RESUMO
This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.
RESUMO
The aim of the study was to search for, isolate and characterize new bacteriolytic enzymes that show promising potential for their use in medicine, agriculture and veterinary. Using a transcriptomic analysis, we annotated in Lysobacter capsici VKM B-2533T the genes of known bacteriolytic and antifungal enzymes, as well as of antibiotics, whose expression levels increased when cultivated on media conducive to the production of antimicrobial agents. The genes of the secreted putative bacteriolytic proteases were also annotated. Two new bacteriolytic proteases, Serp and Serp3, were isolated and characterized. The maximum bacteriolytic activities of Serp and Serp3 were exhibited at low ionic strength of 10 mM Tris-HCl, and high temperatures of, respectively, 80 °C and 70 °C. The pH optimum for Serp was 8.0; for Serp3, it was slightly acidic, at 6.0. Both enzymes hydrolyzed autoclaved cells of Micrococcus luteus Ac-2230T, Proteus vulgaris H-19, Pseudomonas aeruginosa and Staphylococcus aureus 209P. Serp also digested cells of Bacillus cereus 217. Both enzymes hydrolyzed casein and azofibrin. The newly discovered enzymes are promising for developing proteolytic antimicrobial drugs on their basis.
Assuntos
Anti-Infecciosos , Peptídeo Hidrolases , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Transcriptoma , Endopeptidases/metabolismo , Staphylococcus aureus/metabolismoRESUMO
The crystal structure of the Lysobacter capsici VKM B-2533T ß-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.
Assuntos
Endopeptidases , Peptídeo Hidrolases , Peptídeo Hidrolases/metabolismo , AminoácidosRESUMO
Lysobacter capsici VKM B-2533T and Lysobacter gummosus 10.1.1 are promising strains for use in biomedicine as sources of new antimicrobial agents. Here, we report the whole-genome sequences of both strains (total lengths, 6,239,188 bp and 6,056,609 bp, respectively), obtained using the Illumina and Nanopore platforms.
RESUMO
A successful homologous expression system based on Lysobacter capsici VKM B-2533T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, ß-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici PGroEL(A)-blp and L. capsici PT5-blp, the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici PT5-blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533T. The expression of the serp gene in L. capsici PT5-serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains.
Assuntos
Anti-Infecciosos , Lysobacter , Bacteriólise , Lysobacter/genética , Serina Proteases/genéticaRESUMO
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria constitute important factors in defining interactions with the extracellular milieu. Lysobacter sp. XL1 produces OMVs capable of lysing microbial cells due to the presence in their cargo of bacteriolytic protease L5 (AlpB). Although protein L5 has been functionally and biochemically characterized (including aspects of its packing into OMVs), its role in vesicle biogenesis through genetic deletion of alpB had not been studied previously. Here, we have successfully deleted alpB by allelic replacement and show that the alpB deletion mutant produces a significantly lower amount of OMVs that lack bacteriolytic activity and display altered ultrastructural characteristics in relation to the OMVs produced by the wild-type strain. These results confirm that, as previously proposed, protein L5 participates in OMV production through a mechanism that is not yet fully understood.
RESUMO
Lysobacter capsici VKM B-2533T is a promising strain for isolation of new lytic agents. Here, we report a draft genome sequence of this strain, consisting of 131 scaffolds with a total length of 6,196,943 bp. The results obtained will aid in the discovery and study of biologically active compounds important for biomedicine.