Summary The fitness of host‐associated microbes depends on their ability to access nutrients in vivo. Identifying these mechanisms is significant for understanding how microbes have evolved to fill specific ecological niches within a host. Vibrio fischeri is a bioluminescent bacterium that colonizes and proliferates within the light organ of the Hawaiian bobtail squid, which provides an opportunity to study how bacteria grow in vivo. Here, the transcription factor CysB is shown to be necessary for V. fischeri both to grow on several sulfur sources in vitro and to establish symbiosis with juvenile squid. CysB is also found to regulate several genes involved in sulfate assimilation and to contribute to the growth of V. fischeri on cystine, which is the oxidized form of cysteine. A mutant that grows on cystine but not sulfate could establish symbiosis, suggesting that V. fischeri acquires nutrients related to this compound within the host. Finally, CysB‐regulated genes are shown to be differentially expressed among the V. fischeri populations occupying the various colonization sites found within the light organ. Together, these results suggest the biogeography of V. fischeri populations within the squid light organ impacts the physiology of this symbiotic bacterium in vivo through CysB‐dependent gene regulation.

Bacteria that have the capacity to fill the same niche will compete with one another for the space and resources available within an ecosystem. Such competition is heightened among different strains of the same bacterial species. Nevertheless, different strains often inhabit the same host. The molecular mechanisms that impact competition between different strains within the same host are poorly understood. To address this knowledge gap, the type VI secretion system (T6SS), which is a mechanism for bacteria to kill neighboring cells, was examined in the marine bacterium Vibrio fischeri. Different strains of V. fischeri naturally colonize the light organ of the bobtail squid Euprymna scolopes. The genome of FQ-A001, a T6SS-positive strain, features two hcp genes that are predicted to encode identical subunits of the T6SS. Coincubation assays showed that either hcp gene is sufficient for FQ-A001 to kill another strain via the T6SS in vitro. Additionally, induction of hcp expression is sufficient to induce killing activity in an FQ-A001 mutant lacking both hcp genes. Squid colonization assays involving inocula of FQ-A001-derived strains mixed with ES114 revealed that both hcp genes must be deleted for FQ-A001 and ES114 to occupy the same space within the light organ. These experimental results provide insight into the genetic factors necessary for the T6SS of V. fischeri to function in vivo, thereby increasing understanding of the molecular mechanisms that impact strain diversity within a host. IMPORTANCE Different bacterial strains compete to occupy the same niche. The outcome of such competition can be affected by the type VI secretion system (T6SS), an intercellular killing mechanism of bacteria. Here an animal-bacterial symbiosis is used as a platform for study of the genetic factors that promote the T6SS-mediated killing of one strain by another. Identification of the molecular determinants of T6SS function in vivo contributes to the understanding of how different strains interact within a host.

In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, we wished to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, we found Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, we found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gαs, Gαo, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall our data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points.

Sulfur is in cellular components of bacteria and is, therefore, an element necessary for growth. However, mechanisms by which bacteria satisfy their sulfur needs within a host are poorly understood. Vibrio fischeri is a bacterial symbiont that colonizes, grows, and produces bioluminescence within the light organ of the Hawaiian bobtail squid, which provides an experimental platform for investigating sulfur acquisition in vivo. Like other γ‐proteobacteria, V. fischeri fuels sulfur‐dependent anabolic processes with intracellular cysteine. Within the light organ, the abundance of a ΔcysK mutant, which cannot synthesize cysteine through sulfate assimilation, is attenuated, suggesting sulfate import is necessary for V. fischeri to establish symbiosis. Genes encoding sulfate‐import systems of other bacteria that assimilate sulfate were not identified in the V. fischeri genome. A transposon mutagenesis screen implicated YfbS as a sulfate importer. YfbS is necessary for growth on sulfate and in the marine environment. During symbiosis, a ΔyfbS mutant is attenuated and strongly expresses sulfate‐assimilation genes, which is a phenotype associated with sulfur‐starved cells. Together, these results suggest V. fischeri imports sulfate via YfbS within the squid light organ, which provides insight into the molecular mechanisms by which bacteria harvest sulfur in vivo.