Lytic potential of Lysobacter capsici VKM B-2533T: bacteriolytic enzymes and outer membrane vesicles

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.


Results
Dependence of the production of lytic agents in L. capsici VKM B-2533 t on storage and cultivation conditions. The issue of stable production of commercially valuable substances by microorganisms has always been topical. In L. capsici, the dependence of the total production of antimicrobial agents on storage and cultivation conditions has not been investigated earlier.
Upon acquisition of the L. capsici type strain from the All-Russian Collection of Microorganisms RAS, we investigated its ability to lyse live cells of various microorganisms. The strain was found to have a potent lytic action against Gram-positive bacteria, yeasts and fungi. No activity was revealed with respect to Gram-negative bacteria. In the laboratory collection the strain was maintained on agarized LBs and PMA media and in cryopreservation. The culture stored on agarized media was re-inoculated each ten days. A smaller number of passages led to a viability loss.
After a year under the selected storage conditions a 100% antimicrobial activity was preserved only in the cryopreserved culture. In this case, the activity was revealed in cultivation on all tested liquid nutrient media.
In storage on LBs medium, the antimicrobial activity was lost partially. After a year of maintenance on this medium, the activity with respect to all bacterial test objects was revealed on SYM, KSP and RM media (Table 1, Fig. 1a). It is seen from the table that at OD/mL and LU/mL values of the same order of magnitude on different cultivation media the lytic potential with respect to live test objects is not the same. Thus, during the cultivation of the bacterium on PYKM medium the lytic action with respect to live test cultures is totally absent. Herewith, the values of OD/mL and LU/mL on this medium are the same as on SYM and KSP. At high values of LU/mL

Fungi and yeasts
Aspergillus niger Table 1. Dependences of the production of lytic agents in L. capsici VKM B-2533 T . on storage and cultivation conditions ++, strong lytic effect; +, medium lytic effect; -, no lytic effect.
during the cultivation on RM and LB media the lytic action with respect to live test objects is observed only on RM medium. The most potent lytic activity with respect to live test cultures was observed on SYM medium. The yeast-lytic activity was preserved with respect to C. boidinii, being revealed in cultivation on virtually all media. With respect to C. utilis the activity was lost. The antifungal activity was revealed in cultivation on only SYM media (Table 1, Fig. 1b). In storage on PMA medium and subsequent cultivation on various liquid nutrient media, the antimicrobial activity was gradually lost. After a year of this storage, a weak lytic activity was revealed against bacteria M. luteus, M. reseus, S. aureus at the cultivation on RM medium and with respect to M. luteus in cultivation on KSP medium ( Table 1). The yeast-lytic activity was preserved in the same way as during the maintenance of the culture on LBs. The antifungal activity was totally lost. It should be understood that the total lytic activity can be contributed to by bacteriolytic enzymes, peptides and antibiotics, as well as by OMVs that contain these substances. A crucial condition for the antimicrobial properties of L. capsici VKM B-2533 to be retained is its maintenance in cryopreserved state. In this case, the activity is preserved and manifests itself at the cultivation of the bacterium on various nutrient media. The culture maintained on agarized media partially or totally loses its specific activities, which is indicative of a gradual switching-off of individual genes or genes of the whole biosynthetic pathways of some or other agents. However, this feature can be useful in future research into the regulation of the synthesis of these compounds, as well as can facilitate the isolation of specific agents.
isolation of bacteriolytic enzymes. It is known that extracellular bacteriolytic enzymes are mainly alkaline proteins whose molecular weights, as a rule, do not exceed 30 kDa 8,35,36 . For isolation of L. capsici bacteriolytic enzymes, we developed a scheme, which includes fractionation of proteins by ammonium sulphate, cation exchange chromatography and gel filtration (Fig. 2a, Supplementary Table S1). As the result of the developed purification scheme, we succeeded in isolating a mixture of two proteins with MW 29 kDa (Fig. 2b, lane 3) with  Using MALDI-TOF, we found not only the known and earlier characterized proteins among those isolated but also absolutely new proteases not isolated earlier ( Table 2). The mixture of proteins was found to consist of two serine proteases. One of them was identified for the first time. Proteins of МW 21 and 19 kDa are the αand β-lytic proteases well known for Lysobacter 37 . Protein identified as N-acetylglucosaminidase, and one more serine protease of MW 26 kDa, were isolated in Lysobacter for the first time.
