bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 The quorum-sensing systems of campbellii DS40M4 and BB120 are genetically and

2 functionally distinct

3

4 Chelsea A. Simpson†1, Blake D. Petersen†1, Nicholas W. Haas1, Logan J. Geyman1, Aimee H.

5 Lee1, Ram Podicheti2, Robert Pepin3,4, Laura C. Brown4, Douglas B. Rusch2, Michael P.

6 Manzella1, Kai Papenfort5,6, Julia C. van Kessel*1

7

8

9 1. Department of Biology, Indiana University, Bloomington, IN 47405

10 2. Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405

11 3. Mass Spectrometry Facility, Indiana University, Bloomington, IN 47405

12 4. Department of Chemistry, Indiana University, Bloomington, IN 47405

13 5. Friedrich Schiller University, Institute of Microbiology, 07745 Jena, Germany

14 6. Microverse Cluster, Friedrich Schiller University Jena, 07743 Jena, Germany

15

16 †These authors contributed equally to this work.

17

18 * Corresponding author:

19 Email: [email protected]

20 Telephone: 812-856-2235

21 Fax: 812-856-5710

22

23

24 Running Title: in V. campbellii DS40M4

25

26 Keywords: quorum sensing, Vibrio, , natural transformation, biofilm, protease

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27

28 Originality-Significance Statement

29 Wild bacterial isolates yield important information about traits that vary within species.

30 Here, we compare environmental isolate Vibrio campbellii DS40M4 to its close relative, the

31 model strain BB120 that has been a fundamental strain for studying quorum sensing for >30

32 years. We examine several phenotypes that define this species, including quorum sensing,

33 , and biofilm formation. Importantly, DS40M4 is naturally transformable with

34 exogenous DNA, which allows for the rapid generation of mutants in a laboratory setting. By

35 exploiting natural transformation, we genetically dissected the functions of BB120 quorum-

36 sensing system homologs in the DS40M4 strain, including two-component signaling systems,

37 transcriptional regulators, and small RNAs.

38

39 Summary

40 Vibrio campbellii BB120 (previously classified as ) is a fundamental model

41 strain for studying quorum sensing in . A phylogenetic evaluation of sequenced Vibrio

42 strains in Genbank revealed that BB120 is closely related to the environmental isolate V.

43 campbellii DS40M4. We exploited DS40M4’s competence for exogenous DNA uptake to rapidly

44 generate >30 isogenic strains with deletions of genes encoding BB120 quorum-sensing system

45 homologs. Our results show that the quorum-sensing circuit of DS40M4 is distinct from BB120

46 in three ways: 1) DS40M4 does not produce an acyl homoserine lactone but

47 encodes an active orphan LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not

48 solely regulated by autoinducer signaling through the response regulator LuxO, and 3) the

49 DS40M4 quorum-sensing regulon is much smaller than BB120 (~100 genes vs ~400 genes,

50 respectively). Using comparative genomics to expand our understanding of quorum-sensing

51 circuit diversity, we observe that conservation of LuxM/LuxN proteins differs widely both

52 between and within Vibrio species. These strains are also phenotypically distinct: DS40M4

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53 exhibits stronger interbacterial cell killing, whereas BB120 forms more robust biofilms and is

54 bioluminescent. These results underscore the need to examine wild isolates for a broader view

55 of bacterial diversity in the marine ecosystem.

56

57

58

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59 Introduction

60 Quorum sensing is a form of cell-cell communication between in which

61 individual cells synthesize and respond to signaling molecules called . Increasing

62 concentrations of autoinducers trigger changes in gene expression among bacterial populations,

63 resulting in group behaviors. Through this method of cell-cell signaling, bacteria control

64 expression of genes involved in motility, toxin secretion, metabolism, and more (Rutherford and

65 Bassler, 2012). Quorum sensing has been heavily studied in marine Vibrio species, owing to

66 easily observable behaviors such as bioluminescence and biofilm formation (Bassler et al.,

67 1997). Vibrio campbellii BB120 was one of the first Vibrio species to have its quorum-sensing

68 circuit discovered (Bassler et al., 1993, 1994; Freeman et al., 2000). V. campbellii BB120 was

69 historically called Vibrio harveyi BB120 (also known as strain ATCC BAA-1116) until it was

70 recently reclassified (Lin et al., 2010). Although similar quorum-sensing system architectures

71 have been established in other vibrios, BB120 has remained a model organism for studying

72 quorum sensing and signal transduction among the .

73 In the V. campbellii BB120 quorum-sensing system, previous research has identified

74 three membrane-bound histidine kinase receptors, CqsS, LuxPQ, and LuxN (Fig. 1A) (J. M.

75 Henke and Bassler, 2004; Rutherford and Bassler, 2012; Jung et al., 2016). These receptors

76 bind to autoinducers CAI-1 ((S)-3-hydroxytridecan-4-one), AI-2 ((2S, 4S)-2-methyl-2,3,4-

77 tetrahydroxytetrahydrofuran-borate), and HAI-1 (N-(3-hydroxybutyryl)-homoserine lactone),

78 respectively. These autoinducers are constitutively produced by the autoinducer synthases,

79 CqsA, LuxS, and LuxM, respectively. A cytosolic hybrid histidine kinase, termed HqsK (also

80 called VpsS in ) that interacts with a nitric oxide (NO) sensing protein (H-NOX)

81 has also been identified as part of this pathway (Henares et al., 2012; Jung et al., 2015). When

82 cells are at low cell density (LCD), the local concentration of autoinducers produced by V.

83 campbellii is insufficient to bind to the receptors, which results in the receptors functioning as

84 kinases. In the absence of NO, HqsK also acts as a kinase. In this LCD state, all four receptors

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85 phosphorylate LuxU, a phosphotransfer protein that transfers phosphate to the response

86 regulator LuxO (Fig. 1). Phosphorylated LuxO activates transcription of five small RNAs

87 (sRNAs) termed quorum regulatory RNAs (Qrrs). The Qrrs post-transcriptionally repress

88 production of the transcriptional regulator LuxR while activating expression of the transcriptional

89 regulator AphA. The activation of AphA and repression of LuxR combinatorially regulates ~170

90 genes and produces individual behaviors in V. campbellii, including biofilm formation (Fig. 1)

91 (van Kessel et al., 2013). As V. campbellii cells continue to grow and the local concentration of

92 autoinducers produced by cells increases, the autoinducers bind to their cognate receptors and

93 change their kinase activity to act as phosphatases. Likewise, in the presence of NO, the H-

94 NOX protein binds NO, inhibiting the kinase activity of HqsK. In this high cell density (HCD)

95 state, LuxO is not phosphorylated, and the Qrrs are no longer expressed. The result is high

96 expression of LuxR and no production of AphA. Based on transcriptomic data, LuxR regulates

97 >400 genes to produce group behaviors in V. campbellii, including activation of

98 bioluminescence, type VI secretion (T6SS), and protease production, and repression of biofilm

99 production and type III secretion (T3SS, Fig. 1) (van Kessel et al., 2013; Bagert et al., 2016).

