Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13

Kamasaka, Kouhei; Kawamoto, Jun; Chen, Chen; Yokoyama, Fumiaki; Imai, Tomoya; Ogawa, Teiichiro; Kurihara, Tatsuo

doi:10.1016/j.bbrc.2020.03.125

Cited by 14 publications

(12 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To further characterize EMVs and obtain insights into the mechanism of their biogenesis, proteins in EMVs of the gspD2 -disrupted mutant (Δ gspD2 ) were identified by gel-based proteomics. For this experiment, Δ gspD2 , which lacks a putative outer membrane conduit for transport of P49 to EMVs, was used as the disruption of gspD2 does not impair the production of EMVs (Chen et al, 2020; Kamasaka et al, 2020) and facilitates the identification of minor cargo proteins of EMVs owing to the absence of the major cargo protein P49. Indeed, in EMVs from the parental strain, the amount of P49 is much higher than that of other proteins, and this phenomenon prevents the identification of other proteins by 2D-PAGE owing to the limited protein-loading capacity of the gel.…”

Section: Resultsmentioning

confidence: 99%

“…This strain, Shewanella vesiculosa HM13, is a Gram-negative and cold-adapted bacterium that was isolated from horse mackerel intestines (Chen et al, 2020). Vesiculation by this bacterium has two unique features: higher vesicle production than other strains such as Escherichia coli and high purity and productivity of a single specific protein in EMVs; these characteristics make S. vesiculosa HM13 useful for elucidating the mechanisms of bacterial vesiculation and protein transport to EMVs (Chen et al, 2020; Guida et al, 2020; Kamasaka et al, 2020; Kawano et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Yokoyama

Imai

Aoki

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production in a hypervesiculating Gram-negative bacterium, Shewanella vesiculosa HM13. This protein was predicted to possess typical sensing and signaling domains of sensor proteins, such as methyl-accepting chemotaxis proteins. Comparison of vesicle production between the hm1275-disrupted mutant and the parent strain revealed that HM1275 is involved in lysine-induced hypervesiculation. Moreover, HM1275 has sequence similarity to a biofilm dispersion protein, BdlA, of Pseudomonas aeruginosa PAO1, and hm1275 disruption increased the amount of biofilm. Thus, this study showed that the induction of vesicle production and suppression of biofilm formation in response to lysine concentration are under the control of the same putative sensor protein.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Yokoyama

Imai

Aoki

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the P49-gene cluster, the gene designated hm3343 encodes a putative inner membrane flippase, Wzx (9). Wzx family proteins are generally responsible for the biosynthesis of bacterial cell surface polysaccharides, i.e.…”

Section: Wzx-dependent Synthesis Of Emv-associated Cpsmentioning

confidence: 99%

Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium,Shewanella vesiculosaHM13

Kamasaka

Kawamoto

Tsudzuki

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Bacterial extracellular membrane vesicles (EMVs) play various physiologically important roles mediated by cargo proteins. However, our understanding of the molecular mechanism underlying cargo loading onto EMVs is limited. In this study, we analyzed the mechanism of cargo protein loading onto EMVs from a fish intestinal Gram-negative bacterium,Shewanella vesiculosaHM13. This strain secretes EMVs carrying a major cargo protein, P49. Near the P49 gene, we found genes having homology to genes involved in protein secretion and surface polysaccharide-chain synthesis. Among them, the deletion of genes encoding homologs of a flippase involved in bacterial extracellular polysaccharide synthesis (HM3343), phosphoethanolamine transferase (HM3344), and glycerophosphodiester phosphodiesterase (HM3345) resulted in the loss of capsular polysaccharide (CPS) of EMVs. We conducted anin vitroP49 loading assay onto P49-free EMVs to examine whether P49 was loaded onto the EMVs via its interaction with the CPS of the EMVs. We found that purified P49 was loaded onto EMVs harboring CPSin vitro, whereas it was not loaded onto EMVs from the mutants lacking CPS production due to the loss of HM3343, HM3344, and HM3345. Transmission electron microscopy of EMVs loaded with P49in vitroandin vivoshowed spherical nanoparticles around the EMVs, whereas such particles were not observed for EMVs without loaded P49, implying that P49 constitutes those particles on the surface of EMVs. These results indicate that P49 is loaded onto EMVs via its interaction with the CPS of EMVs.

show abstract

“…We recently found that disruption of the genes coding for putative surface glycolipid biosynthesis proteins, including LOS phosphoethanolamine transferase, causes dissociation of P49 from EMVs as well as from the cells [11]. The results suggest that surface glycolipids modified with a phosphoethanolamine group play an essential role in the interaction of P49 with both EMVs and the cells.…”

Section: Possible Involvement Of Los In Interaction Of P49 With Emvs mentioning

confidence: 99%

“…However, P49 is expected to be useful as a carrier to deliver a foreign protein to EMVs in the recombinant protein production system by using this strain as the host. In the transportation of P49 to EMVs, it is reasonable to assume that components of the outer membrane, such as LPSs, play a role because disruption of the gene putatively involved in the synthesis of surface glycolipids caused dissociation of P49 from EMVs [11]. LPSs are amphiphilic macromolecules generally composed of three regions; a glycolipid portion (lipid A) covalently linked to an oligosaccharide moiety (core) which is, in turn, linked to an O-polysaccharide chain (O-chain) [12,13].…”

Section: Introductionmentioning

confidence: 99%

Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of Shewanella vesiculosa HM13

et al. 2020

Self Cite

View full text Add to dashboard Cite

Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria. Shewanella vesiculosa HM13, a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel, is able to produce a large amount of EMVs. S. vesiculosa HM13 has been found to include a cargo protein, P49, in the EMVs, but the entire mechanism in which P49 is preferentially included in the vesicles has still not been completely deciphered. Given these premises, and since the structural study of the components of the EMVs is crucial for deciphering the P49 transport mechanism, in this study the complete characterization of the lipooligosaccharide (LOS) isolated from the cells and from the EMVs of S. vesiculosa HM13 grown at 18 °C is reported. Both lipid A and core oligosaccharide have been characterized by chemical and spectroscopic methods.

show abstract

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13

Cited by 14 publications

References 19 publications

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium,Shewanella vesiculosaHM13

Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of Shewanella vesiculosa HM13

Contact Info

Product

Resources

About