Although all isolated proteins possessed bacteriolytic activities with respect to autoclaved staphylococcal cells, only the β-lytic protease ( enzymogenes is known since the 1960s; 37 no recombinant protein has been produced until now, however.
The complete nucleotide sequence of the gene βl of the L. capsici VKM B-2533 T β-lytic protease was determined and entered into the GenBank under the number MN604699.
The amplified gene of the L. capsici β-lytic protease (1059 bp) was successfully cloned into the expression vector pET19mod, followed by the transformation into cells of E. coli BL21(DE3)/pLysE. As the result of this gene expression, cells of the recombinant strains accumulated the major protein of MW 38 kDa (Fig. 3a, lane 1). This corresponds to the weight of the β-lytic protease pro-protein, calculated in the ExPaSy bioinformatics programme (https://web.expasy.org/compute_pi/). Ultrasonic disruption of cells followed by centrifugation produced a residue and a supernatant, which were analyzed electrophoretically. Cells of E. coli BL21(DE3)/pLysE, which do not induce IPTG, were used as a control. As seen in Fig. 3a, the protein is localized in inclusion bodies (Fig. 3a, lane 3) and is absent in the supernatant (Fig. 3a, lane 5). The use of such approaches as cultivation at low temperature (20 °C), induction by a reduced concentration of IPTG (0.2 mM) as well as the use of the plasmid pT-GroE carrying the gene of the chaperone GroEL failed to transfer the protein from inclusion bodies to a soluble form. Further work was conducted with inclusion bodies.
The recombinant protein was produced according to the developed scheme (Fig. 3b). Special conditions were selected for renaturation of the β-lytic protease. For this, a number of buffers were used (see Materials and methods). The refolding was considered to be successful if a bacteriolytic activity with respect to live and autoclaved S. aureus 209 P cells emerged. The successful refolding was found to occur when using buffers 2, 4, 5 and conditions specified in Fig. 3b.
The recombinant protein was purified to an electrophoretically homogeneous state (Fig. 3c, lane 1). The specific activity on autoclaved and live S. aureus 209 P cells was 4 400 LU/mg. The specific activity of the native β-lytic protease with respect to these substrates was 45 000 LU/mg. Although the recombinant protein was an order of magnitude less active, the produced expression system can be considered successful and applicable for scientific purposes.
Lytic potential of L. capsici VKM B-2533 t outer membrane vesicles. The subject matter of outer membrane vesicles has been intensively developed in recent years. It is clear even now that OMVs play an important role in the activities of Gram-negative bacteria. Strain L. capsici VKM B-2533 T possesses a potent lytic potential, and OMVs can be suggested to make a contribution.
Differential centrifugation of the L. capsici culture liquid yielded a residue, which was analyzed by electron microscopy. The residue was found to consist of whole OMVs 50 up to 200 nm in diameter (Fig. 4).

Proteins identified by MALDI-TOF
Score with protein annotated in NCBI database
Serine trypsin-like protease* 120 with ALN88394. 1 29 28% with lysine-specific serine protease (P15636.1) L. enzymogenes 70 .  www.nature.com/scientificreports www.nature.com/scientificreports/ The lytic potential of vesicles was studied with respect to bacteria, fungi and yeasts (Table 3, Supplementary  Fig. S7). Outer membrane vesicles were found to possess a potent antimicrobial action against all chosen test cultures. Interestingly, the antifungal activity was totally associated with OMVs and disappeared in the surfactant after their precipitation from the culture liquid ( Supplementary Fig. S7a). At the same time, an antibacterial activity was revealed both in OMVs and in the culture liquid after their precipitation ( Supplementary Fig. S7b).

Homogeneous
It is known that bacteria can form outer membrane vesicles that perform various functions. We suggested that L. capsici could form a subpopulation of OMVs containing an antifungal agent. To confirm this suggestion, the total preparation of OMVs was fractionated in a 30-55% sucrose density gradient. As the result, we obtained 22 fractions: 2-6, fractions distributed in a 30% sucrose; 7-10, fractions distributed in a 35% sucrose; 11-14, fractions distributed in a 40% sucrose; 15-18, fractions distributed in a 45% sucrose; 19-22, fractions distributed in a 50% sucrose. All fractions were analyzed by electron microscopy (Fig. 5).
Comparative analysis of OMV lytic action in the obtained fractions showed fractions 8-13 to possess antibacterial activities (Fig. 6a). An antifungal activity was found predominantly in fraction 9 and insignificantly in fraction 10 (Fig. 6b).
Thus, an important result was obtained confirming that L. capsici cells formed a group of OMVs responsible for antifungal activity.

Discussion
By the 1970s, the Canadian microbiologists Christensen and Cook proposed to single out from myxobacteria a group of microorganisms that differ by a number of properties. The main unifying feature by which this group was attributed to myxobacteria is their ability to lyse cells of prokaryotic and eukaryotic microorganisms. In a paper published in 1978, these investigators proposed a new genus Lysobacter. The novel genus then included  Table 3. Lytic action of OMVs produced by L. capsici VKM B-2533 T . ++, strong lytic effect; +, medium lytic effect; -, no lytic effect. www.nature.com/scientificreports www.nature.com/scientificreports/ four species: L. enzymogenes ATCC 29487 named so for its ability to produce two important proteolytic enzymes; Lysobacter antibioticus ATCC 29479, for its ability to produce the antibiotic myxin; L. brunescens ATCC 29482; L. gummosus ATCC 29489 1 . In the period from 1980 to 2000, the biochemistry of the Lysobacter bacteria had been dealt with by separate groups of investigators. Starting from the 2000s, research interest in these bacteria began to rise gradually, and in the recent five years the number of publications exceeded the previous years. A significant part of these publications has been research into the isolation of novel species; genetic studies have been published; to a smaller extent, biologically active substances have been described. It is evident to date that not all isolated species of Lysobacter possess expected antimicrobial activities [38][39][40] . Among relatively new species of this genus, L. capsici possesses an explicitly pronounced antimicrobial activity.
It is known that the production of virtually all microbial substances strongly depends on storage and cultivation conditions. This is especially important for commercially valuable products of microbial synthesis. For L. capsici, the preservation of antimicrobial properties is complicated by the fact that the activity in question can be due to the combined effect of substances from different classes of compounds (proteins, antibiotics, peptides). Optimal conditions should be chosen for the preservation and efficient production of all these substances.
Earlier, we have shown that, to preserve the production of bacteriolytic enzymes in Lysobacter sp. XL1, the culture should be maintained on super-rich nutrient media 43 . In this work, we used the accumulated experience, and L. capsici was maintained on super-rich LBs, rich with PMA and cryopreserved. The maintenance of the culture in cryopreserved state was shown to be optimal for the preservation of the total lytic activity of L. capsici. This preserves the antifungal, antibacterial and yeast-lytic activities for the cultivation on various media. The storage of the culture on LBs leads to a virtually complete loss of antifungal activity. Herewith, the antibacterial activity is preserved at the cultivation on SYM, KSP and RM media. Storage on PMA leads to an almost complete loss of the antimicrobial properties on the whole.
If we analyze the component composition of the cultivation media, we can see that the concentration of protein hydrolysate (peptone, tryptone, casitone, aminopeptide) affects the exhibition of antibacterial activity. At a hydrolysate content of 10 g/L and higher this activity is observed to decrease. We should note here that this dependence is most probably determined by the production of bacteriolytic proteases. Thus, to preserve the production of these enzymes, it is important that the culture be maintained on super-rich media, and for the optimal revelation of their activity, the other way round, the cultivation on media with the low content of protein hydrolysate is required.
Also, we observe that at the maintenance and cultivation of L. capsici on rich media the antifungal activity is virtually lost. This activity could be suggested to be due to the production of an antibiotic. For this class of compounds it is known that on rich nutrient media their biosynthesis is not stable. For this reason, only cryopreservation is an optimal variant for the antibiotic activity to be retained. Herewith, a synthetic medium should be developed for the optimal manifestation of this activity.
Thus, L. capsici VKM B-2533 T is a potent producer of antimicrobial agents, which can include bacteriolytic enzymes.
A purification scheme was developed for isolation of L. capsici bacteriolytic enzymes. We succeeded in isolating four proteins in a homogeneous form and a mixture of two proteins ( Table 2). According to MALDI-TOF, the isolated proteins comprise those identical to the αand β-lytic proteases and the lysine-specific protease of L. enzymogenes. The serine proteases of MW 26 kDa and 29 kDa, and the protein identified as N-acetylglucosaminidase were isolated for the first time. Thus, these proteins are novel not only for the species L. capsici but also for the genus Lysobacter and require a detailed investigation.
Noteworthy among the isolated proteins is the β-lytic protease, which efficiently digests live cells of the methicillin-resistant staphylococcus. As a matter of fact, the β-lytic protease, along with the α-lytic protease, are the first bacteriolytic enzymes isolated in Lysobacter 37 . Both proteins have been characterized to various extents [44][45][46][47] . However, information about the recombinant β-lytic protease is absent in the literature. In this work, we succeeded in producing the recombinant protein in active state owing to the developed scheme of refolding. The produced expression system for the β-lytic protease based on E. coli BL21(DE3)/pLysE is of scientific value. Subsequently it can be used in studies of functionally significant sites for the antimicrobial activity of this protein to be revealed. At present, work is under way to develop a commercially valuable expression system for the β-lytic protease.
It is indisputable that bacteriolytic enzymes are promising for their use as the basis for new-generation antimicrobial agents. The problem of their active application is the complexity of developing biotechnologically valuable expression systems. Production of recombinant soluble bacteriolytic enzymes is problematic as it leads to the lysis of the producing strain. As the Lysobacter bacteria on the whole and the species L. capsici in particular can be considered to be a fount of such proteins, a solution of the problem can be to create homologous expression systems. Lysobacter genetic studies are now at an early stage of their development, so an efficient expression system for these proteins would hopefully appear in the near future.
As we already mentioned, the antimicrobial potential can be contributed to, besides enzymes and antibiotics, by outer membrane vesicles, which can contain all these agents. The vesicular subject area in Gram-negative bacteria was founded by the Americans Jagath L. Kadurugamuwa and Terry J. Beveridge, who carried out a comprehensive research into Pseudomonas aeruginosa vesicles. Those investigators laid the modern trends in vesicular studies, including the prospects of treating cancer and other diseases caused by pathogenic microorganisms [48][49][50] . At our laboratory, OMVs of Lysobacter sp. have been investigated for over ten years now 8,24,51 . Our research was pioneering in the field of OMV studies in Lysobacter bacteria. These days, one of the directions of our studies is the biogenesis of Lysobacter sp. XL1 OMVs and the role of the secreted bacteriolytic enzyme L5 in this process 51,52 .
This work established the ability to form outer membrane vesicles for L. capsici VKM B-2533 T . The preparation of isolated OMVs possessed a potent antimicrobial action with respect to bacteria, fungi and yeasts. This activity can be due to both bacteriolytic enzymes and other antimicrobial agents. For instance, for Lysobacter sp. XL1 it has been shown that the bacteriolytic protease L5 gets into the medium by means of vesicles 8 . As a constituent of the vesicles, this enzyme lyses a broad range of bacteria, in contrast with its soluble form 53 . It proved noteworthy that all the antifungal activity of L. capsici concentrated in OMVs. A similar result has been obtained earlier for OMVs of L. enzymogenes C3 25 . The polycyclic tetramate macrolactam antibiotics have been identified in the vesicular preparation of this bacterium. It is known that vesicles can perform various functions in the bacterial cell 54,55 . We assume that particular groups responsible for various, including antifungal, functions, can be singled out in the population of OMVs produced by L. capsici. Indeed, during the separation of total OMVs in a sucrose density gradient we succeeded in producing a separate vesicular fraction that possessed an antifungal activity. Herewith, an antibacterial activity was found in all vesicular fractions. It can be assumed that different antimicrobial agents are responsible for antifungal and antibacterial activities. That said, there is a subpopulation of antifungal vesicles, too. This result is, in our view, very important for understanding the issues of vesicle biogenesis in Gram-negative bacteria. One of the possible biogenesis mechanisms is the involvement of the secreted product in this process 51,56 . It can be suggested that the antifungal agent of L. capsici affects the biogenesis of the OMV population by means of which it gets into the medium. This is an issue for further studies.
It is especially worth pointing out that the development of the L. capsici vesicular subject area has an evident biomedical trend. Vesicles that contain antimicrobial agents are a valuable model for the development of new-generation medicines on their basis 49,57 .
Thus, the research we did enabled important initial results for understanding the production of antimicrobial agents in L. capsici, for isolating bacteriolytic enzymes, including those not investigated earlier, for producing the recombinant β-lytic protease, for proving the occurrence of a group of outer membrane vesicles responsible for the antifungal activity. On the whole, the antimicrobial potential of L. capsici VKM B-2533 T can be characterized as significant and requiring thorough research.

Materials and methods
Strains and cultivation conditions. The work used strain L. capsici VKM В-2533 Т .
For cultivation of L. capsici, we used liquid nutrient media of the following composition: KSP (casitone, 2.5 g/L; sucrose, 2.5 g/L; peptone, 2. www.nature.com/scientificreports www.nature.com/scientificreports/ Spot assay of lytic action. The lytic action of preparations was determined by the spot test of live bacteria.
A preparation in the amount of 10 μL was applied onto a grown lawn of bacterial target cells. Dishes were dried for 30 min and incubated at 29 °C overnight. In the case of live mycelial fungi, 35 μL of the preparation each was introduced into wells made in a medium with a lawn of target cells. Dishes were incubated for 48 h at room temperature. The lytic action on test cultures was indicated by the appearance of lysis zones in sample application spots. A strong lytic effect manifested itself as a clearcut visible area of lysis in the application spot. A weak lytic action showed as a weakly transparent lysis area in the application spot. Sterile 10 mM Tris-HCl, pH 8.0, was used as a control.
isolation of bacteriolytic enzymes. The culture of L. capsici cells was grown for 20 h with aeration. Cells were discarded by centrifugation at 5000×g and 4°С for 20 min. Culture liquid proteins were fractionated by ammonium sulphate stepwise at 0-60% and 60-80% saturation. Residues of the fractions were produced by centrifugation at 25 960×g and 4°С for 60 min. The residue of the fraction at 60-80% saturation was dissolved in 50 mM Tris-HCl, pH 8.0, and dialyzed using a dialysis tubing of 10 kDa MW cut-off against the same buffer at 4°С overnight.
The dialysis was followed by cation exchange chromatography on a Toyopearl CM-650М column (Tosoh, Japan) equilibrated with 50 mM Tris-HCl, pH 8.0. After washing the column, isocratic elution with 0.3 M NaCl in the same buffer was done. Lytically active elution fractions were combined and dialyzed against 50 mM Tris-HCl, pH 8.0. As the wash-out had a lytic activity, it was also dialyzed against the same buffer. The subsequent stages of purifying elution and wash-out enzymes were the same. After the dialysis, cation exchange chromatography on an ENrich S column (Bio-Rad, USA) equilibrated with the same buffer was conducted again, using an NGC chromatographic system (Bio-Rad, USA). After washing the column, elution in a NaCl linear gradient from 0 to 1 M was done. Lytically active fractions were combined and subjected to gel filtration using the same chromatographic system and a HiLoad 16/60 (Superdex 75) column (Amersham Biosciences, Sweden) equilibrated with 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0.

MALDI-TOF mass spectrometry.
