O. A. Stepnaya

Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles

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.

Identification and characterization of the metal ion-dependent l-alanoyl-d-glutamate peptidase encoded by bacteriophage T5

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.

Isolation and Characterization of a New Extracellular Bacteriolytic Endopeptidase of Lysobacter sp. XL1

The previously unstudied bacteriolytic enzyme L4 was isolated from the culture liquid of the bacterium Lysobacter sp.XL1 in electrophoretically homogeneous state. The enzyme L4 is a diaminopimelinoyl-alanine endopeptidase relative to peptidoglycan of Lysobacter sp. XL1. The enzyme is an alkaline protein of ∼ 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°C. The half-inactivation temperature of the enzyme is 52°C. The endopeptidase L4 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.

Structural investigations and identification of the extracellular bacteriolytic endopeptidase L1 from Lysobacter sp. XL1.

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 α-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 α-lytic proteinase EC 3.4.21.12.

Extracellular yeast-lytic enzyme of the bacterium Lysobacter sp. XL 1.

An enzyme exhibiting yeast-lytic activity has been isolated from the culture liquid of the bacterium Lysobacter sp. XL. 1. The optimal conditions for the hydrolysis of Saccharomyces cerevisiae cells by the enzyme have been established: 0.15 M sodium acetate buffer, pH 6.0, 50°C. The yeast-lytic activity of the enzyme is inhibited by EDTA, p-chloromercuribenzoate, and phenylmethylsulfonyl fluoride. According to the data of SDS-PAGE, the molecular weight of the protein is 36 kD. The enzyme hydrolyzes casein, hemoglobin, and synthetic peptide Abz-Ala-Ala-Phe-pNA, i.e. it exhibits proteolytic activity. The properties of the enzyme and its molecular weight correspond to those of a previously isolated extracellular metalloproteinase. The N-terminal amino acid sequence of the protein exhibits 67% homology with the N-terminal sequence of achromolysine of Achromobacter lyticus (EC 3.4.24.-).

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.

Lytic Peptidase L5 of Lysobacter sp. XL1 with Broad Antimicrobial Spectrum

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.

Specificity of the action of lysoamidase on Staphylococcus aureus 209P cell walls.

Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase.

Structure of an acidic polysaccharide present in the bacteriolytic complex lysoamidase

The structure of an acidic polysaccharide component of a bacteriolytic complex (lysoamidase), isolated from a bacterium of the genus Xanthomonas, was studied. On the basis of sugar analysis and one‒ and two‒dimensional 1H and 13C NMR spectroscopic study of the initial polysaccharide and its O‒deacetylated and carboxyl‒reduced derivatives, the following structure of the trisaccharide repeating unit of the polysaccharide was established where ManNAcA and GalNAcA are 2‒acetamido‒2‒deoxymannuronic acid and 2‒acetamido‒2‒deoxygalacturonic acid, respectively.

Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads

Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms.