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PubMed1975

Susceptibility to mitomycin C and lecithinase activities of Clostridium oedematiens (C. novyi) type B and D.

In tests with broth-culture products of Clostridium oedematiens, none of 15 type-B strains showed beta-toxin lecithinase activities exceeding 20 egg-units per ml, whereas 12 of 13 type-D strains consistently produced much greater amounts of the lecithinase. The types also differed in their susceptibility to lysis by mitomycin C (MC). Of 13 type-D strains tested, 12 were sensitive to MC at a concentration of 1 or 2 mug per ml, whilst 14 of 15 type-B strains were insensitive. Phage-like particles were observed in the MC-lysates of some type-D strains. No type-specific differences in the production of indole or the fermentation of maltose were demonstrated.

Bacteriolysis↗
PubMed1994

The Bacillus subtilis lipoprotein LplA causes cell lysis when expressed in Escherichia coli.

A gene called lplA (lipoprotein-like) has been isolated from a genomic library of Bacillus subtilis expressed in Escherichia coli. Clones carrying the lplA gene were selected by the ability of the colonies to give visible haloes of starch hydrolysis. The cloned fragment contains an open reading frame (ORF) of 1509 bp encoding a protein of 56 kDa. The protein contains a typical N-terminal signal sequence, a putative transmembrane anchor domain and a leucine zipper at the C-terminus. The expression of this protein in E. coli causes cell lysis, only the N-terminal domain of the LplA protein being responsible for this phenotype. The mechanism of cell lysis is similar to that previously suggested for the expression in E. coli of the lipoproteins encoded by the Streptococcus pneumoniae genes malX and amiA. The protein is modified with palmitic acid when secreted in E. coli, confirming that it is a typical lipoprotein.

Amino Acid Sequence↗
PubMed1994

Lytic enzymes associated with defective prophages of Bacillus subtilis: sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX.

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.

Amino Acid Sequence↗
PubMed1995

Characterization of cryptic prophages (monocins) in Listeria and sequence analysis of a holin/endolysin gene.

Monocins in Listeria were induced by UV-irradiation of liquid cultures, and defective phage particles were purified from the lysates. Electron microscopy showed flexible, non-contractile bacteriophage-tail-like particles, consisting of specific proteins of molecular mass 20-45 kDa and pI 4.6-6.7. These particles were able to lyse listerial cells. DNA sequence homologies between chromosomal DNA of monocin-producing strains and labelled Listeria phage DNAs were inferred from DNA/DNA hybridizations, suggesting that most of the prophage DNA is still present in the listerial chromosome. An endolysin gene cpl2438 was cloned from listerial chromosomal DNA and was identified by its expression of lytic activity against Listeria cells in a bioassay. The gene consists of 864 nt encoding a protein of 287 aa with a calculated molecular mass of 32975 Da (CPL2438). This is in good agreement with the size of a protein observed in SDS-PAGE after overexpression of the lytic protein in Escherichia coli. The nucleotide sequence of a putative holin gene (hol2438, 291 nt) upstream of cpl2438 was determined after PCR-amplification of listerial DNA and it shows typical features common to the holin gene family. Expression of the encoded protein (HOL2438, 95 aa, 10.1 kDa) in E. coli was found to be lethal for the host cells. The results underline the close relationship between monocins and intact Listeria bacteriophages, indicating that monocins are incompletely assembled phage particles derived from cryptic prophages of Listeria, probably including the phage lysin.

Amino Acid Sequence↗
PubMed1995

A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization.

Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the proform is processed to release the active enzyme during germination.

Amino Acid Sequence↗
PubMed1996

Gene expression in the cos region of the Streptomyces temperate actinophage phi C31.

A transcription map of a 5.12 kb region containing the cos ends of actinophage phi C31 was determined using RNA prepared from induced and uninduced cultures of the temperature-sensitive lysogen, Streptomyces coelicolor A3(2) (phi C31cts1). In induced cultures, RNA synthesis was detected only late in the lytic cycle. A late operon initiated downstream of the int gene on the right-hand end of the genome traversed the cos ends and extended at least 3.6 kb along the left-hand end. Shorter, possibly processed, mRNAs were also present. The map was superimposed on the DNA sequence of 2.8 kb of the region, part of which had been determined previously and part of which is presented here. The late-expressed transcripts contained a tRNA(Thr)-like gene and four ORFs (1, 2, 3 and 5) detected on the basis of codon bias. Analysis of the putative protein products showed that one of the ORFs could encode a lysis protein and at least one may be involved in DNA maturation. Transcription mapping of RNA from uninduced cultures demonstrated a 620 nt transcript, xRNA1, of ORF6. So far this is the only gene in phi C31 found to be expressed right to left with respect to the standard map of phi C31; its function during lysogenic growth could not be deduced from database searches.

Amino Acid Sequence↗
PubMed1999

Structural and functional analysis of pCI65st, a 6.5 kb plasmid from Streptococcus thermophilus NDI-6.

The 6.5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S. thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of gram-positive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 degrees C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant.

Amino Acid Sequence↗
PubMed1999

Partial characterization of a major autolysin from Mycobacterium phlei.

Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.

Amino Acid Sequence↗
PubMed1999

Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD.

A gamma-D-glutamate-meso-diaminopimelate muropeptidase was detected in the vegetative growth phase of Bacillus subtilis 168. It is encoded by the monocistronic lytF operon expressed by the alternative vegetative sigma factor, sigmaD. Sequence analysis of LytF revealed two domains, an organization common to exoproteins of B. subtilis as well as to those from other organisms. The N-terminal domain contains a fivefold-repeated motif attributed to cell wall binding, whilst the C-terminal domain is probably endowed with the catalytic activity. Overexpression of LytF allowed its purification and biochemical characterization. Inactivation of lytF led to the loss of the cell-wall-bound protein 49' (CWBP49') and of the corresponding lytic activity as revealed by renaturation gel assay. Native cell walls prepared from the multiple lytC lytD lytE lytF-deficient mutant did not exhibit any autolysis, whereas walls prepared from a strain endowed with LytF but not with the other three enzymes underwent a slight lysis. Analysis of degradation products of cell wall devoid of teichoic-acid-bound O-esterified D-alanine unambiguously confirmed that LytF cuts the gamma-D-glutamate-mesodiaminopimelate bond.

Amino Acid Sequence↗
PubMed1999

A theoretical and empirical investigation of the invasion dynamics of colicinogeny.

A mathematical model describing the dynamics of a colicinogenic and a colicin-sensitive population propagated under serial transfer culture conditions was formulated. In addition, a series of in vitro invasion experiments using six representatives of the E colicin group was undertaken, together with the estimation of the growth rates and colicinogenic characteristics of the strains. Growth rates among the strains varied by up to 44%. There were 14-fold differences among strains in their lysis rates and there were up to 10-fold differences in the amount of colicin produced per lysed cell. The in vitro serial transfer invasion experiments revealed that regardless of initial frequency all colicinogenic strains succeeded in displacing the sensitive cell populations. The amount of time required for the colicin-sensitive cell population to be displaced declined as the initial frequency of the colicinogenic population increased and strains producing higher titres of colicin tended to displace the sensitive strain more rapidly. Overall, the observed dynamics of the invasion of colicinogenic strains was adequately described by the theoretical model. However, despite there being substantial differences among the strains in their growth rates and colicinogenic characteristics there were relatively few differences, observed or predicted, in the invasion dynamics of the six colicinogenic strains. These results suggest that the characteristics of different colicinogenic strains cannot be used to explain the extensive variation in the relative abundance of different colicins in natural populations of bacteria.

Bacteriolysis↗
PubMed1999

Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus.

The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. Immunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of lytM expression in two global regulatory mutants, agr and sar, showed that expression of lytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X11-Val-X12/20-Gly-X5-6-His. Atomic absorption spectrometric analysis of LytM revealed the presence of 0.9 mol zinc (mol LytM)(-1).

Animals↗
PubMed2003

Expression of heterologous O-antigen in Yersinia pestis KIM does not affect virulence by the intravenous route.

All strains of Yersinia pestis examined have been found to lack an O-antigen. In other members of the Enterobacteriaceae, the rough phenotype often results in attenuation. However, Y. pestis is the aetiological agent of bubonic plague. In evolving from the ancestral enteropathogenic Yersinia pseudotuberculosis, and with the development of an arthropod-vectored systemic pathogenesis, smooth LPS production is not necessary for Y. pestis virulence and the metabolic burden has been alleviated by inactivation of the O-antigen biosynthetic operon. To investigate this, Y. pestis strain KIM D27 was transformed with a plasmid carrying the operon encoding the O-antigen of Yersinia enterocolitica O : 3. Expression of the O-antigen could be detected in silver-stained gels. The receptor for bacteriophage phiYeO3-12 has been shown to be O-antigen, and infection by this bacteriophage results in lysis of Y. enterocolitica O : 3. Expression of the O-antigen in Y. pestis conferred sensitivity to lysis by phiYeO3-12. The O-antigen-expressing clone was shown to be as virulent in mice by the intravenous route of challenge as the rough wild-type. Assays showed no alteration in the ability of Y. pestis to resist lysis by cationic antimicrobial peptides, serum or polymyxin.

Animals↗
PubMed2004

One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.

The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.

Animals↗
PubMed2003

Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis.

A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The encoded AcmB protein has a modular structure with three domains: an N-terminal domain, especially rich in Ser, Thr, Pro and Asn residues, resembling a cell-wall-associated domain; a central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a C-terminal domain of unknown function. A recombinant AcmB derivative, devoid of its N-terminal domain, was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of several Gram-positive bacteria, including L. lactis. Though showing sequence similarity with enterococcal muramidase, AcmB has N-acetylglucosaminidase specificity. The acmB gene was inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell separation but contributes to cellular autolysis.

Acetylglucosaminidase↗
PubMed2003

Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis.

Staphylococcus epidermidis biofilm formation on polymer surfaces is considered a major pathogenicity factor in foreign-body-associated infections. Previously, the 148 kDa autolysin AtlE from S. epidermidis, which is involved in the initial attachment of the cells to polymer surfaces and also binds to the extracellular matrix protein vitronectin, was characterized. Here, the characterization of a novel autolysin/adhesin (Aae) in S. epidermidis is described. Aae was identified as a 35 kDa surface-associated protein that has bacteriolytic activity and binds vitronectin. Its N-terminal amino acid sequence was determined and the respective gene, aae, was cloned. DNA-sequence analysis revealed that aae encodes a deduced protein of 324 amino acids with a predicted molecular mass of 35 kDa. Aae contains three repetitive sequences in its N-terminal portion. These repeats comprise features of a putative peptidoglycan binding domain (LysM domain) found in a number of enzymes involved in cell-wall metabolism and also in some adhesins. Expression of aae by Escherichia coli and subsequent analysis revealed that Aae possesses bacteriolytic activity and adhesive properties. The interaction of Aae with fibrinogen, fibronectin and vitronectin was found to be dose-dependent and saturable and to occur with high affinity, by using the real-time Biomolecular Interaction Analysis (BIA). Aae binds to the Aalpha- and Bbeta-chains of fibrinogen and to the 29 kDa N-terminal fragment of fibronectin. In conclusion, Aae is a surface-associated protein with bacteriolytic and adhesive properties representing a new member of the staphylococcal autolysin/adhesins potentially involved in colonization.

Adhesins, Bacterial↗
PubMed2004

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles.

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; rho=1.18 g cm(-3)) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Amino Acid Sequence↗
PubMed2004

DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential.

Natural membrane vesicles (n-MVs) produced by Pseudomonas aeruginosa PAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were purified and analysed for DNA content. The MVs were isolated by a procedure designed to ensure no cellular contamination from the parent MV-producing cells. Fluorometry analysis revealed that p-MVs were associated with 7.80 ng DNA (20 microg MV protein)(-1). PCR analysis using specific primers for pAK1900 sequences and a chromosomal target, oprL, indicated that only plasmid DNA was contained within the lumen of p-MVs after exogenous DNA was digested by DNase. MVs have previously been shown to be capable of fusing into the outer membrane (OM) of PAO1 and Escherichia coli DH5 alpha. Accordingly, p-MVs should deliver the plasmid into the periplasm, where it would only have to by-pass the plasma membrane (PM) for effective transformation. It was speculated that p-MVs should increase transformation efficiency but the data suggested otherwise. p-MVs did not transform PAO1 nor DH5 alpha under a variety of transforming conditions. To characterize p-MVs and to ensure that membrane-encapsulated pAK1900 was not derived from a small proportion of lysed cells within the culture and bound by PM instead of OM, which typically forms n-MVs, the physical and ultrastructural differences between n- and p-MVs were determined. Cryo-transmission electron microscopy (cryo-TEM) revealed that n-MVs and p-MVs closely resembled isolated OM. Buoyant density measurements using isopycnic sucrose gradients on isolated PM, OM, n- and p-MVs demonstrated that isolated OM and n-MVs both fractionated into two bands (rho=1.240 and 1.260 g ml(-1)). p-MVs also produced two bands but at two different densities (rho=1.250 and 1.265 g ml(-1)) which may be attributed to the presence of DNA. SDS-PAGE showed that p-MVs possessed most major OM proteins and also contained 43.70 nmol 3-deoxy-d-manno-octulosonic acid (KDO) (mg protein)(-1) as an LPS marker. The amount of NADH oxidase activity, a PM enzyme, in the p-MVs was barely detectable. These data strongly suggest that p-MVs are OM-based, with little if any PM material associated with them. The possibility of whether exogenous plasmid DNA could enter n-MVs once the vesicles had departed from cells was also tested; surprisingly, a small amount of DNA could. Accordingly, the data suggest that DNA can be taken up by MVs using two separate routes: (1) via a periplasmic route and (2) via an extracellular, exogenous route.

Bacteriolysis↗