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PubMed2003

Taxonomic positioning of two biological control agents for plant diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics.

AIMS: To taxonomically position two bacterial strains conferring biological control activity towards plant diseases. METHODS AND RESULTS: Key phenotypic characteristics, including gliding motility and a high percentage of G + C content, indicated biocontrol strains N4-7 and C3 were essentially identical to those described for Lysobacter enzymogenes. Cellular fatty acid analysis confirmed a close relatedness of strains N4-7 and C3 to L. enzymogenes and a more distant relatedness to L. antibioticus. The 16S rDNA phylogenetic analysis revealed a distinct Lysobacter clade that included both strains within the gamma-proteobacteria. CONCLUSIONS: The combined taxonomic methods provide clear evidence that N4-7 and C3 should be grouped as strains of L. enzymogenes and not Stenotrophomonas maltophilia or a novel taxon. Phylogenetic analysis of 16S rDNA formed a Lysobacter clade that included several other environmentally diverse bacterial strains obtained from databases and confirmed relatedness of strains N4-7 and C3 to L. enzymogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Inclusion of N4-7 and C3 as strains of L. enzymogenes is among the first description of members of this genus as biocontrol agents of plant diseases. These results suggest that members of the Lysobacter group might provide a new source as plant-associated microbes that display biocontrol activity.

Bacterial Typing Techniques↗
PubMed1996

The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini.

A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.

Bacteriolysis↗
PubMed1996

Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis.

For most large phages of both Gram-positive and Gram-negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage-encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non-specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual-start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N-terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysiseffector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a 'hole' in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual-start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.

Amino Acid Sequence↗
PubMed1996

The two-step lysis system of pneumococcal bacteriophage EJ-1 is functional in gram-negative bacteria: triggering of the major pneumococcal autolysin in Escherichia coli.

The holin function Ejh of the pneumococcal bacteriophage EJ-1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli, and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.

Amino Acid Sequence↗
PubMed1998

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release.

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H-19B genome located the genes encoding shiga-like toxin I (Stx-I) downstream of the gene encoding the analogue of the phage lambda Q transcription activator with its site of action, qut at the associated pR' late promoter, and upstream of the analogues of lambda genes encoding lysis functions. Functional studies, including measurement of the effect of H-19B Q action on levels of Stx expressed from an H-19B prophage, show that the H-19B Q acts as a transcription activator with its associated pR'(qut) by promoting readthrough of transcription terminators. Another toxin-producing phage, 933W, has the identical Q gene and pR'(qut) upstream of the stx-II genes. The H-19B Q also activates Stx-II expression from a 933W prophage. An ORF in H-19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx-I expression can be enhanced by transcription from pR' as well as a model for toxin release through cell lysis mediated by action of phage-encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H-19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N-like system differs significantly from those of other lambdoid phages.

Amino Acid Sequence↗
PubMed1998

Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties.

A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-beta-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.

Adhesins, Bacterial↗
PubMed2001

The puzzle of zmpB and extensive chain formation, autolysis defect and non-translocation of choline-binding proteins in Streptococcus pneumoniae.

Choline-binding proteins (CBPs) from Streptococcus pneumoniae are involved in several important processes. Inactivation of zmpB, a gene that encodes a surface-located putative zinc metalloprotease, in a S. pneumoniae serotype 4 strain was recently reported to reveal a composite phenotype, including extensive chain formation, lysis defect and transformation deficiency. This phenotype was associated with the lack of surface expression of several CBPs, including the major autolysin LytA. LytA, normally 36 kDa in size, was reported to form an SDS-resistant 80 kDa complex with CinA. ZmpB was therefore proposed to control translocation of CBPs to the surface, possibly through the proteolytic release of CBPs (and RecA) from CinA. Based on the use of 12 independent mariner insertions in the zmpB gene of the well-characterized R6 laboratory strain, we could not confirm several of these observations. Our zmpB mutants: (i) did not form chains; (ii) lysed normally in the presence of deoxycholate, which indicates the presence of a functional autolysin; (iii) transformed at normal frequency; and (iv) contained bona fide CinA and LytA species. Polymorphism of ZmpB between R6 and the serotype 4 isolate could not account for the discrepancy, as inactivation of zmpB (through replacement by transposon-inactivated zmpB R6 alleles) in the latter strain did not affect separation of daughter cells and autolysis. The conflicting observations could be explained by our finding that the reportedly serotype 4 zmpB 'mutant' differed from its S. pneumoniae parent in lacking capsule and in exhibiting characteristic traits of the Streptococcus viridans group, including resistance to optochin.

Amino Acid Sequence↗
PubMed2001

Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli.

N-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein cross-bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.

Anti-Bacterial Agents↗
PubMed2002

The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174.

Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn. All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.

Amino Acid Sequence↗
PubMed2003

DNA gyrase requirements distinguish the alternate pathways of Mu transposition.

The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.

Bacteriolysis↗
PubMed2003

Characterization of RAT, an autolysis regulator in Staphylococcus aureus.

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.

Amino Acid Sequence↗
PubMed1999

A comparative study on the structure and function of a cytolytic alpha-helical peptide and its antimicrobial beta-sheet diastereomer.

Antimicrobial peptides which adopt mainly or only beta-sheet structures have two or more disulfide bonds stabilizing their structure. The disruption of the disulfide bonds results in most cases in a large decrease in their antimicrobial activity. In the present study we examined the effect of d-amino acids incorporation on the structure and function of a cytolytic alpha-helical peptide which acts on erythrocytes and bacteria. The influence of a single or double d-amino acid replacement in alpha-helical peptides on their structure was reported previously in 50% 2,2,2, trifluoroethanol/water [Krause et al. (1995) Anal. Chem. 67, 252-258]. Here we used Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy and found that the predominant structure of the wild-type peptide is alpha-helix in phospholipid membranes, whereas the structure of the diastereomer is beta-sheet. However, the linear, beta-sheet diastereomer preserved its cytolytic activity on bacteria but not on erythrocytes. Previous studies have shown that the ability of antimicrobial peptides to lyse bacteria but not normal mammalian cells correlated with their ability to disintegrate preferentially negatively charged, but not zwitterionic phospholipid membranes. In contrast, the diastereomer described here disrupts zwitterionic and negatively charged vesicles with similar potencies to those of the hemolytic wild-type peptide. Interestingly, whereas addition of a positive charge to the N-terminus of the wild-type peptide (which caused a minor effect on its structure) increased activity only towards some of the bacteria tested, similar modification in the diastereomer increased activity towards all of them. Furthermore, the modified wild-type peptide preserved its potency to destabilize zwitterionic and negatively charged vesicles, whereas the modified diastereomer had a reduced potency on zwitterionic vesicles but increased potency on negatively charged vesicles. Overall our results suggest that this new class of antimicrobial diastereomeric peptides bind to the membrane in 'carpet-like' manner followed by membrane disruption and breakdown, rather than forming a transmembrane pore which interfere with the bacteria potential. These studies also open a way to design new broad-spectrum antibacterial peptides.

Amino Acid Sequence↗
PubMed2004

Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey.

Predation at the lowest trophic level, i.e. between bacteria, is poorly understood, hindering efforts to assess its impact on the structure of bacterial communities. The interaction of Bdellovibrio and Bacteriovorax (Bdellovibrio and like organisms, BLOs), a group of obligate, ubiquitous predatory bacteria, with their Gram-negative bacterial prey results in the multiplication of the predator and in the lysis, but not in the eradication, of the prey. We show that the residual, surviving populations of prey cells exposed to predation stress differ from the populations before exposure, as they exhibit increased resistance to predation. This resistance was demonstrated in a number of Gram-negative prey. Moreover, predation resistance is not specific for the BLO strain experienced by the prey. The phenomenon does not stem from a mutation but is a plastic response associated with a phenotypic change, and it disappears upon removal of the predator. As resistance to predation is not total, this mechanism can ensure survival of both predator and prey.

Adaptation, Physiological↗
PubMed1998

Effects of tea tree oil on Escherichia coli.

Tea tree oil (TTO) stimulates autolysis in exponential and stationary phase cells of Escherichia coli. Electron micrographs of cells grown in the presence of TTO showed the loss of electron dense material, coagulation of cell cytoplasm and formation of extracellular blebs. Stationary phase cells demonstrated less TTO-stimulated autolysis and also had greater tolerance to TTO-induced cell death, compared to exponentially grown cells. It was also revealed that subpopulation of stationary phase cells demonstrated increased tolerance to TTO-bactericidal effects.

Bacteriolysis↗
PubMed1998

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa.

The presence of divalent (Mg2+) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7.8 or 9.0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg2+ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.

Bacteriolysis↗
PubMed2001

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria.

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.

Adenylate Kinase↗
PubMed2002

In-well cell lysis technique reveals two new megaplasmids of 103.0 and 212.6 MDa in the multiple plasmid-containing strain V517 of Escherichia coli.

AIMS: Identification of two new plasmids in the multiple plasmid-containing strain V517 of Escherichia coli. METHOD AND RESULTS: By using an in-well mild cell lysis technique suitable for megaplasmids observation, two plasmids of 103.0 and 212.6 MDa were detected in the multiplasmid-containing E. coli V517. CONCLUSIONS: The two new megaplasmids that were completely overlooked when standard disruptive procedures were used, can now be added to the list of eight plasmids with molecular size from 1.36 to 35.84 MDa reported earlier. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding allows to use the strain V517 not only as a size reference of small and moderately large plasmids but as a size reference of megaplasmids as well.

Bacteriological Techniques↗