Protein bands were excised from acrylamide gels after electrophoresis under denaturing conditions (SDS-PAGE), washed in 50% (v/v) acetonitrile, 50% (v/v) 50-mM ammonium bicarbonate at 37 °C and dehydrated in acetonitrile. After that, a 10-μl trypsin solution (20 μg/mL) in 50-mM bicarbonate buffer was added (sequencing grade, Promega USA), and the mixture was incubated for 16 h at 37 °C. The hydrolysate in the amount of 0.5 μL was mixed on a mass spectrometer (Bruker, Germany) target with an equal volume of a solution of 2,5-dihydroxybenzoic acid in 50% acetonitrile and 3% TFA, and air-dried.
The mass spectra of trypsin-digested proteins were acquired using a MALDI-TOF/TOF mass spectrometer (Ultraflex, Bruker Daltonics, Germany) equipped with an Nd:YAG laser in the reflector mode. Monoisotopic [MH + ] ions were measured in the m/z range of 700-3500 with a tolerance of 50 ppm. Fragment ion spectra were obtained in the LIFT mode. The accuracy of fragment ion mass peak measurements was within 1 Da 61 .
The proteins were identified using the MASCOT search software (peptide fingerprinting combined with the ion search option; www.matrixscience.com 62 . The search was carried out using the NCBI.nr database with no taxonomy limits. Candidate proteins were considered as reliably identified when the score was greater than 83 (p < 0.05). For the search of candidate proteins in combined ms + (ms-ms) data, Biotools 3.0 (Bruker Daltonics) was used 61 . Homologues of isolated proteins were searched for using the BLAST databases (https://blast.ncbi. nlm.nih.gov) 63 .
Bacteriolytic activity assay. The bacteriolytic activities of the preparations were determined by turbidimetry. As substrates, autoclaved or live S. aureus 209 P cells were used. Cells of the staphylococcus were suspended in 10 mM Tris-HCl, pH 8.0, to an optical density of OD 540 = 0.5. An enzyme preparation in the amount of 50 μL was added to a 0.950-mL substrate, and the mixture was incubated at 37 °C for 5 min. The reaction was arrested by placing test tubes on ice; the absorption of the suspension was measured at 540 nm. An amount of enzyme leading to a decrease in the absorption of the cell suspension by 0.01 optical units at 37 °C per 1 min was taken as a unit of bacteriolytic activity (LU). production of oMVs. The preparation of outer membrane vesicles was produced by differential centrifugation from the culture liquid of L. capsici grown on RM medium. Cells from a 0.3-L culture were discarded by centrifugation at 7 500×g for 20 min at 4 °C. OMVs were precipitated from the produced culture liquid by centrifugation at 113 000×g for 2 h at 4 °C. The OMV residue was washed with 50 mM Tris-HCl, pH 8.0, by centrifugation at the same speed for 1 h. The produced OMV residue was resuspended in equal volumes of 50 mM Tris-HCl, pH 8.0, and stored at -20 °C. fractionation of oMVs in sucrose density gradient. The preparation of L. capsici OMVs was fractionated in a sucrose density gradient according to an earlier developed protocol. The OMV residue was washed with 50 mM Tris-HCl, pH 8.0, followed by centrifugation at the same speed; the residue was then dissolved in 2 mL of 25% sucrose in 10 mM Tris-HCl, 5 mM EDTA, pH 8.0. The OMV preparation thus produced was layered on a stepwise sucrose density gradient (30-55% sucrose in 10 mM Tris-HCl, 5 mM EDTA, pH 8.0) at a step of 5% sucrose. Centrifugation was run at 106 500×g for 12 h. Fractions were collected in equal volumes of 500 μL, www.nature.com/scientificreports www.nature.com/scientificreports/ starting from the meniscus. As the result, 22 OMV fractions were taken. The fractions were diluted to a volume of 30 mL with 10 mM Tris-HCl pH 8.0 and centrifuged at 115 000×g for 2 h. The residues were resuspended in equal volumes of 50 mM Tris-HCl, pH 8.0 51 . cloning and expression of L. capsici VKM B-2533 t β-lytic protease. The β-lytic protease gene (βl) was amplified with the chromosomal DNA isolated using a Genomic DNA Purification Kit (Thermo Scientific, USA) according to the manufacturer's recommendations. To amplify the target sequence (NCBI reference sequence of protein: ALN84242.1), oligonucleotides were designed in OligoAnalizer3.1 (http://eu.idtdna.com/ calc/analyzer) using the bioinformatics data of the L. capsici 55 genome in the GenBank. The signal peptide was searched for in SignalP 4.1 package (http://www.cbs.dtu.dk/services/SignalP/). Oligonucleotides (Table 4) were synthesized by Evrogen (Moscow, Russia). Oligonucleotides containing restriction endonuclease recognition sites were used for cloning into the vector pET19(mod) 64 . The target gene was amplified on a Mastercycler ProS (Eppendorf, Germany). The protocol for the PCR with the DNA-dependent DNA polymerase Q5 (New England Biolabs, USA) was as follows: primary heating, 98°С for 30 s; 35 cycles: 98°С for 10 s, 60°С for 20 s, 72°С for 45 s; additional elongation for 2 min in the last cycle.

