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Bacteria sense population density via the cell–cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell–cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in which Vibrio cells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically “locked” at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild-type, but as glucose is depleted, wild-type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants.

Kelsey Barrasso, Samit Watve, Chelsea A. Simpson Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America Logan J. Geyman Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America ORCID logo https://orcid.org/0000-0001-6697-6725 Julia C. van Kessel * E-mail: jcvk@indiana.edu (JCVK); wai-leung.ng@tufts.edu (WLN) Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America Wai-Leung Ng * E-mail: jcvk@indiana.edu (JCVK); wai-leung.ng@tufts.edu (WLN) Affiliations Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Program in Molecular Microbiology, Graduate School of Biomedical Sciences, Tufts University, Boston, Massachusetts, United States of America ORCID logo https://orcid.org/0000-0002-8966-6604 Introduction Quorum-sensing (QS) systems, which rely on the production and detection of chemical signals called autoinducers (AIs) made by the bacteria themselves, are classically thought to be employed as a means to sense “self,” ensuring that bacteria cooperate and share resources to benefit their kin. [...]most QS receptors are found to be specific for their cognate AIs. Vibrio QS systems that detect host-generated signals Many Vibrio species including Vibrio cholerae, Vibrio harveyi, and Vibrio fischeri spend part of their life cycle inside animal hosts either as a pathogen or as a symbiont. Phosphorylated LuxO promotes and inhibits the production of master transcriptional regulators AphA and HapR, respectively, resulting in the activation of virulence and biofilm gene expression at LCD, which is critical for V. cholerae host colonization [18]. The VqmA/DPO pathway inhibits biofilm formation in V.c. AI-2, autoinducer-2; CAI-1, cholera autoinducer-1; CP, cytoplasm; DPO, 3,5-dimethyl-pyrazin-2-ol; HAI-1, harveyi autoinducer-1; HK, histidine kinase; H-NOX, heme nitric oxide/oxygen binding; NO, nitric oxide; PP, periplasm; QS, quorum sensing.

Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus . In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi ). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae , expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus . To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus. IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus .

SmcR family proteins were discovered in the 1990s as central regulators of quorum-sensing gene expression and later discovered to be conserved in all studied Vibrio species. SmcR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each SmcR-type protein has a predicted ligand-binding pocket. However, no native ligand has been identified for these proteins that control their function as regulators. Here, we used SmcR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo , providing the first demonstration of SmcR function connected to ligand binding for this historical protein family.

In species, quorum sensing signaling culminates in the production of the master transcription factor SmcR that regulates group behavior genes in a density-dependent manner. Previously, we identified a small molecule thiophenesulfonamide inhibitor called PTSP that targets the SmcR family of proteins and blocks activity . Here, we used structure-function analyses to identify eight PTSP-interacting residues in the ligand binding pocket that are required for PTSP inhibition of SmcR. Binding of PTSP to SmcR drives allosteric unfolding of the N-terminal DNA-binding domain and, in this state, SmcR is degraded by the ClpAP protease. SmcR degradation controls the timing of the phenotypic switch between high and low cell density, and strains expressing degradation-resistant alleles are impervious to changes in cell density state. These studies implicate ligand binding as a mediator of SmcR protein stability and function, which dictates the timing of quorum sensing gene expression in three pathogens. SmcR family proteins were discovered in the 1990s as central regulators of quorum sensing gene expression and later discovered to be conserved in all studied species. SmcR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each SmcR type protein has a predicted ligand binding pocket. However, no ligand has been identified for these proteins that control their function as regulators. Here, we used SmcR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation , the first demonstration of SmcR function connected to ligand binding for this historical protein family.

Bacteria sense population density via the cell-cell communication system called quorum sensing (QS). Some QS-regulated phenotypes ( , secreted enzymes, chelators), are public goods exploitable by cells that stop producing them. We uncovered a phenomenon in which cells optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically 'locked' at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating-mutations in (methylenetetrahydrofolate reductase) and (the master QS transcriptional regulator). Methionine/THF synthesis genes are repressed at low cell density when glucose is plentiful and are de-repressed by LuxR at high cell density as glucose becomes limiting. In mixed cultures, QS mutant strains initially co-exist with wild-type, but as glucose is depleted, wild-type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links private and public goods within the QS regulon, preventing accumulation of QS-defective mutants.

In marine Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome via homologous recombination. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in two Vibrio campbellii strains, DS40M4 and NBRC 15631, enables high frequencies of natural transformation. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4. This result is in contrast to Vibrio cholerae that requires the quorum-sensing regulator HapR to activate the competence regulator QstR. However, similar to V. cholerae, QstR is necessary for transformation in DS40M4. To investigate the difference in transformation frequencies between BB120 and DS40M4, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. BB120 encodes homologs of all known competence genes, but most of these genes were not induced by ectopic expression of TfoX, which likely accounts for the non-functional natural transformation in this strain. Comparison of transformation frequencies among Vibrio species indicates a wide disparity among even closely related strains, with Vibrio vulnificus having the lowest functional transformation frequency. We show that ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus.

Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi. We describe a new method called RAIL (Rapid Arbitrary PCR Insertion Libraries) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of the luxCDABE and betIBA-proXWV operons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method to gfp, mCherry, and lacZ reporters and multiple promoters in V. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.

Vibrio campbellii BB120 (previously designated as Vibrio harveyi) is a fundamental model strain for studying population density-based cell-to-cell communication, known as quorum sensing. In V. campbellii BB120, sensing of autoinducers at high cell densities activates the expression of the master transcriptional regulator, LuxR, which controls the expression of genes involved in group behaviors. The environmental isolate Vibrio campbellii DS40M4 was recently shown to be capable of natural transformation, a process by which bacteria take up exogenous DNA and incorporate it into their genome via homologous recombination. In contrast, BB120 is not naturally transformable. Here, we compare additional phenotypes between these two V. campbellii strains. DS40M4 has a faster growth rate and stronger type VI secretion-mediated cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. To explore the function of DS40M4-encoded homologs of the BB120 quorum-sensing system, we exploited the power of natural transformation to rapidly generate >30 mutant strains. Our results show that DS40M4 has a similar quorum-sensing circuit to BB120 but with three distinct differences: 1) DS40M4 lacks the canonical HAI-1 autoinducer LuxM synthase but has an active LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signaling through the response regulator LuxO, and 3) the DS40M4 LuxR regulon is <100 genes, which is relatively small compared to the >400 genes regulated in BB120. This work illustrates that DS40M4 is a tractable and relevant model strain for studying quorum-sensing phenotypes in Vibrio campbellii.

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 >30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system homologs. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120 in three ways: 1) DS40M4 does not produce an acyl homoserine lactone autoinducer but encodes an active orphan LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signaling through the response regulator LuxO, and 3) the DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs ~400 genes, respectively). Using comparative genomics to expand our understanding of quorum-sensing 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. Wild bacterial isolates yield important information about traits that vary within species. Here, we compare environmental isolate Vibrio campbellii DS40M4 to its close relative, the model strain BB120 that has been a fundamental strain for studying quorum sensing for >30 years. We examine several phenotypes that define this species, including quorum sensing, bioluminescence, and biofilm formation. Importantly, DS40M4 is naturally transformable with exogenous DNA, which allows for the rapid generation of mutants in a laboratory setting. By exploiting natural transformation, we genetically dissected the functions of BB120 quorum-sensing system homologs in the DS40M4 strain, including two-component signaling systems, transcriptional regulators, and small RNAs.