100 Recently, we showed that natural transformation is achievable in two wild isolates of V.

101 campbellii, DS40M4 and NBRC 15631, by ectopically inducing expression of the V. cholerae

102 competence regulator TfoX (Simpson et al., 2019). By exploiting this process, researchers are

103 able to make marked and unmarked gene deletions, point mutations, and insertions using

104 transformation (Meibom et al., 2005; Yamamoto et al., 2011; Sun et al., 2013). Importantly,

105 multiple mutations can be generated simultaneously in V. cholerae through a technique termed

106 MuGENT: multiplex genome editing by natural transformation (Dalia et al., 2017; Dalia, 2018).

107 While V. campbellii strains all contain homologs of the known competence genes, TfoX

108 overexpression does not stimulate expression of these genes in BB120, unlike in the wild

109 isolates DS40M4 and NBRC 15631 (Simpson et al., 2019). This finding suggested that BB120

110 may have lost regulation by TfoX due to domestication in laboratory conditions. We

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111 hypothesized that environmental V. campbellii strains may exhibit differences in other behaviors

112 such as growth and quorum sensing-controlled phenotypes. Using natural transformation, we

113 systematically constructed isogenic mutant strains of DS40M4 with deletions in predicted

114 quorum-sensing components. With these mutants, we assessed the regulatory circuit of

115 DS40M4 compared to BB120. In addition, we tested differences in quorum sensing-controlled

116 phenotypes between DS40M4 and BB120, including biofilm formation, interbacterial cell killing,

117 protease activity, and bioluminescence. Altogether, our data show that while DS40M4 is similar

118 to BB120 in the presence of specific genes, it has a distinct quorum-sensing circuit that

119 produces different downstream behaviors.

120

121 Results

122

123 V. campbellii DS40M4 has an active quorum-sensing system

124 We had previously shown that DS40M4 encodes a LuxR and LuxO homolog that

125 function to regulate bioluminescence similarly to the epistasis observed in BB120 (Simpson et

126 al., 2019). We have also shown that DS40M4 responds to autoinducers AI-2 and CAI-1

127 produced by BB120. However, we had not examined the presence and function of other core

128 quorum-sensing components. DS40M4 is closely related to BB120; for example, chromosome I

129 of both organisms share 97.45% nucleotide identity. Thus, we predicted that much of the

130 quorum sensing circuit would be conserved in DS40M4. As a comparison, most of the BB120

131 quorum sensing circuit architecture is conserved in V. parahaemolyticus, which is more distantly

132 related to BB120 (chromosome I of both organisms share 83.58% nucleotide identity) (Bassler

133 et al., 1997; J. Henke and Bassler, 2004; Gode-Potratz and McCarter, 2011). Thus, we

134 searched the DS40M4 genome for homologs of the proteins and sRNAs previously identified in

135 the V. campbellii BB120 quorum-sensing system. DS40M4 encodes genes with high amino acid

136 or nucleotide identity to all the components except for LuxM, for which there was no homolog in

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137 DS40M4 (Table 1). We also identified homologs of the HqsK/VpsS and H-NOX system from

138 BB120 (Fig. 1, Table 1). There have been additional quorum-sensing system components

139 identified in V. cholerae, including the CqsR receptor, VqmA receptor, and DPO autoinducer

140 synthase Tdh (Fig. 1, Table 2) (Henares et al., 2012; Jung et al., 2015; Papenfort et al., 2017).

141 However, while BB120 encodes homologs to some of these, their function in BB120 has not yet

142 been tested. In order to focus specifically on similarities/differences between DS40M4 and

143 BB120 with regard to verified functional autoinducer signaling pathways, we chose not to assay

144 the function of these additional proteins in this manuscript. We include these as putative

145 pathways for both BB120 and DS40M4 in Figure 1.

146 To determine whether homologs of BB120 quorum-sensing sRNAs and proteins have

147 similar functions in DS40M4, we generated numerous isogenic mutant strains, including ΔcqsA

148 ΔluxS, ΔluxO, ΔluxR, luxO D47E (a LuxO phosphomimic allele (Freeman and Bassler, 1999a)),

149 in addition to every combination of qrr deletion (single or combined) (Table S2). Our goal was to

150 assay quorum sensing gene regulation in these DS40M4 strains by assessing bioluminescence

151 production as a direct output of quorum sensing-driven LuxR production, which is a standard

152 assay in the field (Jung et al., 2015, 2016; Kim et al., 2018). However, we observed that, unlike

153 BB120, DS40M4 produces no bioluminescence at HCD (Fig. S1B). A genome search of the

154 LuxCDABE protein sequences showed that DS40M4 only encodes a homolog of BB120 LuxB,

155 one of the monomers of the bioluminescence protein luciferase (Meighen, 1991). DS40M4 lacks

156 the luxCDAE genes, which encode the second monomer (LuxA) of the luciferase heterodimer,

157 and the enzymes required for the production of tetradecanal, the substrate for luciferase (Fig.

158 S1C) (Meighen, 1991). However, since most Vibrio species lack homologs to the luxCDABE

159 genes and are incapable of producing bioluminescence, the use of a multi-copy plasmid

160 expressing the BB120 luxCDABE genes under their native promoter is a standard method for

161 assaying quorum sensing regulatory networks in heterologous Vibrio species, including Vibrio

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162 cholerae and Vibrio vulnificus (Lenz et al., 2005; Liu et al., 2006; Jung et al., 2015; Kim et al.,

163 2018). We previously showed, using a PluxCDABE-gfp reporter plasmid, that wild-type DS40M4

164 activates the luxCDABE promoter from BB120, and this is dependent on DS40M4 LuxR

165 (Simpson et al., 2019). We have since constructed a plasmid encoding the BB120 luxCDABE

166 operon under control of its endogenous promoter (pCS38) for use in DS40M4, which enables us

167 to monitor bioluminescence of various DS40M4 strains and assess LuxR regulation as a

168 function of upstream quorum-sensing components.