Minimal inhibitory concentration assay.
The restriction treatment and DNA ligation were carried out according to the standard techniques described in ref. 65 . The restriction treatment was done using FastDigest NdeI and FastDigest BamHI (Thermo Fisher Scientific, USA). The T4 DNA Ligase (Thermo Fisher Scientific, USA) was used for ligation.
The recombinant plasmid pET19(mod)-Bl was transformed into chemically competent E. coli BL21(DE3)/ pLysE cells produced according to the protocol in ref. 66 . The cultures were grown on an antibiotics-containing LB medium to OD 540 = 0.7, were induced with 1 mM IPTG, then the cultivation was continued at 37 °C for 4 h.
To establish the complete nucleotide sequence of the gene βl, this gene with the genomic DNA flanking sequence (the N-end of the βl gene product was flanking) was cloned into the plasmid pET19(mod). The oligonucleotides were designed ( Table 4).
The sequences of all cloned DNA fragments were confirmed by sequencing at Evrogen (Moscow). Development of renaturation conditions for β-lytic protease. The following buffers were chosen for renaturation: buffer 1-50 mM Tris-HCl, 0.5 M arginine, 0.05% Chaps, pH 8.5; buffer 2-50 mM Tris-HCl, 0.5 М arginine, 5 mM cysteine, 1 мМ cystine, 0.05% Chaps, pH 8.5; buffer 3-50 mM Tris-HCl, 0.5 М arginine, 0.25 М NaCl, 0.40 М sucrose, 5 mM 2-mercaptoethanol, 0.05% Chaps, pH 8.5; buffer 4-0.8 М Tris-HCl, 5 mM cysteine, 1 мМ cystine, 0.05% Chaps, pH 8.5; buffer 5-0.8 М tricine, 5 mM GSH, 1 mM GSSG, 0.05% Chaps, pH 8.5. The enzyme preparation was dialyzed against the chosen buffers for 44 h without mixing at 7 °C. After that, aggregated proteins were discarded by centrifugation at 10 000×g for 20 min. Then the dialysis was repeated against 50 mM Tris, pH 8.0. Newly formed aggregated protein molecules were discarded by centrifugation under the same conditions. The produced samples were incubated at room temperature for 44 h. The efficiency of renaturation was assessed by electrophoresis under denaturing conditions and by measurement of the bacteriolytic activity with respect to autoclaved and live cells of S. aureus 209 P. Purification of the β-lytic protease recombinant protein to an electrophoretically homogeneous state was done using an ENrich S column (Bio-Rad, USA) and an NGC chromatographic system (Bio-Rad, USA). electrophoresis of proteins in polyacrylamide gel. For the electrophoretic assay, proteins from the analyzed preparations were sedimented with trichloroacetic acid at a final concentration of 10% or were used without sedimentation in the case of their subsequent staining with zinc 67 . Protein residues were analyzed by