SummaryMembrane vesicle (MV) release remains undefined, despite its conservation among replicating Gramnegative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane proteinpeptidoglycan (OM-PG) and OM protein-inner membrane protein (OM-PG-IM) interactions within the envelope structure. Modulation of OM-PG and OM-PG-IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM-PG and OM-PG-IM associations within the envelope structure, thus releasing OM as MVs.

The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism.

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-α, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4+ T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4+ T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

Rhizobwum meliloti strain SU47 produces both high molecular weight (HMW) and low molecular weight (LMW) forms of an acidic exopolysaccharide, succinoglycan.Genetic studies have shown that succinoglycan is required for alfalfa root nodule invasion. We found that LMW succinoglycan, when applied exogenously to alfalfa roots, restored nodule invasion to exoA, exoB, exoF, and exoH mutants. Nodule initiation signals were not involved, since LMW succinoglycan from R. melilot nodDID2D3 and nodA mutants and from luteolin-induced wild-type cultures elicited effects similar to LMW succinoglycan from the uninduced wild-type strain. In contrast, LMW fractions from an exoA mutant, nonsuccinylated LMW succinoglycan, and HMW succinoglycan did not promote invasion, nor did LMW exopolysaccharides from R.leguminosarum bv. trifoiji and Rhizobium sp. strain NGR234. LMW succinoglycan could be separated by anion-exchange chromatography into several distinct subfractions differing in repeating subunit multiplicities (monomer, trimer, and tetramer) and charge. When tested singly, only the most charged, tetrameric form was active. These results show that a specific oligosaccharide form of succinoglycan promotes nodule invasion in alfalfa. The implications for the mode of action of succinoglycan are discussed.Exo-mutants of Rhizobium sp. strain NGR234 (9), Rhizobium leguminosarum bv. trifolii (10), and R. leguminosarum bv. viciae (11) were defective on their respective hosts.Evidence that nodule invasion relies on structurally specific forms of succinoglycan came from the observation that R. meliloti exoH mutants, which produce succinoglycan lacking the normal succinyl substituent (12) and also lacking the LMW fraction (8), also induce empty nodules (12). Furthermore, nodule invasion by Exo-mutants of Rhizobium sp. strain NGR234 or of R. leguminosarum bv. trifolii could be restored by the exogenous addition of EPS from the parental strain of the same species but not by EPS of different structure from the other species (13). We report here that invasion of alfalfa by R. meliloti Exomutants was restored by the exogenous addition of succinoglycan from R. meliloti. A variety of EPSs of different structure all failed to substitute for normal succinoglycan. Furthermore, only a specific subfraction of LMW succinoglycan was active. Preliminary results were presented at the