169 In a wild-type DS40M4 strain containing pCS38 expressing luxCDABE, as the cells grow

170 from LCD to HCD, bioluminescence is maximally produced at ~OD600=1.0 (Fig. 2A, 2B). The

171 luxO D47E phosphomimic constitutively expresses Qrr sRNAs throughout the curve to inhibit

172 LuxR production, resulting in very low bioluminescence, which we observe in both BB120 and

173 DS40M4 (Fig. 2A, 2B). Conversely, we observed that a DS40M4 strain lacking all of the qrr1-5

174 genes does not inhibit LuxR production and is constitutively bright, similar to BB120 (Fig. 2A,

175 2B). Deletion of luxR results in no bioluminescence, and complementation of luxR restores light

176 production. Each of these phenotypes matches the analogous BB120 mutant phenotypes as

177 published (Tu and Bassler, 2007). However, the DS40M4 ΔluxO strain has a distinct difference

178 compared to BB120. In BB120, a ΔluxO mutant strain is constitutively bright and is similar to the

179 Δqrr1-5 strain because ΔluxO cannot direct expression of the Qrrs (Fig. 2A). Likewise,

180 complementation of luxO decreases bioluminescence at LCD compared to the ΔluxO strain (Fig.

181 2A). Curiously, in DS40M4, the ΔluxO strain is indistinguishable from wild-type (Fig. 2B),

182 suggesting that in the absence of LuxO, this strain can still regulate bioluminescence production

183 in a density-dependent manner. Complementation of luxO in the DS40M4 ΔluxO strain delayed

184 light production, suggesting that LuxO regulation does still repress bioluminescence but is not

185 required for density-dependent regulation of bioluminescence (Fig. 2B). One possibility to

186 explain this result is that another functional LuxO is present in V. campbellii. Both BB120 and

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187 DS40M4 encode a second homolog with 49% identity to LuxO (VIBHAR_02228 and

188 DSB67_07165, respectively). However, deletion of the DS40M4 luxO homolog in both the wild-

189 type and ΔluxO strains did not alter the curve (Fig. S1D), indicating that this luxO homolog does

190 not regulate bioluminescence. Given the epistatic relationship of the Qrrs to LuxO in control of

191 bioluminescence, we propose that regulation of bioluminescence is occurring via an additional

192 regulatory circuit outside the LuxO quorum-sensing pathway that also converges on the Qrrs.

193

194 DS40M4 produces and responds to CAI-1 and AI-2 but not HAI-1

195 DS40M4 lacks a LuxM homolog (Fig. 3A), which is the synthase for the acyl homoserine

196 lactone (AHL) autoinducer HAI-1 in BB120 (Bassler et al., 1993). However, DS40M4 encodes a

197 LuxN homolog that is 63% identical to BB120 LuxN (Fig. 3A, 3B, Table 1, Fig. S2A). In BB120,

198 LuxN is the HAI-1 receptor that exhibits kinase activity in the absence of HAI-1 ligand and

199 phosphatase activity when HAI-1 is bound. Curiously, there is high sequence conservation (91%

200 amino acid identity) between the BB120 and DS40M4 LuxN proteins in the C-terminal receiver

201 domain (amino acids 466-849), whereas the N-terminal region of BB120 LuxN (amino acids 1-

202 465) that binds the HAI-1 autoinducer shares only 40% identity with DS40M4 LuxN (Fig. 3B,

203 Fig. S2A). We therefore hypothesized that DS40M4 LuxN senses a different molecule than HAI-

204 1, possibly produced by a different synthase. DS40M4 does not encode a LuxI homolog, which

205 is another enzyme capable of producing AHLs (Waters and Bassler, 2005). However, DS40M4

206 encodes close homologs of BB120 CqsS and LuxPQ (98% and 99% amino acid identity,

207 respectively; Table 1), which bind CAI-1 and AI-2, respectively (Schauder et al., 2001; Ng et al.,

208 2011). We investigated the activity of the autoinducers produced by DS40M4 by collecting cell-

209 free supernatant from DS40M4 and adding it to autoinducer-sensing reporter strains in the

210 BB120 background in which a single autoinducer synthase is deleted. For example, a ΔcqsA

211 BB120 strain does not produce CAI-1 and has a lower level of bioluminescence compared to

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212 wild-type (Fig. 3C). Addition of a supernatant containing CAI-1 such as that from wild-type

213 BB120 results in an increase in bioluminescence (Fig. 3C). We observed that addition of

214 DS40M4 supernatant increases bioluminescence in the AI-2 and CAI-1 BB120 sensing strains

215 (ΔluxS and ΔcqsA, respectively), but does not affect bioluminescence in the ΔluxM HAI-1

216 sensing strain (Fig. 3C). From these experiments, we conclude that DS40M4 produces AI-2 and

217 CAI-1 molecules that can be sensed by BB120, but not an HAI-1 molecule.

218 We previously assessed the molecules sensed by DS40M4 receptors CqsS and LuxPQ

219 (Simpson et al., 2019). Our data showed that DS40M4 senses the AI-2 and CAI-1 molecules

220 produced by BB120 but does not respond to BB120 HAI-1, even though DS40M4 encodes a

221 LuxN homolog. From both sets of data regarding autoinducers produced by BB120 and

222 DS40M4, we conclude that DS40M4 does not produce or respond to BB120 HAI-1 under our

223 lab conditions. To investigate whether LuxN responds to a different autoinducer, we first

224 assayed for the presence of AHLs in DS40M4 supernatants. Using liquid chromatography -

225 mass spectrometry (LCMS), we observed a clear signal for HAI-1 in the supernatant of BB120,

226 which aligned as expected with the retention time of the HAI-1 peak from the synthetic control

227 sample (Fig. 3D, 3E; hydroxy-C4-homoserine lactone (HSL); HC4). However, no signal was

228 observed in the supernatant of DS40M4 above background intensity (Fig. 3D, S2B). To further

229 assay for AHLs, we utilized an AHL reporter strain of Agrobacterium tumefaciens (strain

230 KYC55). This reporter strain relies on the overexpression of the innate A. tumefaciens TraR

231 protein that promiscuously detects AHL variants at nanomolar concentrations using β-

232 galactosidase activity as a standard reporter (Zhu et al., 2003; Joelsson and Zhu, 2006).

233 However, TraR does not respond to other classes of autoinducers, and thus β-galactosidase

234 activity should not be induced by AI-2 or CAI-1. We observe significant activity when KYC55 is

235 supplemented with exogenous C8 or hydroxy-C8 HSL (Fig. 3F). Surprisingly, the KYC55

236 reporter strain does not respond to C4 nor HAI-1, either in the supernatant of BB120 or

237 synthetically produced (Fig. 3F). Importantly, no β-galactosidase activity is observed when the

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238 reporter strain is grown in medium supplemented with cell-free supernatants from wildtype

239 DS40M4 relative to the LM medium control (Fig. 3F). In contrast, cell-free supernatant from

240 Vibrio fischeri MJ1, which produces both C8 HSL and oxo-C6 HSL, results in a significant

241 increase in β-galactosidase activity. Further, addition of HC8-HSL to BB120 supernatant prior to

242 filtration also produced β-galactosidase activity, confirming that HSLs from either biological or

243 synthetic sources can be recovered and detected from our supernatant filtration process. These

244 data, along with conclusions drawn from the LCMS experiments, suggest that DS40M4 does not

245 produce a detectable AHL autoinducer.

246 To assess whether LuxN binds to a different AHL ligand other than HAI-1, we

247 constructed mutant strain backgrounds to specifically focus on the kinase/phosphatase activity

248 contributed solely by LuxN. In V. campbellii BB120, deletion of the LuxPQ and CqsS receptors

249 and the LuxM synthase (ΔluxPQ ΔluxM ΔcqsS) is sufficient to abolish quorum sensing

250 throughout all cell densities and shut off bioluminescence (Fig. 3G). Constitutive light production

251 can be induced in this strain when exogenous HAI-1 is supplemented (Fig. 3G). Thus, we made

252 a ΔluxPQ ΔcqsS DS40M4 strain because we hypothesized that, in the absence of these two

253 receptors, if LuxN is not detecting a ligand, this strain could not respond to changes in cell

254 density. Surprisingly, the ΔluxPQ ΔcqsS strain produces bioluminescence in response to

255 increases in cell density much like wild-type throughout the entire curve (Fig. 3G, 3H). To

256 examine if addition of exogenous AHLs could stimulate earlier light production, we added

257 synthetic AHLs to cultures of DS40M4 ΔluxPQ ΔcqsS at LCD and monitored bioluminescence.

258 Neither the addition of HAI-1 nor any of the other AHL derivatives results in earlier light

259 production for the DS40M4 ΔluxPQ ΔcqsS mutant (Fig. 3G). From these data, we conclude that

260 DS40M4 is producing and sensing another molecule outside the LuxSPQ and CqsAS pathways.

261 Further, if LuxN is detecting a molecule, it is not likely an AHL produced by DS40M4.

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262 To determine what pathway(s) were functional for quorum sensing signaling, we used

263 MuGENT to construct combinations of deletions in all genes encoding predicted quorum

264 sensing receptors: cqsS, luxPQ, luxN, cqsR, and hqsK. However, the sextuple mutant strain

265 (ΔcqsS, ΔluxPQ, ΔluxN, ΔcqsR, ΔhqsK) still responds to increases in cell density similar to wild-

266 type (Fig. 3H). These data suggest that another signal-receptor pair are functioning to drive the

267 quorum sensing response in the absence of all predicted pathways.

268

269 DS40M4 LuxN positively regulates bioluminescence and biofilm formation at HCD

270 Our collective data from our previous publication and this study show that DS40M4 does

271 not sense or produce an AHL, but did not address whether the DS40M4 LuxN protein is a

272 functional protein (Simpson et al., 2019). To determine whether DS40M4 LuxN is functional to

273 alter regulation of gene expression downstream, we generated numerous mutants in the

274 DS40M4 background and examined bioluminescence and biofilm formation. First, we examined

275 the biofilm phenotype in various DS40M4 isogenic strains compared to BB120 strains using

276 culture tubes and 96-well plates. We normalized the optical density reading after crystal violet

277 staining (OD590) to the optical density for the culture prior to processing (OD600) because there is

278 a large difference in growth in cultures of BB120 and DS40M4 grown statically or with shaking

279 (Fig. 4A, S1A). In both BB120 and DS40M4, the luxO D47E phosphomimic allele increases

280 biofilms above wild-type levels (Fig. 4A, 4B). Deletion of luxN in BB120 significantly decreases

281 biofilms, whereas we observe that the DS40M4 ΔluxN strain produces the same biofilm levels

282 as wild-type (Fig. 4A, 4B). However, based on the visual inspection of the crystal violet staining,

283 strains BB120 ΔluxN, DS40M4 wild-type, and DS40M4 ΔluxN are nearing the lower limit of

284 detection for this assay, thus it is possible we would not be able to detect a significant decrease

285 in biofilm formation in DS40M4.

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286 To further assess the phenotypic effects of deleting luxN in the DS40M4 strain, we

287 introduced the ΔluxN allele into a ΔcqsA ΔluxS strain. Our logic was that deletion of these two

288 autoinducer synthases would lead to increased kinase activity of CqsS and LuxPQ, which we

289 predicted would result in higher levels of phosphorylated LuxO and increased biofilm formation.

290 Thus, we predicted that the effect of deleting luxN would be more easily observable in this

291 background. In DS40M4, deletion of luxR does not maximize biofilm production to the level of

292 the luxO D47E strain (Fig. 4C), possibly due to production of AphA and/or Qrrs that may still

293 control expression of biofilm genes in a ΔluxR background but would not in the luxO D47E

294 strain. As we predicted, the DS40M4 ΔcqsA ΔluxS mutant is significantly increased in biofilm

295 formation compared to wild-type (Fig. 4C). Deletion of cqsA but not luxS increases biofilm

296 formation; complementation of cqsA restores biofilm levels to that of wild-type DS40M4 (Fig.

297 4C). We observe that deletion of luxN in the wild-type background or the ΔcqsA ΔluxS

298 background does not significantly alter biofilm formation, though there is a modest decrease

299 (Fig. 4C). However, over-expression of luxN in the ΔluxN background significantly increases

300 biofilm formation. This result, together with the observed decrease in biofilm formation in ΔluxN

301 strains, indicates that LuxN is acting to positively regulate biofilm formation. However,

302 complementation of luxN in the ΔcqsA ΔluxS ΔluxN background does not significantly alter

303 biofilm formation (Fig. 4C), possibly because the kinase activities of CqsS and LuxPQ are

304 dominant under the conditions in this assay.

