The quorum-sensing systems of Vibrio campbellii DS40M4 and BB120 are genetically and functionally distinct

Abstract

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 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.

Figure 1:. Model for quorum-sensing regulation in V. campbellii strains BB120 and DS40M4.

The quorum-sensing models in V. campbellii BB120 (A) and DS40M4 (B). Darker colored circuits represent pathways that have been tested in each strain. Lighter colored circuits include putative homologs to other Vibrio systems that have not yet been tested in these strains. (A) In the established BB120 pathway, the autoinducers CAI-1, HAI-1, AI-2, are produced by autoinducer synthases CqsA, LuxM, and LuxS and sensed by receptors CqsS, LuxN, and LuxPQ, respectively. At LCD, autoinducers are at low concentrations, resulting in the receptors acting as kinases. The receptors phosphorylate LuxU (phosphorelay protein), which transfers the phosphate to LuxO. Phosphorylated LuxO activates qrr expression through Sigma-54. The Qrrs together with Hfq bind to the aphA and luxR mRNAs, and AphA is expressed and LuxR production is minimal. The combination of AphA and LuxR protein levels leads to LCD behaviors, such as T3SS and biofilm formation. As autoinducers accumulate at HCD, the receptors bind autoinducers and in this state act as phosphatases. De-phosphorylated LuxO does not activate the qrr genes, thus leading to maximal LuxR production and absence of AphA. This ultimately leads to HCD behaviors such as bioluminescence, proteolysis, and type VI secretion (T6SS). In the absence of nitric oxide (NO), HqsK acts as a kinase. In the presence of NO, NO-bound H-NOX inhibits the kinase activity of HqsK contributing to the de-phosphorylation of LuxO. In V. cholerae, the CqsR receptor senses an unknown autoinducer from an unidentified synthase. In the V. cholerae DPO-dependent QS pathway, the autoinducer DPO is produced from threonine catabolism which is dependent on the Tdh enzyme. Extracellular DPO binds to the receptor VqmA. The VqmA-DPO complex activates transcription of the VqmR sRNA, which represses genes required for biofilm formation and toxin production. (B) Our proposed model for quorum-sensing regulation in V. campbellii DS40M4. The CqsA/CqsS and LuxS/LuxPQ receptor pairs are conserved and act as described above. The LuxN receptor is active either in the absence of a ligand or in the presence of an unidentified ligand other than an AHL, but the mechanism by which it functions is undetermined. We propose that there is an additional regulator outside of the LuxO pathway that also converges on the Qrrs. At HCD, LuxR controls fewer behaviors than in BB120. Image created with BioRender.com.

Figure 2.. Quorum sensing regulation of bioluminescence in BB120 and DS40M4.

(A, B) Bioluminescence production is shown normalized to cell density (OD₆₀₀) during a growth curve for wild-type and mutant strains of BB120 (A) and DS40M4 containing pCS38 (B) (see Table S2 for strain names). For each graph, the data shown are from a single experiment that is representative of at least three independent biological experiments. In panel A, luxR and luxO are complemented on plasmids pKM699 and pBB147, respectively, compared to the pLAFR2 empty vector control. In panel B, luxR and luxO are complemented on the chromosome at the luxB locus.

Figure 3.. V. campbellii DS40M4 produces and responds to CAI-1 and AI-2 autoinducers, but not HAI-1.

(A) Diagram of the luxN loci in BB120 and DS40M4 strains. Shaded areas indicate the percent amino acid identity shared between the strains for each gene. (B) Diagram of the predicted structure of the DS40M4 LuxN monomer. The conserved C-terminal domain is colored purple, and the N-terminal region that is not conserved is colored orange. (C) Bioluminescence production normalized to cell density (OD₆₀₀) for strains in which supernatants from the indicated strains were added to BB120 strains. Different letters indicate significant differences in 2-way analysis of variance (ANOVA) of log-transformed data followed by Tukey’s multiple comparison’s test (n = 4, p < 0.05). Statistical comparisons were performed comparing the effects of the three supernatant conditions for each reporter strain; data were not compared between reporter strains. (D) Superimposed total ion chromatograms for extracts of supernatant from strains BB120 and DS40M4. (E) Superimposed extracted ion chromatograms of 10 μM AHLs. Each color corresponds to a different AHL species. (F) β-galactosidase activity as measured by Modified Miller Units in A. tumefaciens reporter strain KYC55 whole-cell lysate. KYC55 was grown in cell-free supernatant (sup) from Vibrio strains or supplemented with an exogenous AHL (10 μM) as indicated. Data shown represent at least 2 independent biological replicates except BB120 sup with spiked-in HC8 (10 μM) and V. fischeri sup which represent a single replicate each. (G) Bioluminescence production normalized to cell density (OD₆₀₀) for BB120 ΔluxPQM ΔcqsS (TL25) and DS40M4 ΔluxPQ ΔcqsS (cas322) with or without the supplementation of exogenous AHLs (10 μM). The data shown in panels G and H are representative of three independent experiments. (H) Bioluminescence production normalized to cell density (OD₆₀₀) for wild-type DS40M4 (cas291), ΔluxO (BDP065), luxO D47E (BDP062), Δqrr1–5 (cas269), ΔluxPQ ΔcqsS (cas322), and ΔluxPQ ΔcqsS ΔluxN ΔcqsR ΔhqsK (cas399).

Figure 4.. DS40M4 LuxN positively regulates biofilm formation and bioluminescence.

(A) Biofilm formation measured by crystal violet staining (OD₅₉₀) normalized to cell density (OD₆₀₀). Culture tubes grown statically and assayed for biofilm formation by crystal violet staining. (B, C) Biofilm formation measured by crystal violet staining (OD₅₉₀) normalized to cell density (OD₆₀₀). Strains containing complementation plasmids or empty vector were induced with 10 μM IPTG. (D) Bioluminescence production normalized to cell density (OD₆₀₀). For panels B, C, and D, different letters indicate significant differences in 2-way ANOVA of log-transformed data followed by Tukey’s multiple comparison’s test (n = 3 or n = 4, p < 0.05). See Table S2 for strain names for all panels.

Figure 5.. Conservation of LuxM/LuxN in Vibrio strains.

(A) Phylogenetic tree of 287 sequenced Vibrio genomes from Genbank. The assembly number, genus, species, and strain are indicated for each organism. The red shading indicates the amino acid identity of a protein in these strains to either LuxM or LuxN in BB120. The presence of pseudogenes for luxN or luxM, or the presence of multiple LuxN genes is indicated by symbols. (B) Enlarged inset (from the gray region in panel A) of the V. campbellii region of the tree in panel A. The scale of 0.1 indicated in both panels refers to genetic distance between nodes based on nucleotide identity.

Figure 6.. The Qrrs regulate bioluminescence in DS40M4.

(A) Bioluminescence production is shown normalized to cell density (OD₆₀₀) during a growth curve for wild-type BB120, ΔluxR (KM669), qrr1+ (KT234), qrr2+ (KT212), qrr3+ (KT225), qrr4+ (KT215), qrr5+ (KT133), and Δqrr1–5 (KT220). (B) Bioluminescence production is shown normalized to cell density (OD₆₀₀) during a growth curve for wild-type DS40M4, ΔluxR (BP64), qrr1+ (cas0266), qrr2+ (cas0265), qrr3+ (cas0368), qrr4+ (cas0264), qrr5+ (cas0268), and Δqrr1–5 (cas0269). For both panels, a strain expressing only a single qrr is labeled with a ‘+’ sign. For example, qrr4+ expresses only qrr4, and the qrr1, qrr2, qrr3, and qrr5 genes are deleted. The data shown for both panels are from a single experiment that is representative of at least three independent experiments.

Figure 7.. Identification of the LuxR regulon in DS40M4.

(A, B) Data shown are from qRT-PCR analyses of transcripts of the exsB and tssC genes in the DS40M4 wild-type, ΔluxR, or Δqrr1–5 strains. Reactions were normalized to the internal standard (hfq).

Figure 8.. Virulence phenotypes in DS40M4 and BB120 differ in quorum-sensing regulation.

For all panels, assays were performed with V. campbellii DS40M4 wild-type, ΔluxO (cas197), luxO D47E (BDP060), and ΔluxR (cas196). (A) Survival of E. coli in interbacterial killing assays (n = 9). (B) Exoprotease activity measured by HPA digestion and normalized to cell density (OD₆₀₀). (n = 3). (C) Biofilm formation measured by crystal violet staining (OD₅₉₅) normalized to cell density (OD₆₀₀) (n = 6). For all panels, different letters indicate significant differences in 2-way ANOVA of log-transformed data followed by Tukey’s multiple comparison’s test (p < 0.05).