants are blocked in murein (peptidoglycan) recycling and accumulate cytoplasmic muropeptides that interact with the transcriptional activator ampR, which de-represses beta-lactamase expression. Salmonella enterica serovar Typhimurium, an extensively studied enteric pathogen, was used to show that mutations in ampD decreased the ability of S. typhimurium to enter a macrophage derived cell line and made the bacteria more potent as inducers of inducible nitric oxide synthase (iNOS), as compared with the wild-type. ampG mutants, defective in the transport of recycled muropeptides across the cytoplasmic membrane, behaved essentially as the wild-type in invasion assays and in activation of iNOS. As ampD mutants also have reduced in vivo fitness in a murine model, we suggest that the cytoplasmic accumulation of muropeptides affects the virulence of the ampD mutants.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1992

Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage.

The development of an appropriate technique for the identification of autolysin-defective mutants of pneumococcus has been a fundamental step to carry out studies on the molecular characteristics of the lytic enzymes of Streptococcus pneumoniae and its bacteriophage. Our results show that the principal pneumococcal autolysin (an amidase) is responsible for the separation of the daughter cells at the end of the cell division. On the other hand, this system provides a reliable experimental model to support the extended idea concerning the modular organization of most proteins. The comparative analyses of the deduced amino acid sequences of these enzymes, as well as the construction of functional chimeric phage-bacterial enzymes, demonstrate that the C-terminal domain, which contains a large number of repeated amino acid motifs, is the substrate-binding domain, whereas the N-terminal domain provides enzymatic specificity. We propose that the pneumococcal lytic enzymes have evolved by modular exchange providing examples of the types of novel genes that the bacteria or the phage might create to allow them to become adapted to new environmental situations.

Amino Acid Sequence↗

PubMed1995

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin.

We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus. In the presence of 0.02% Triton-X-100, 17 formerly methicillin-resistant strains exhibited enhanced sensitivity to oxacillin. One homogenous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N-acetylglucosaminidase and 62 kDa N-acetylmuramoyl-L- alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus influencing a factor (s) other than PBPs, bacteriolytic enzymes, or femAB products.

Acetylglucosaminidase↗

PubMed2002

Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD.

The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal beta-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC beta-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC beta-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive beta-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135-->Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal beta-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal beta-lactamase.

Amino Acid Sequence↗

PubMed2002

The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains.

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage phi3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage lambda S holin in lambdadeltaSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA(Arg) and tRNA(Ile) genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.

Bacterial Proteins↗

PubMed1981

Role of autolysin in generating the pneumococcal purpura-producing principle.

The results of four studies described in this paper support the idea that pneumococcal autolysin (N-acetylmuramyl-L-alanine amidase) plays a key role in generating the pneumococcal purpura-producing principle (PPP) from the bacterial cell wall. This conclusion is based on the observation that (i) concentrates of pneumococcal cell-free extracts prepared at a pH which is inhibitory to autolysin activity contained significantly less PPP activity than extract concentrates prepared at a pH which is optimal for autolysin activity; (ii) PPP activity was not detectable in concentrates of cell-free extracts prepared from cells which contained ethanolamine in the cell wall, but a normal amount of activity was found in extract concentrates derived from cells containing choline in the cell wall; (iii) PPP activity was not detectable in concentrates of cell-free extracts prepared from an autolysin-defective mutant, but a normal amount of activity was found in extract concentrates derived from the wild-type strains; and (iv) incubation of purified wild-type strain cell walls with pneumococcal autolysin resulted in a significant increase in the amount of purpurogenic activity in the supernatant fluids of the incubation mixtures.

Amidohydrolases↗