305 Second, we examined the bioluminescence phenotypes of the DS40M4 isogenic

306 deletion strains. Because the Ptac-luxN complementation experiment utilized the same plasmid

307 backbone as the bioluminescence reporter pCS38, we were unable to assay bioluminescence in

308 the complementation strains. As shown previously (Fig. 2B), we see complete loss of

309 bioluminescence in the DS40M4 ΔluxR strain. Deletion of cqsA or luxS alone does not

310 significantly alter bioluminescence, but we note a general decrease in bioluminescence in the

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311 ΔcqsA strain as well as an increase in variability in the assay that we also see in the ΔcqsA

312 ΔluxS strain (Fig. 4D). In BB120, a ΔluxN strain does not alter bioluminescence compared to

313 wild-type at any point in the growth curve, but if LuxN is the only receptor expressed (ΔluxQ

314 ΔcqsS), bioluminescence increases at LCD (Bassler et al., 1993; J. M. Henke and Bassler,

315 2004). Thus, if the DS40M4 LuxN protein functions similarly to BB120 LuxN, we predicted that

316 deletion of luxN would result in decreased bioluminescence due to loss of phosphatase activity.

317 We assayed this in the ΔcqsA ΔluxS ΔluxN background, which is not producing AI-2 or CAI-1,

318 and thus the LuxPQ and CqsS receptors are predicted to be functioning as kinases at HCD.

319 Indeed, deletion of luxN in the ΔcqsA ΔluxS background significantly decreases

320 bioluminescence (Fig. 4D). From these collective data, we conclude that DS40M4 LuxN

321 positively regulates both bioluminescence and biofilm formation. However, these experiments

322 cannot conclusively determine whether LuxN is acting solely as a phosphatase or has kinase

323 activity in other conditions.

324

325 Conservation of LuxM and LuxN is widely different among vibrios

326 The collective results of the supernatant assays and examination of the luxN locus

327 suggested that DS40M4 evolved a different quorum-sensing pathway than BB120. We were

328 therefore curious as to the conservation of LuxMN across the Vibrionaceae. We used

329 comparative genomics to identify the presence or absence of these proteins in other Vibrio

330 strains. First, we used the genomes of 287 fully sequenced Vibrio strains from Genbank to

331 assemble a phylogenetic tree. Strains were grouped based on a shared set of 214 complete

332 and non-duplicated genes identified in each genome (Fig. 5A). The organization of this tree

333 remained the same each time we randomly chose 100 genes from the set of 214 genes to

334 reconstruct the tree, which substantiates this assembly. We observed that the species that

335 comprise the Harveyi clade (V. harveyi, V. campbellii, V. parahaemolyticus, V. natriegens, V.

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336 jasicida, V. rotiferianus, and V. owensii) are grouped similarly to previous taxonomic studies

337 (Urbanczyk et al., 2013). This phylogenetic tree indicates that the most closely related strains to

338 V. campbellii BB120 are DS40M4 and NBRC 15631 (also known as CAIM 519, ATCC 25920)

339 (Fig. 5B).

340 Next, we used a Hidden Markov Model to compare protein sequences from annotated

341 and predicted genes to determine which vibrios encode either LuxM and/or LuxN (Fig. 5A). The

342 analysis revealed that LuxMN are broadly conserved across many different Vibrio species and

343 are most often found as a syntenic pair. Similar to DS40M4, our analysis showed 15 other Vibrio

344 species encode LuxN but lack LuxM (2 V. campbellii, 1 V. parahaemolyticus, 7 V. natriegens, 2

345 V. taketomensis, 1 V. ponticus, 1 V. panuliri, and 2 V. scophthalmi strains). The NBRC 15631

346 strain also does not encode LuxM, similar to DS40M4 (Fig. 5B). In addition, several Vibrio

347 strains contain LuxM or LuxN pseudogenes due to frame-shift mutations or transposon

348 insertions, suggesting that these strains are going through reductive evolution (Fig. 5A).

349 Conversely, some Vibrio strains encode multiple luxN genes. Together, these data show that

350 conservation of the LuxMN system varies widely across Vibrionaceae. Furthermore, DS40M4 is

351 one of only a few Vibrio species that encode a luxN homolog but not luxM.

352

353 The Qrrs in DS40M4 have different activities compared to BB120

354 DS40M4 encodes five Qrrs with high percent nucleotide identity and similar genome

355 position compared to BB120 (Fig. S3). Further, the Sigma-54 and LuxO binding sites identified

356 in BB120 qrr promoters are conserved in DS40M4 qrr promoters, and only the promoter of qrr5

357 diverges from BB120 at its two LuxO binding sites (Fig. S3) (Tu and Bassler, 2007). To examine

358 the regulatory role of the Qrrs, we constructed strains containing different combinations of

359 deletions of the qrr1-5 genes (Table S2). We exploited the power of natural transformation in

360 DS40M4 to construct every combination of qrr gene deletion, with up to four unmarked deletions

361 in a single reaction (Fig. S4). Using MuGENT, we constructed strains expressing only a single

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362 Qrr, e.g., qrr1+, qrr2+, etc. and assessed their bioluminescence phenotype. Surprisingly, these

363 phenotypes also did not match previous data from BB120 (Tu and Bassler, 2007). In BB120

364 strains that contain only a single qrr, the curve exhibits increasing levels of bioluminescence

365 depending on the Qrr gene expressed, such that the curves for each Qrr-expressing strain lie

366 between the wild-type curve and the Δqrr1-5 constitutively bright curve (Fig. 6A). Specifically, in

367 BB120, expression of Qrr2 and Qrr4 alone exhibit the strongest effect on bioluminescence

368 repression, followed by Qrr3, then Qrr1, and finally Qrr5, which has no effect when deleted

369 because it is not expressed under tested conditions (Fig. 6A) (Tu and Bassler, 2007). Thus, our

370 results match those previously published in BB120: Qrr2/Qrr4>Qrr3>Qrr1>Qrr5 with regard to

371 strength of bioluminescence.

