Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles

Abstract

The Gram‐negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1–L5), into the culture medium. These proteins are able to degrade Gram‐positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram‐positive and Gram‐negative bacteria: Staphylococcus aureus 209‐P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram‐negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram‐positive and Gram‐negative bacteria.

Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

“…Lysobacter sp. strain XL1 releases ϳ20 times as much endopeptidase L1 as L5 into its culture liquid (51). To explain the difference, we analyzed the translation and transcription signals for their ORFs.…”

“…Endopeptidase L5 is secreted via outer membrane vesicles, whereas L1 is not found in them. Secretion via vesicles promotes the delivery of L5 to peptidoglycans of Gramnegative bacteria through the outer membrane, which significantly expands the antimicrobial action spectrum of L5 (51).…”

“…The bacteriolytic enzymes of the lysoamidase preparation are represented by three endopeptidases, L1, L4, and L5, as well as N-acetylmuramoyl-L-alanine amidase L2 and muramidase L3 (42,45,47,51). It is obvious that the occurrence of lytic enzymes specific to different bonds in peptidoglycan determines a broad range of lysoamidase antibacterial activities.…”

“…It is obvious that the occurrence of lytic enzymes specific to different bonds in peptidoglycan determines a broad range of lysoamidase antibacterial activities. From inhibition data, endopeptidases L1, L4, and L5 have been assigned to the serine protease catalytic type (42,51). Endopeptidase L1, a small protein with a molecular mass of about 22 kDa, has a broad substrate specificity and hydrolyzes the amide bonds in proteins, peptides, and microbial cell walls.…”

Meiothermus ruber H328 enhances the production of membrane vesicles for feather degradation

“…The diameter of the vesicles was about 100-400 nm, similar to those reported for other gram-negative bacteria. 4,8) To obtain an approximation for the frequency of vesicle buddings, 1103 cells were examined in ultrathin section samples, and 71 images of possible budding sites were found. In the absence of feathers, on the other hand, vesicle buddings were also observed but less frequently; 21 images of possible budding sites were counted in 1920 cells.…”