Deletion of alpB Gene Influences Outer Membrane Vesicles Biogenesis of Lysobacter sp. XL1

Abstract

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.

The First Homologous Expression System for the β-Lytic Protease of Lysobacter capsici VKM B-2533T, a Promising Antimicrobial Agent

“…After a successful gene knockout of the Lysobacter sp. XL 1 bacteriolytic enzyme L5 [ 27 ], we commenced the creation of a homologous expression system for L. capsici VKM B-2533 T Blp.…”

“…Highly competent E. coli XL1-Blue cells were transformed using a ligation mixture using the RbCl method [ 41 ]. The electroporation of L. capsici VKM B-2533 T using constructed plasmids ( Table 3 ) was conducted in accordance with the method of Lin, with a modification [ 27 ]. Briefly, a 20 µL volume of competent L. capsici cells (in 10% ice-cold glycerol) was mixed with 500 ng of constructed plasmids and transferred to 0.2 cm electroporation cuvettes (Bio-Rad, USA).…”

Lysobacter gummosus 10.1.1, a Producer of Antimicrobial Agents

“…It is known that Lysobacter bacteria are capable of forming outer membrane vesicles that may contain antimicrobial agents [6,12,13]. For L. gummosus species, the ability to form vesicles has not been previously shown.…”

“…Cells of L. capsici and L. enzymogenes are capable of forming outer membrane vesicles (OMVs) that contain antibiotics and/or bacteriolytic enzymes. This significantly expands the antimicrobial potential of these species [4,12,13].…”

Whole-Genome Sequencing of Lysobacter capsici VKM B-2533 T and Lysobacter gummosus 10.1.1, Promising Producers of Lytic Agents

“…L. gummosus 10.1.1 was supplied by Joeke Postma (Wageningen University and Research, Netherlands). Both strains were cultivated in a modified LB medium ( 13 ) at 29°C with aeration for 18 h. DNA was extracted using the Wizard genomic DNA purification kit (catalog number A1125) according to the manufacturer’s instructions.…”