372 In contrast, in DS40M4, we observe that deletion of qrrs has a much different effect (Fig.

373 6B). A strain expressing only qrr1, qrr4, or qrr5 produces very low bioluminescence, much less

374 than wild-type and similar to a ΔluxR strain (Fig. 2B, 6B). Importantly, this occurs even at HCD,

375 when the Qrrs are not expected to be expressed based on data in BB120. Expression of only

376 qrr2 or qrr3 modestly decreases bioluminescence compared to wild-type, and these strains

377 appear to respond to the change from LCD to HCD with increasing bioluminescence production

378 over time. (Fig. 6B). These data collectively indicate that expression of all Qrrs is not controlled

379 only by the defined quorum-sensing pathways. However, expression of luxO D47E completely

380 represses bioluminescence (Fig. 2B), suggesting that phosphorylated LuxO is epistatic to other

381 regulatory inputs that activate bioluminescence. Further, deletion of all qrr genes results in

382 constitutive bioluminescence, supporting the conclusion that the Qrrs repress bioluminescence

383 production.

384

385 RNA-seq defines the quorum sensing-regulated genes in DS40M4

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386 To examine the genes regulated by quorum sensing in DS40M4, we used RNA-seq to

387 compare gene expression of wild-type and ΔluxR cultures grown to OD600 = 1.0. We chose this

388 optical density for several reasons: 1) this is the point at which bioluminescence is maximal and

389 the cells have reached quorum for this phenotype in this medium (Fig. 2B), 2) this is the optical

390 density at which RNA was collected and analyzed by RNA-seq in BB120, and 3) this density is

391 near the end of exponential growth phase but not yet in stationary phase where stringent

392 response might occur (Fig. S1A). Our RNA-seq analysis shows that LuxR regulates 90 genes

393 >2-fold (p > 0.05, Table S1), including those involved in metabolism, cyclic di-GMP synthesis,

394 transport, and transcriptional regulation (Table 3). This is a smaller regulon than those observed

395 in other vibrios, including previous RNA-seq data from BB120 in which LuxR regulates >400

396 genes (van Kessel et al., 2013; Chaparian et al., 2016). Certain genes that are regulated by

397 LuxR in other vibrios, such as most T6SS genes or osmotic stress genes (Wang et al., 2013;

398 Shao and Bassler, 2014; Zhang et al., 2017; Hustmyer et al., 2018), are not regulated by LuxR

399 in DS40M4. Further, the fold-change in gene activation and repression by LuxR is smaller. For

400 example, the T3SS operons in DS40M4 are all repressed <2-fold by LuxR, whereas these are

401 repressed >10-fold in BB120.

402 To extend the results of the RNA-seq analysis, we performed qRT-PCR on wild-type,

403 ΔluxR, and Δqrr1-5 DS40M4 strains to examine the transcript levels for T3SS and T6SS, both

404 gene classes previously shown to be regulated by LuxR in BB120 (van Kessel et al., 2013;

405 Bagert et al., 2016; Chaparian et al., 2016). RNA samples were collected from these three

406 strains at LCD (OD600 = 0.1), HCD (OD600 = 1.0), and late stationary phase (OD600 = 3.25), and

407 measured by qRT-PCR. Similar to what is observed in BB120, the T3SS gene exsB is more

408 highly expressed at LCD, repressed by LuxR, and activated by qrr1-5 (Fig. 7A). In the RNA-seq

409 data, only one T6SS gene, tssC, was modulated by LuxR. While qRT-PCR showed that tssC

410 expression is mediated by LuxR at HCD, tssC is much more highly activated at late stationary

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411 phase, and this activation is not mediated by LuxR (Fig. 7B). Together, our qRT-PCR results

412 confirmed our RNA-seq results. We conclude that the LuxR regulon in DS40M4 at HCD is

413 smaller than that of LuxR in BB120, and the fold-change of gene regulation by LuxR is generally

414 reduced in DS40M4.

415

416 Virulence-associated phenotypes are differentially regulated by quorum sensing in DS40M4 and

417 BB120

418 Quorum sensing plays an important role in regulating several behaviors in Vibrio strains

419 that are important for Vibrio survival in various niches (Croxatto et al., 2002; Miyata et al., 2010;

420 Rutherford and Bassler, 2012; Kim et al., 2013). To assess the impact of quorum sensing in

421 DS40M4 and BB120, we compared three phenotypes that have previously been described as

422 controlled by quorum sensing: interbacterial killing, exoprotease activity, and biofilm formation

423 (Waters et al., 2008; Anetzberger et al., 2012; Shao and Bassler, 2014). BB120 exhibited

424 stronger changes in interbacterial killing assays with quorum-sensing mutants (Fig. 8A). The

425 LCD mutant strains, which are the luxO D47E strain and the ΔluxR strain, both showed

426 increased killing of E. coli compared to wild-type. This is unexpected given previous findings in

427 V. campbellii BB120 and V. cholerae which show that LuxR/HapR activate T6SS operons while

428 the Qrrs repress them (Zheng et al., 2010; Shao and Bassler, 2014; Bagert et al., 2016).

429 However, DS40M4 quorum-sensing mutants showed no significant changes in E. coli killing

430 relative to the wild-type, suggesting that quorum sensing does not control this behavior in this

431 strain (Fig. 8A). The interbacterial killing observed is likely due to the expression of T6SS genes,

432 which is not regulated by LuxR or quorum sensing as observed in the RNA-seq and qRT-PCR

433 data. However, it could also include production of antibacterial compounds and enzymes. Thus,

434 we conclude that quorum sensing does not regulate interbacterial killing in DS40M4.

435 When we examined relative exoprotease activity between BB120 and DS40M4 strains,

436 we observed analogous functions of the quorum-sensing proteins between the two strains.

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437 While BB120 exhibits stronger exoprotease activity than DS40M4, activity significantly drops in

438 LCD mutants of both strains, suggesting this behavior is dependent on LuxR in both BB120 and

439 DS40M4 (Figure 8B). Comparing biofilm formation between DS40M4 and BB120 mutants

440 showed that LCD mutants exhibit increased biofilm formation compared to the wild-type parent

441 strain (Figure 8C). Interestingly, however, while BB120 overall has significantly stronger biofilm

442 formation than DS40M4, quorum sensing appears to regulate biofilm formation more strongly in

443 DS40M4 than in BB120. From these experiments, we conclude that quorum sensing controls

444 protease production and biofilm formation in both BB120 and DS40M4, whereas interbacterial

445 killing is only regulated by quorum sensing in BB120.

446

447 Discussion

448

449 V. campbellii BB120 has historically served as a useful model strain for quorum-sensing

450 experiments in Vibrio species. BB120 was isolated as a conjugation-proficient mutant of parent

451 strain BB7 that arose after multiple cycles of conjugation and laboratory passaging (Bonnie

452 Bassler, personal communication). Although BB120 was originally selected as a mutant, the

453 field has referred to BB120 as the “wild type” for ~25 years; therefore, we have kept this

454 nomenclature (Bassler et al., 1997; Freeman and Bassler, 1999a, 1999b; Mok et al., 2003). It is

455 perhaps not surprising that the wild isolate DS40M4 strain isolated by Haygood and colleagues

456 (Haygood et al., 1993) exhibits different physiological and functional characteristics compared to

457 BB120. DS40M4 grows at a faster rate and exhibits increased interbacterial killing of E. coli

458 cells. Interestingly, however, we found that BB120 exhibits much stronger biofilm formation than

459 DS40M4, a trait often lost by laboratory propagation (White and Surette, 2006; Davidson et al.,

460 2008; McLoon et al., 2011), as well as a high levels of exoprotease production (Steensels et al.,

461 2019). In addition, DS40M4 is one of many dark (non-bioluminescent) V. campbellii isolates that

462 contain lesions in the lux locus that abrogate bioluminescence production (O’Grady and

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463 Wimpee, 2008). As an environmental isolate, DS40M4 likely serves as a more relevant model

464 for comparing behaviors of V. campbellii to Vibrio strains in their native marine environment.

465 One such behavior is the interbacterial killing activity of DS40M4. It will be informative to assess

466 the effects of interbacterial killing and growth rate on virulence in host systems in future studies,

467 as well as other virulence-associated characteristics such as T3SS and motility.

468 Although BB120 still remains a core model strain for Vibrio studies, it is one of several

469 Vibrio strains that is not capable of natural transformation, at least not under any conditions we

470 have tested (Simpson et al., 2019). Thus, making mutations in BB120 is laborious and requires

471 construction of suicide plasmids and the use of counter-selection and colony screening. Due to

472 this process, it can take up to three months to make a single genetic alteration. This not only

473 drains time and resources, but disincentivizes genetic-heavy experiments as a tool for

474 evaluating gene functions. Here we have also expanded upon our previous study of natural

475 transformation in DS40M4 (Simpson et al., 2019) to show the power of natural transformation

476 for constructing up to four unmarked mutations simultaneously (Fig. S4). The Qrr genes in the

477 quorum-sensing circuit are only one example of a gene set for which MuGENT is useful in

478 studying combinatorial mutant phenotypes. The ease with which mutants are constructed in

479 DS40M4 allowed us to quickly generate deletion mutants of several core quorum-sensing

480 genes.

481 Our model of the quorum-sensing circuit of DS40M4 is presented in Figure 1B based on

482 the experiments performed in this study. DS40M4 shares the AI-2/CAI-1 pathway of BB120, but

483 it lacks a LuxM (HAI-1 synthase) homolog, and its LuxN (HAI-1 receptor) homolog is only 63%

484 identical. Although the DS40M4 LuxN homolog cannot sense HAI-1 from BB120, our data show

485 that LuxN is functional and activates biofilm formation and bioluminescence. It is yet unclear if

486 LuxN senses a ligand, even if one is produced by V. campbellii DS40M4. In BB120, LuxM

487 synthesizes the AHL autoinducer HAI-1. V. fischeri has a LuxI synthase that produces an AHL

488 autoinducer 3-oxo-C6; however, DS40M4 does not have a clear homolog to this protein either.

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489 Further, a protein blast against the receptor, AinR, of V. fischeri, which senses the autoinducer

490 C8 AHL produced by the AinS synthase, showed 39.71% identity to the DS40M4 LuxN receptor,

491 and no AinS homolog is present in DS40M4. Thus, it is unlikely that 3-oxo-C6 AHL or C8 AHL is

492 the autoinducer to which DS40M4 LuxN responds. We were unable to detect any AHLs in the

493 supernatant of DS40M4 through LCMS or in vivo sensor assays. Thus, we propose that

494 DS40M4 does not produce AHLs and that LuxN activity is independent of AHL sensing.

495 Although our experiments show that LuxN is active and regulates biofilms and bioluminescence,

496 we could not decipher whether LuxN functions predominantly as a phosphatase or kinase using

497 these assays. Further, the observation that BB120 ΔluxN exhibits low biofilm formation

498 contradicts our prediction for this mutant based on what is seen in the literature. LuxN has been

499 shown to be more phosphatase-biased such that when LuxN is the sole receptor expressed

500 (ΔluxQ ΔcqsS), the strain produces more bioluminescence at LCD (J. M. Henke and Bassler,

501 2004). Thus, we predicted that deletion of luxN would yield more biofilm formation because

502 there would be less phosphatase activity in the system, and this could yield more biofilm

503 formation. Although we do not understand this result, it is possible that this type of assay for

504 biofilm formation (end-point assay performed at HCD) is not appropriate for determining the

505 function of receptors as kinases and phosphatases in which the phenotype being measured is

506 controlled at LCD. Rather, microfluidics experiments might be a more valuable and insightful

507 tool for this purpose as has been used for V. cholerae (Bridges and Bassler, 2019).

508 When comparing the synteny of luxMN across different vibrio species, we found luxMN

509 are conserved as a pair in many Vibrio species. DS40M4 is one of a few strains, both across

510 vibrios and more specifically within the Harveyi clade, that encodes LuxN but not LuxM. There

511 are several evolutionary possibilities for this. One possibility is that DS40M4 never gained luxM

512 compared to other members of the V. harveyi clade. However, another possibility is that

513 DS40M4 lost its luxM gene. In our phylogeny, DS40M4 and V. campbellii NBRC 15631 both

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514 branch off from other members of the V. harveyi clade. NBRC 15631 also encodes a luxN but

515 not luxM, and like DS40M4 its LuxN is only 63% identical to the BB120 homolog. One

516 hypothesis suggests that DS40M4 and NBRC 15631 both lost luxM but maintained their luxN

517 homolog. Without a cognate LuxM synthase for the LuxN receptor, it is possible that the luxN

518 mutated from other V. campbellii homologs in such a way that it no longer responds to an

519 autoinducer. While this hypothesis remains to be tested, future work may more clearly establish

520 whether DS40M4 lost luxM from its genome.

