Cloning and expression analysis of genes encoding lytic endopeptidases L1 and L5 from Lysobacter sp. strain XL1.

Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the Lysobacter sp. strain XL1 alpA and alpB genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB). In silico analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the alpA and alpB open reading frames (ORFs) in Escherichia coli confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The alpA and alpB mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the alpA and alpB mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of alpA mRNA in cells of Lysobacter sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

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 degrees 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.-).

Substrate specificity and some physicochemical properties of autolytic enzymes of the bacterium Lysobacter sp. XL 1.

The substrate specificity of autolytic enzymes of the bacterium Lysobacter sp. XL 1 has been established. The periplasmic enzyme A8, the cytosolic enzyme A1, and the enzyme A10 solubilized from the cell walls and membranes with Triton X-100 exhibit glucosaminidase activity; the cytosolic enzyme A4 and the enzyme A9 solubilized from the cell walls and membranes with LiCl exhibit the muramidase activity. The cytosolic enzymes A3 and A6 have N-acetylmuramoyl-L-alanine amidase activity, and the enzyme A5 exhibits the diaminopimelinoyl-alanine endopeptidase activity. Some physicochemical properties of the most active autolytic cytosolic enzymes of Lysobacter sp. XL 1 (endopeptidases A5 and A7 and N-acetylmuramoyl-L-alanine amidase A6) were studied. The enzymes exhibit maximal activity over a wide range of buffer concentrations in weakly alkaline medium and moderate temperatures. The investigated enzymes are comparatively thermolabile proteins.

Isolation and characterization of a new extracellular bacteriolytic endopeptidase of Lysobacter sp. XL1.

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.

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 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.

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.

Intracellular peptidoglycan hydrolases of the bacterium Xanthomonas campestris XL-1.

A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A(1)-A(7)) are localized in cytosol, one enzyme (A(8)) in periplasm, and two enzymes (A(9), A(10)) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A(1) significantly increases, and A(5) and A(6) decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A(7) present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation.

Influence of acidic exopolysaccharide of Xanthomonas campestris IBPM 124 on the kinetic parameters of extracellular bacteriolytic enzymes.

Interactions of a negatively charged exopolysaccharide of Xanthomonas campestris IBPM 124 with its extracellular enzymes (muramidase, endopeptidase, and neutral phosphatase) and also with egg lysozyme, lysostaphin, muramidase of Streptomyces globisporus, and a bacteriolytic enzyme complex of Streptomyces albus were studied. All these enzymes were positively charged under the conditions of their maximal activity. It was shown that interaction of the acidic exopolysaccharide from X. campestris with these enzymes changed their kinetic parameters. The change was either positive (increase in reaction rate) or negative (decrease in reaction rate) and depended on the enzyme and type of substrate cleaved. Due to such interactions, the acidic exopolysaccharide secreted by X. campestris into the environment not only retained and transported positively charged exoenzymes into the near-cellular space, but also regulated their activity.

Intracellular glucosaminidase of the bacterium Xanthomonas campestris IBPM B-124: purification and properties.

A system of intracellular autolytic enzymes of the bacterium Xanthomonas campestris IBPM B-124 was found to include enzymes with muramidase and glucosaminidase activities, while a system of extracellular bacteriolytic enzymes of the same bacterium includes muramidase, muramoylalanine amidase, and endopeptidase. Using a purification technique including fractional precipitation with ammonium sulfate, gel-filtration on Toyopearl HW-55F, and FPLC ion-exchange chromatography on Mono Q, a preparation of intracellular glucosaminidase was purified 435-fold with 16% yield (SDS-PAGE data indicated the presence of minor protein contaminants). Some physicochemical properties of the purified enzyme were determined: molecular mass 26 kD, Km = 5.6 x 10(-4) M with p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as the substrate, and pH optimum 8.0-8.5. The enzyme is active over a wide range of Tris-HCl buffer concentrations (0.01-0.5 M) and has temperature optimum at 37-40 degrees C. The glucosaminidase activity is sensitive to p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), and the disodium salt of ethylenediamine tetraacetic acid (EDTA). The properties of this glucosaminidase markedly differ from those of all extracellular bacteriolytic enzymes of Xanthomonas campestris. These findings indicate that the system of autolytic enzymes of this bacterium functions independently and is not connected with the system of extracellular bacteriolytic enzymes.

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 [formula: see text] where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively.