Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes. We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism. IMPORTANCE Beneficial bacteria communicate with their hosts through a variety of means. These communications are often carried out by a combination of molecules that stimulate responses from the host and are necessary for development of the relationship between these organisms. Naturally produced bacterial outer membrane vesicles (OMVs) contain many of those molecules and can stimulate a wide range of responses from recipient organisms. Here, we describe how a marine bacterium, Vibrio fischeri, changes the makeup of its OMVs under conditions that it experiences as it goes from its free-living lifestyle to associating with its natural host, the Hawaiian bobtail squid. This work improves our understanding of how bacteria change their signaling profile as they begin to associate with their beneficial partner animals.

Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. Shewanella oneidensis strain MR-1 is a versatile, facultatively anaerobic bacterium that lives in aquatic environments and is capable of respiring numerous organic and inorganic compounds in the absence of oxygen. The respiratory diversity of S. oneidensis has widespread effects on biogeochemical cycling (1) and has therefore been a focus for applications in biotechnology and bioremediation (2). Terminal electron acceptors that S. oneidensis can use, aside from oxygen, include dimethyl sulfoxide (DMSO), trimethylamine N-oxide, fumarate, nitrate, and sulfite (3-6), as well as oxidized metals, such as iron and manganese (hydr)oxides (3, 7), which are abundant in the types of sediments (8) in which Shewanella spp. are often found (1). The molecular mechanisms that allow dissimilatory metal-reducing bacteria to survive under iron-rich conditions, however, are not fully understood.Respiration of ferric iron (Fe 3ϩ ) results in the production of ferrous iron (Fe 2ϩ ), which can remain as aqueous Fe 2ϩ ions or become incorporated into solid-phase minerals (9, 10), depending on the environmental conditions. As iron respiration by S. oneidensis continues, the local concentration of aqueous Fe 2ϩ may increase, and Fe 2ϩ ions can be taken up by cells through transition metal ion uptake systems, primarily the iron transport complex FeoAB (11). At higher concentrations, however, Fe 2ϩ is toxic to cells. Aerobically, Fe 2ϩ toxicity is thought to be caused by oxidative damage from hydroxyl radicals produced through the Fenton reaction (12), but the cause of damage under anaerobic conditions is not well understood. Several possible causes of anaerobic Fe 2ϩ toxicity have been proposed, such as the production of reactive nitrogen species (13) or inhibition of the F o F 1 ATPase (14). Regardless of the basis for toxicity, microorganisms have evolved means of minimizing the cellular damage caused by high concentrations of Fe 2ϩ and other metal ions. One of the well-characterized mechanisms that microorganisms use to prevent metal toxicity is efflux via membrane transporters. Metal efflux proteins are widespread in all three domains of life and comprise multiple protein families and superfamilies.For example, the major facilitator family includes the tetracyclinemetal ion transporter TetL in Bacillus subtilis (15) and the iron citrate exporter IceT in Salmonella enterica serovar Typhimurium (16). P-type ATPases, which couple the uptake or efflux of cations to ATP hydrolysis, include the cadmium exporter CadA in Staphylococcus aureus and B. subtilis (17,18) and the copper transporter CopA in Escherichia coli (19). To date, however, no proteins mediating Fe 2ϩ resistance have been described in S. oneidensis. A transposon scre...