Molecular Characterization and Functional Analysis of Outer Membrane Vesicles from the Antarctic Bacterium Pseudomonas syringae Suggest a Possible Response to Environmental Conditions

Abstract

Outer membrane vesicles (OMVs) of Gram-negative bacteria form an important aspect of bacterial physiology as they are involved in various functions essential for their survival. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz4W were isolated, and the proteins and lipids they contain were identified. The matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis revealed that phosphatidylethanolamines and phosphatidylglycerols are the main lipid components. The proteins of these vesicles were identified by separating them by one-dimensional gel electrophoresis and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). They are composed of outer membrane and periplasmic proteins according to the subcellular localization predictions by Psortb v.3 and Cello V2.5. The functional annotation and gene ontology of these proteins provided hints for various functions attributed to OMVs and suggested a potential mechanism to respond to the extracellular environmental changes. The OMVs were found to protect the producer organism against the membrane active antibiotics colistin and melittin but not from streptomycin. The 1-N-phenylnapthylamine (NPN)-uptake assay revealed that the OMVs protect the bacterium from membrane active antibiotics by scavenging them and also showed that membrane and protein packing of the OMVs was similar to the parent bacterium. The sequestering depends on the composition and organization of lipids and proteins in the OMVs.

Protective role of E. coli outer membrane vesicles against antibiotics

“…Recent research revealed that the OMVs prepared from E. coli protect them against membrane active antibiotic molecules like colistin, melittin, polymixin B by sequestering them (Manning & Kuehn, 2011;Kulkarni et al, 2014). In the environment, several bacterial populations co-exist.…”

“…The protein content was determined by using Bio-rad Bradford proteins estimation kit and lipid content was measured by using FM4-64 fluorescence (Kulkarni et al, 2014). The number of CFUs in the culture being harvested was determined by dilution plating and colony counting.…”

“…The growth of each bacterial strain was studied in the presence of antibiotic with increasing concentration of OMVs. M a n u s c r i p t 6 Quantitative plate assay was performed as described earlier by incubating the bacteria at 37 0 C (Kulkarni et al 2014). This assay was carried out on LB agar plates to study the effect of OMVs of E. coli on the growth of E. coli, P. aeruginosa and A. radioresistens in the presence of different antibiotics.…”

“…The proteins from the OMVs from E. coli MG1655 were identified as described earlier (Kulkarni et al, 2014). In brief, the proteins from OMVs were separated on SDS-PAGE (12%) ( Fig.…”

“…The presence of antibiotics in the medium and stress caused by host environment resulted in increased vesiculation in some bacterial strains (Dutta et al, 2004;Irazoqui et al, 2010). The OMVs were also found to protect the bacterial cells from antibiotic stress when challenged with lethal concentrations of membrane perturbing peptides (Manning & Kuehn, 2011;Kulkarni et al, 2014). Multiple functions of OMVs such as toxin delivery to host, lyses of other bacteria, interspecie's communication from a Pseudomonas sp have been reported (Tashiro et al, 2012).…”