Hybrid Histidine Kinase BinK Represses Vibrio fischeri Biofilm Signaling at Multiple Developmental Stages

Abstract

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light-organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually-beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCE Bacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization

“…The accommodation phenotype is unlikely to be due to flagellar motility, as most bacteria lose their flagella during the mature association ( 8 ). Therefore, these effects may be due to regulation of biofilm genes in the host ( 7 ) or may be due to other unrecognized effects in the High cdG strain.…”

“… Vibrio fischeri is the exclusive, beneficial light organ symbiont of the Hawaiian bobtail squid ( Euprymna scolopes ). V. fischeri undergoes the motility-to-biofilm lifestyle transition during the initiation of host colonization ( 4 – 7 ). While V. fischeri is solitary and expresses polar flagella in seawater, upon encountering the juvenile bobtail squid light organ the bacteria require the symbiosis polysaccharide (Syp) to aggregate in the host mucus prior to commencing further colonization ( 5 , 8 – 11 ).…”

High levels of cyclic diguanylate interfere with beneficial bacterial colonization

“…Therefore, these effects may be due to regulation of biofilm genes in the host (7) or may be due to other unrecognized effects in the High cdG strain.…”

“…Vibrio fischeri is the exclusive, beneficial light organ symbiont of the Hawaiian bobtail squid (Euprymna scolopes). V. fischeri undergoes the motility-to-biofilm lifestyle transition during the initiation of host colonization (Ludvik et al, 2021;Nyholm et al, 2000;Visick, 2009;Yip et al, 2006). While V. fischeri is solitary and expresses polar flagella in seawater, upon encountering the juvenile bobtail squid light organ the bacteria require the symbiosis polysaccharide (Syp) to aggregate in the host mucus prior to commencing further colonization (Koehler et al, 2019;Rotman et al, 2019;Ruby and Asato, 1993;Yip et al, 2006Yip et al, , 2005.…”

Two enhancer binding proteins activate σ54-dependent transcription of a quorum regulatory RNA in a bacterial symbiont

“…Enhanced crypt colonization by the binK mutant is independent of Qrr1 Qrr1 and BinK are significant factors in the life cycle of V. fischeri because they each impact how V. fischeri cells initially establish symbiosis with E. scolopes. BinK inhibits the aggregation that occurs among environmental V. fischeri cells collected by the light organ, such that cells lacking BinK form large aggregates prior to light organ entry (21,23). In addition, animals exposed to an inoculum mixed evenly with a ΔbinK mutant and its wild-type parental strain result in more ΔbinK cells than wild-type cells within their light organs (21), which suggests that BinK inhibits the ability of a cell to establish symbiosis in the context of other colonizing bacteria.…”