Swimming motility is a critical virulence factor in pathogenesis for numerous Vibrio species. Vibrio campbellii DS40M4 is a wild isolate that has been recently established as a highly tractable model strain for bacterial genetics studies. We sought to exploit the tractability and relevance of this strain for characterization of flagellar gene regulation in V. campbellii . Using comparative genomics, we identified homologs of V. campbellii flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core regulators of the flagellar hierarchy established in other vibrios: RpoN (σ 54 ), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other motile Vibrio species. However, there are several critical differences in V. campbellii : (i) the σ 54 -dependent regulator FlrA is dispensable for motility, (ii) the flgA , fliEFGHIJ , flrA , and flrBC operons do not require σ 54 for expression, and (iii) FlrA and FlrC co-regulate class II genes. Our model proposes that the V. campbellii flagellar transcriptional hierarchy has three classes of genes, in contrast to the four-class hierarchy in Vibrio cholerae . Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae. Importance Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments and is an important global pathogen of fish and shellfish. Disruption of the flagellar motor significantly decreases host mortality of V. campbellii , suggesting that motility is a key factor in pathogenesis. Using this model organism, we identified >60 genes that encode proteins with predicted structural, mechanical, or regulatory roles in function of the single polar flagellum in V. campbellii . We systematically tested strains containing single deletions of each gene to determine the impact on motility and flagellum production. Our studies have uncovered differences in the regulatory network and function of several genes in V. campbellii as compared to established systems in Vibrio cholerae and Vibrio parahaemolyticus .

Vibrio campbellii BB120 (previously classified as Vibrio harveyi) is a fundamental model strain for studying quorum sensing in vibrios. A phylogenetic evaluation of sequenced Vibrio strains in Genbank revealed that BB120 is closely related to the environmental isolate V. campbellii DS40M4. We exploited DS40M4's competence for exogenous DNA uptake to rapidly generate greater than 30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologues. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: (i) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, (ii) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signalling through the response regulator LuxO and (iii) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs.~400 genes, respectively). Using comparative genomics to expand our understanding of quorumsensing circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both between and within Vibrio species. These strains are also phenotypically distinct: DS40M4 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. These results underscore the need to examine wild isolates for a broader view of bacterial diversity in the marine ecosystem.

We present the complete genome sequence of Vibrio campbellii DS40M4, assembled from Illumina and Oxford Nanopore data. This effort improves upon a previous draft assembly to resolve this organism’s two-chromosome and one-plasmid genetic structure and to provide valuable context for evaluating the gene arrangement and evolution of this species.

Vibrio campbellii DS40M4 is a tractable model strain that diverges from the canonical quorum-1 sensing regulatory circuit in vibrios 2 3 4 5 Abstract 33 Vibrio campbellii BB120 (previously designated as Vibrio harveyi) is a fundamental 34 model strain for studying population density-based cell-to-cell communication, known as quorum 35 sensing. In V. campbellii BB120, sensing of autoinducers at high cell densities activates the 36 expression of the master transcriptional regulator, LuxR, which controls the expression of genes 37 involved in group behaviors. The environmental isolate Vibrio campbellii DS40M4 was recently 38 shown to be capable of natural transformation, a process by which bacteria take up exogenous 39 DNA and incorporate it into their genome via homologous recombination. In contrast, BB120 is 40 not naturally transformable. Here, we compare additional phenotypes between these two V. 41 campbellii strains. DS40M4 has a faster growth rate and stronger type VI secretion-mediated 42 cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. To explore the 43 function of DS40M4-encoded homologs of the BB120 quorum-sensing system, we exploited the 44 power of natural transformation to rapidly generate >30 mutant strains. Our results show that 45 DS40M4 has a similar quorum-sensing circuit to BB120 but with three distinct differences: 1) 46DS40M4 lacks the canonical HAI-1 autoinducer LuxM synthase but has an active LuxN 47 receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer 48 signaling through the response regulator LuxO, and 3) the DS40M4 LuxR regulon is <100 49 genes, which is relatively small compared to the >400 genes regulated in BB120. This work 50 illustrates that DS40M4 is a tractable and relevant model strain for studying quorum-sensing 51 phenotypes in Vibrio campbellii. 52 53 Importance 54 Wild isolates of bacterial type strains can yield important information about traits that 55 vary within species. Here, we compare the recently sequenced isolate of Vibrio campbellii 56 DS40M4 to the canonical lab type strain BB120 and examine several phenotypes that define 57 this species, including quorum sensing, bioluminescence, and biofilm formation. Importantly, 58DS40M4 is naturally transformable with exogenous DNA, which allows for the rapid generation 59 of mutants in a laboratory setting. By exploiting natural transformation, we genetically dissected 60 the functions of BB120 quorum-sensing system homologs in the DS40M4 strain, including two-61 component signaling systems, transcriptional regulators, and small RNAs. Our results show 62 important distinctions between the quorum-sensing circuits of these two strains that underscore 63 the need to examine wild isolates alongside type strains. 64 65 66 3 Introduction 67 Quorum sensing is a form of cell-cell communication between bacteria in which 68 individual cells synthesize and respond to signaling molecules called autoinducers. High 69 concentrations of autoinducers trigger changes in gene...

Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments, and it is a pathogen of fish and shellfish. Swimming motility via a single polar flagellum is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. To examine V. campbellii flagellar gene regulation, we identified homologs of flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other motile Vibrio species but with two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.