Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.

How bacteria colonise surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multi-purpose class of cell surface polymers. Here we directly visualise the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth upon its natural habitat - chitinous surfaces. As predicted, these pili are highly dynamic and retract prior to DNA-uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA, present in environmental isolates, creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces, are required for chitin colonisation under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonisation. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the beststudied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

24 ORIGINALITY-SIGNIFICANCE STATEMENT 25A recent study highlighted the role of a herpes virus as primary etiological agent of Pacific oyster 26 mortality syndrome (POMS), which affects juveniles of the oyster Crassostrea gigas. We show 27 here that the selection of virulent bacteria in oyster farms is also an important piece of the POMS 28 puzzle. This bacteria taxonomically assigned to Vibrio crassostreae species, carries a plasmid 29 that encodes a Type 6 Secretion System (T6SS), which increases its ability to kill the major 30 cellular players of oyster immunity, the hemocytes. This T6SS was identified in two additional 31 species that infect mollusks, suggesting a parallel evolution of these pathogens. Finally, our 32 results indicate that broad range of pathogens kill their hosts via hemocyte cytotoxicity. 33 34 ABSTRACT 35Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the 36 sustainability of commercial and natural stocks of this species. Vibrio crassostreae has been 37 repeatedly isolated from diseased animals and the majority of the strains have been demonstrated 38 to be virulent for oysters. In this study we showed that oyster farms exhibited a high prevalence 39 of a virulence plasmid carried by V. crassostreae while oysters, at an adult stage, were reservoirs 40 of this virulent population. The pathogenicity of V. crassostreae depends on a novel 41 transcriptional regulator, which activates the bidirectional promoter of a Type 6 Secretion System 42 (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are 43 necessary for virulence but act independently to cause to hemocyte (oyster immune cell) 44 cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. 45 tapetis, which infect adult oysters and clams, respectively. We propose that hemocyte 46 cytotoxicity, is a lethality trait shared by a broad range of mollusk pathogens and we speculate 47 107 108 RESULTS 110The virulence plasmid is widespread in V. crassostreae population occurring in oyster farms 111We previously hypothesized that the introgression of the virulence plasmid pGV into 112 V. crassostreae might have been favored by elevated host density in farming areas (Bruto et al., 113 2017). However, wild oyster beds can also reach high densities, as exemplified by the recent 114 invasion of C. gigas oysters into the Wadden sea (North Sea) (Reise et al., 2017). To date, no 115Vibrio-associated mass mortalities have been observed in this area, in contrast to observations in 116 heavily farmed areas. We thus investigated the presence and frequency of the pGV plasmid in 117 V. crassostreae strains sampled from Sylt. For this, 910 Vibrio strains were isolated from 118 seawater fractions and oysters from Sylt, genotyped by partial hsp60 gene sequencing and 119 assigned to Vibrio populations as described previously ( Figure S1). Multi Locus Sequencing 120Typing (MLST) further confirmed the taxonomic assignment of 47 V. crassostreae str...

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host’s osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae’s intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

Summary Bacteria of the genus Vibrio are common members of aquatic environments where they compete with other prokaryotes and defend themselves against grazing predators. A macromolecular protein complex called the type VI secretion system (T6SS) is used for both purposes. Previous research showed that the sole T6SS of the human pathogen V. cholerae is induced by extracellular (chitin) or intracellular (low c‐di‐GMP levels) cues and that these cues lead to distinctive signalling pathways for which the proteins TfoX and TfoY serve as master regulators. In this study, we tested whether the TfoX‐ and TfoY‐mediated regulation of T6SS, concomitantly with natural competence or motility, was conserved in non‐cholera Vibrio species, and if so, how these regulators affected the production of individual T6SSs in double‐armed vibrios. We show that, alongside representative competence genes, TfoX regulates at least one T6SS in all tested Vibrio species. TfoY, on the other hand, fostered motility in all vibrios but had a more versatile T6SS response in that it did not foster T6SS‐mediated killing in all tested vibrios. Collectively, our data provide evidence that the TfoX‐ and TfoY‐mediated signalling pathways are mostly conserved in diverse Vibrio species and important for signal‐specific T6SS induction.