The Gram‐negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1–L5), into the culture medium. These proteins are able to degrade Gram‐positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram‐positive and Gram‐negative bacteria: Staphylococcus aureus 209‐P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram‐negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram‐positive and Gram‐negative bacteria.

Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin‐encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity). The protein purified was shown to be an l‐alanoyl‐d‐glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers (25–50 mm) at pH 8.5. The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium/manganese chlorides. It was found that, along with E. coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram‐negative microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis). This endolysin is the first example of an l‐alanoyl‐d‐glutamate peptidase in a virulent phage infecting Gram‐negative bacteria. There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic l‐alanoyl‐d‐glutamate peptidases. The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution.

The Gram-negative bacterium Lysobacter sp. XL1 secretes lytic enzymes (L1-L5) into the culture medium. Enzyme L5 is the most recently found extracellular lytic enzyme of this bacterium. The paper presents the results of the isolation and characterization of some properties of this enzyme. Thus, enzyme L5 of Lysobacter sp. XL1 is a lytic serine protease. Earlier, the enzyme was shown to be secreted into the culture medium by means of outer membrane vesicles, which possess a lytic effect towards living cells of Erwinia caratovora B15 [Vasilyeva et al., FEBS J 2008;15:3827-3835]. This work shows the action of enzyme L5 either as a vesicle component or the homogeneous enzyme L5 on a broad range of Gram-positive and Gram-negative microorganisms. Moreover, the vesicles containing this enzyme were shown to lyze the selected test cultures more efficiently than the soluble enzyme L5. It appears to be one of the first precedents of a bacteriolytic effect mediated by the action of outer membrane vesicles filled with extracellular lytic enzymes. The results suggest that the enzyme L5 of Lysobacter sp. XL1 and the vesicles containing this enzyme can be used as an antimicrobial drug.

The previously unstudied bacteriolytic enzyme L(4) was isolated from the culture liquid of the bacterium Lysobacter sp. XL1 in electrophoretically homogeneous state. The enzyme L(4) is a diaminopimelinoyl-alanine endopeptidase relative to peptidoglycan of Lysobacter sp. XL1. The enzyme is an alkaline protein of approximately 21 kD. The N-terminal amino acid sequence of the enzyme has been determined - A V V N G V N Y V Gx T T A ... The maximal activity of the enzyme was observed in 0.05 M Tris-HCl at pH 8.0 and 50-55 degrees C. The half-inactivation temperature of the enzyme is 52 degrees C. The endopeptidase L(4) is not a metalloenzyme since it is not affected by EDTA. The enzyme is inhibited by p-chloromercuribenzoic acid by 72% and by phenylmethylsulfonyl fluoride by 43%, which indicates the involvement of serine and thiol groups in its functioning.

Membrane vesicles produced by bacteria have been intensively studied in the recent years. Investigators have noted their roles in essential processes in the bacterial cell including secretion of proteins by the ‘eukaryotic' vesicular mechanism. To date, formation of vesicles is not considered to be a spontaneous event. Many believe it to be a programmed process that can be guided by several mechanisms. Vesicles are derivatives of the cell envelope, which in turn is a supramolecular structure where the functioning and biogenesis of all components are interrelated. Proteins secreted beyond the cell in their translocation are also part of the cell envelope. This also suggests their role in vesicle biogenesis. This review presents the results of vesicle studies in the Gram-negative bacterium Lysobacter sp. This bacterium is of interest as it secretes a number of proteins to the environment, including bacteriolytic enzymes. Bacteriolytic enzymes, on the one hand, are important for studies from a medical point of view as they can form the basis of new generation antimicrobial means. On the other hand, they are a convenient subject for studies of vesicle functions in the vital activities of the bacterial cell.

The N-terminal amino acid sequence (23 amino acid residues) and the amino acid composition of the extracellular bacteriolytic enzyme L1 of 21 kD from the bacterium Lysobacter sp. XL1 have been determined. The enzyme was hydrolyzed by trypsin, the resulting peptides were isolated, and their primary structures were determined. A high extent of homology (92%) of the N-terminal amino acid sequence and the primary structure of isolated peptides of the enzyme L1 (62 amino acid residues or 31% of protein sequence) to the corresponding sites of alpha-lytic proteinases (EC 3.4.21.12) of Lysobacter enzymogenes and Achromobacter lyticus was found. These data allowed identification of the endopeptidase L1 of Lysobacter sp. XL1 as alpha-lytic proteinase EC 3.4.21.12.

No abstract

No abstract