Recent recurrent outbreaks of bacterial resistance to antibiotics have shown the critical need to identify new lytic agents to combat them. The species Lysobacter capsici VKM B-2533T possesses a potent antimicrobial action against a number of bacteria, fungi and yeasts. Its activity can be due to the impact of bacteriolytic enzymes, antibiotics and peptides. This work isolated four homogeneous bacteriolytic enzymes and a mixture of two proteins, which also had a bacteriolytic activity. The isolates included proteins identical to L. enzymogenes α- and β-lytic proteases and lysine-specific protease. The proteases of 26 kDa and 29 kDa and a protein identified as N-acetylglycosaminidase had not been isolated in Lysobacter earlier. The isolated β-lytic protease digested live methicillin-resistant staphylococcal cells with high efficiency (minimal inhibitory concentration, 2.85 μg/mL). This property makes the enzyme deserving special attention. A recombinant β-lytic protease was produced. The antimicrobial potential of the bacterium was contributed to by outer membrane vesicles (OMVs). L. capsici cells were found to form a group of OMVs responsible for antifungal activity. The data are indicative of a significant antimicrobial potential of this bacterium that requires thorough research.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

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.

Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a β-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533T. This BlpLc possesses a valuable property, not described for β-lytic proteases (Blps) earlier, of hydrolyzing living cells of Staphylococcus aureus 55 MRSA clinical isolate. This work phylogenetically characterized the BlpLc and investigated its properties. Analysis revealed a variability of pre-/pro-parts of Blp precursors. The mature BlpLc is the closest to the earlier annotated but not isolated Blp from Lysobacter sp. Root690. The biochemical characterization found conditions for the BlpLc general bacteriolytic activity relative to autoclaved S. aureus 209P cells to differ from that of earlier isolated Blp. Unexpected was the effect of serine (phenylmethylsulfonyl fluoride (PMSF)) and cysteine (p-chloromercuribenzoate (p-CMB)) protease inhibitors on BlpLc bacteriolytic and proteolytic activities. The specificity of BlpLc proteolytic action relative to hemoglobin, elastin, gelatin, collagen, azofibrin, myoglobin, ovalbumin, and ovamucoid was found. New types of peptide bonds—Gly-X, Ser-X, Lys-X, Ala-X, Val-X, Glu-X, and Phe-X—hydrolyzed by the enzyme in protein substrates were first revealed using MALDI-TOF. Turbidimetrically, the BlpLc was found to lyze living cells of S. aureus 209P, Micrococcus luteus B1819, and M. roseus B1236, which is important for expanding the enzyme’s applied properties.

The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained.