Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1.

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-L-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

[Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies].

The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

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.

[Mechanism of action of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-positive bacteria: role of the cell wall anionic polymers of the target bacteria]. / Rol' anionnykh polimerov kletochnykh stenok grampolozhitel'nykh bakterii-mishenei v mekhanizme deistviia na nikh vnekletochnykh bakterioliticheskikh fermentov Lysobacter sp.

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of teichoic and teichuronic acids, rather than on their chemical composition.

[The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria]. / Deistvie vnekletochnykh bakteriologicheskikh fermentov Lysobacter sp. na gramotritsatel'nye bakterii.

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 had been altered by treating them with polymyxin B.

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.

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.

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.

[Bacteriolytic enzyme preparation lysoamidase. Purification and some properties of bacteriolytic peptidase L1]. / Bakterioliticheskii fermentnyi preparat lizoamidaza. Ochistka i nekotorye svoistva bakteriolithceskoi peptidazy L1.

The bacteriolytic peptidase L1 has been isolated from the enzyme preparation of lysoamidase capable to lyze cell walls of gram-positive bacteria using ion-exchange chromatography and gel filtration. Some physico-chemical properties of the enzyme have been established. The molecular mass of L1 is 21 kDa, the pH optimum for Staphylococcus aureus cell lysis is 7-11. The optimal concentration of the buffer is 50 mM; the temperature optimum is 70 degrees C; the half-inactivation temperature is 55 degrees C.

[Changes in biogenesis and secretion of periplasmic alkaline phosphatase, coded by the phoA gene included in the Escherichia coli DH1 plasmid]. / Izmeneniia v biogeneze i sekretsiia v sredu periplazmaticheskoi shchelochnoi fosfatazy, kodiruemoi genom phoA v sostave plazmidy u Escherichia coli DH1.

Augmented synthesis of periplasmic alkaline phosphatase from E. coli DH1 coded by the phoA gene within the composition of plasmid pHI-7 results in alterations in the enzyme biogenesis, such as accumulation of intermediate enzyme forms corresponding to various stages of its posttranslational modification, and alternative localization. The cell cytoplasm was found to contain a large proportion of alkaline phosphatase precursors in the form of insoluble aggregates. The mature enzyme was detected in both the periplasm and the cultural fluid in the form three methazymes with an increased (as compared with the original strain) content of methazymes I and II. The cell envelope of E. coli DHI transformed by plasmid pHI-7 with the phoA gene differed from that of the original strain by an increased acid phospholipid (cardiolipin/phosphatidylglycerol) ratio as well as by increased distribution density of intramembrane particles on the surface of the cytoplasmic membrane facing the cytoplasm. Changes in the biogenesis of the enzyme during its augmented synthesis are due to disturbances in the equilibrium between the synthesis of polypeptide chains, on the one hand, and their translocation and processing, on the other, apparently as a result of restricted secretion sites and lack of inhibition of translation of secreted proteins in bacteria.

[Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykh form u Escherichia coli. II. Vliianie zamen aminokislot v saite protsessinga i N-kontsevom domene zreloi polipeptidnoi tsepi shchelochnoi fosfatazy na ee biogenez.

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of in vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

[Features of the biogenesis of Escherichia coli alkaline phosphatase during its supersynthesis]. / Osobennosti biogeneza shchelochnoi fosfatazy Escherichia coli pri ee sverkhsinteze.

The oversynthesis of the secreted alkaline phosphatase (PhoA) in E.coli K12802 cells due to transformation with the PhoA+ plasmid pHI-7 leads to a change in its biogenesis--alternative localization and accumulation of the enzyme intermediate forms corresponding to different stages of the its post-translational modification. Instead of the soluble PhoA available in the parent strain mostly as a completely processed mature metazyme III localized in the periplasm, five enzyme forms were discovered in the PhoA overproducer: a cytoplasmic PhoA precursor (prePhoA) as insoluble aggregates; three soluble metazymes of a mature active form localized in the periplasm as in well as in culture medium; and a soluble high-molecular form in the periplasm. PrePhoA was isolated and purified by removal of soluble cell fractions using differential centrifugation, solubilization of membrane proteins with Triton X100, dissolution of the aggregates in the buffer with 8M urea and FPLC on MonoQ. Extracellular PhoA was purified by ultrafiltration, thermal treatment, and gel chromatography on Sepharose CL-4B. It was shown that the isolated prePhoA can be transformed into a mature form in the presence of a leader peptidase in 0.8 urea and is completely cleaved with proteinase K. Three forms of the mature PhoA vary in resistance to proteinase K and trypsin. Metazyme I, the unprocessed mature PhoA, is the most resistant to proteolysis.

[Secretion of periplasmic PhoA protein during its supersynthesis into the medium using outer membrane vesicles. Features of chemical composition of the vesicles and membranes of Escherichia coli secreting cells]. / Sekretsiia periplazmaticheskogo PhoA-belka pri ego sverkhsinteze v sredu s pomoshch'iu bezikul vneshnikh membran. Osobennosti khimicheskogo sostava vezikul i membran sekretirushchikh kletok Escherichia coli.

E. coli K12802 cells transformed by multicopy plasmid with phoA gene acquire the ability to oversynthesize alkaline phosphatase, secrete it into the cultural medium, and accumulate the precursor of this enzyme. The dynamics of enzyme production and secretion as well as cytomorphological changes revealed the existence of a mechanism of selective enzyme secretion into the medium. It is characterized by a decrease of enzyme specific activity in periplasm and its increase in cultural medium, appearance of numerous local zones of adhesion of cytoplasmic and outer membranes, formation of large extracellular outer membrane vesicles containing PhoA protein on the cell poles, and their release into the medium. We isolated the vesicles and found that they contain PhoA (in dominating quantity), several other periplasmic proteins, and matrix proteins of outer membranes. By their phospholipid and protein composition, they correspond to the fraction of outer membranes which have the largest density and sedimentation rate and, apparently, contain no lipoprotein.

[Transformation of various strains of Escherichia coli by multicopy plasmids with the phoA gene leads to secretion of a periplasmatic alkaline phosphatase into the media and accumulation of its precursor]. / Transformatsiia razlichnykh shtammov Escherichia coli mul'tikopiinymi plazmidami s genom phoA pribodit k sekretsii periplazmaticheskoi shchelochnoi fosfatazy v sredu i nakopleniiu ee predshestvennika.

Various E. coli strains have been transformed by multicopy plasmids pHI-1, pHI-7 and pPHO I carrying the entire regulatory and structural phoA sequences. All the transformants with the intact pho regulatory system have been shown to be capable of alkaline phosphatase oversynthesis and secretion into the medium. They are also able to accumulate the alkaline phosphatase precursor localized in the outer membrane fraction. The transformants of the restriction or recombination mutants are the most efficient producers of the extracellular enzyme and its precursor.