521 With regard to other signaling proteins in the circuit, DS40M4 encodes an HqsK homolog

522 with 98.29% identity to BB120 (Fig. 1B, Table 1). In BB120, HqsK has been shown to feed into

523 the quorum-sensing pathway through interaction with a NO-sensitive H-NOX protein. Although

524 at HCD it contributes to the dephosphorylation of LuxU, the effect of the other three receptors

525 overwhelms the effect that NO has on bioluminescence (Henares et al., 2012). Since we

526 focused this study on the effects of AI-sensing receptors on the QS pathway, we have not yet

527 assessed the role of the HqsK homolog in DS40M4. A recently identified quorum sensing

528 receptor protein called CqsR in V. cholerae has not yet been characterized in BB120. CqsR is

529 an additional histidine sensor kinase in V. cholerae that phosphorylates LuxU, and thus far the

530 ligand for this receptor has not been identified (36, 37). BB120 and DS40M4 both appear to

531 encode homologs of V. cholerae CqsR with a percent identity of 54.76% and 55.03%

532 respectively (Table 2). Another quorum-sensing system in V. cholerae has been identified in

533 which the molecule DPO (3,5-dimethylpyrazin-2-ol, synthesized by threonine dehydrogenase) is

534 bound by transcription factor VqmA (Papenfort et al., 2017). VqmA-DPO activates expression of

535 a sRNA (VqmR) that represses biofilm formation. A homolog of VqmA is present in both BB120

536 and DS40M4 (Fig. 1, Table 2), and a putative VqmR lies directly upstream of these vqmA

537 genes. Both BB120 and DS40M4 encode Tdh, which is not surprising given its role in

538 metabolism (Table 2). However, the production of DPO, the function of VqmA and VqmR, and

539 any connection to downstream genes still remains to be tested in most vibrios, including BB120

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540 and DS40M4. Based on our data and the recent identification of novel autoinducers/receptors in

541 V. cholerae (Jung et al., 2015; Papenfort et al., 2017), it is very possible that there are other

542 quorum-sensing components that act in parallel or integrate into the known circuit that have yet

543 to be identified in various Vibrio species. Further, the roles of HqsK, VpsS, CqsR, and LuxPQ in

544 sensing signals from “other” cells (not exclusively self-produced) has yet to be explored in

545 BB120 and DS40M4 (Barrasso et al., 2020).

546 Our data also suggest that DS40M4 contains a regulator that converges on the quorum-

547 sensing circuit at the Qrrs (Fig. 1B). Contrary to BB120, deletion of luxO in DS40M4 does not

548 result in maximal quorum sensing (monitored via bioluminescence production). The ∆luxO strain

549 bioluminescence phenotype is quite similar to wild-type DS40M4, whereas the ∆qrr1-5 mutant

550 has maximal bioluminescence at all cell densities. Further complicating quorum-sensing

551 regulation, our data also show that when either qrr4 or qrr5 is the only qrr gene remaining in

552 DS40M4, cells have constitutively low bioluminescence levels. We propose that LuxO is not the

553 sole regulator for the Qrrs, and that an additional regulator acts to influence Qrr expression in

554 DS40M4. Although DS40M4 and BB120 have an additional LuxO homolog, the transcript levels

555 of these genes are very low in both strains, and deletion of the homolog in the DS40M4 strain

556 did not result in a constitutively bright strain. Genetic screens will likely uncover any additional

557 regulator(s) of the Qrrs.

558 Previous studies of quorum sensing in V. campbellii have typically been performed in

559 BB120. However, while it is understood and accepted that strains of the same bacterial species

560 can vary widely in nature, it isn’t often investigated how the canonical models diverge amongst

561 these strains. Although BB120 and DS40M4 are very closely related (Colston et al., 2019), we

562 have shown that these two strains differ widely from one another in natural transformation,

563 quorum-sensing pathways, and various other phenotypes. It is important to consider strain to

564 strain variations and how bacteria in the environment may differ from the models established

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565 from laboratory strains. Here we have shown that the environmental isolate DS40M4 may serve

566 as another useful model strain when studying behaviors in V. campbellii.

567

568

569 Experimental Procedures

570

571 Bacterial strains and media

572 All bacterial strains and plasmids are listed in Tables S2 and S3. V. campbellii strains were

573 grown at 30°C on Luria marine (LM) medium (Lysogeny broth supplemented with an additional

574 10 g NaCl per L). Instant water (IOW) medium was used in the chitin-independent

575 transformations; it consists of Instant Ocean sea salts (Aquarium Systems, Inc.) diluted in sterile

576 water (2X=28 g/l). Transformations were outgrown in LBv2 (Lysogeny Broth medium

577 supplemented with additional 200mM NaCl, 23.14mM MgCl2, and 4.2mM KCl). When

578 necessary, strains were supplemented with kanamycin (100μg/ml), spectinomycin

579 (200μg/ml), gentamicin (100 μg/ml), or trimethoprim (10μg/ml). Plasmids were transferred

580 from E. coli to Vibrio strains by conjugation on LB plates. Exconjugants were selected on LM

581 plates with polymyxin B at 50U/ml and the appropriate selective antibiotic.

582

583 Construction of linear tDNAs

584 All PCR products were synthesized using Phusion HF polymerase (New England

585 Biolabs). Sequencing of constructs and strains was performed at Eurofins Scientific. Cloning

586 procedures and related protocols are available upon request. Oligonucleotides used in the study

587 are listed in Table S4. Linear tDNAs were generated by splicing-by-overlap extension (SOE)

588 PCR as previously described (Ankur B. Dalia et al., 2014). Briefly, an UP arm containing ~3kb

589 homology of the area upstream of the gene to be deleted was PCR amplified via a F1 and R1

590 primer set. Similarly, a DOWN arm containing ~3kb homology of the area downstream of the

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591 gene to be deleted was PCR amplified via a F2 and R2 primer set. For selected linear

592 transforming DNA products, a PCR using the UP and DOWN products and an antibiotic

593 resistance cassette as template was amplified using the F1 and R2 primers to make a selected

594 SOE product. For unselected tDNA products, the UP and DOWN arms were used as template

595 and amplified via the F1 and R2 primers to make a SOE product that lacked an antibiotic

596 marker.

597

598 Natural Transformation

599 DS40M4 can be induced for competence by overexpression of the master competence

600 regulator, Tfox (chitin-independent transformation) (Simpson et al., 2019). Here, chitin-

601 independent transformations were performed following the protocol established in Dalia et al.

602 2017 (Dalia et al., 2017). Natural competence was induced via ectopic expression of Tfox on a

603 plasmid under an IPTG inducible promoter. A strain containing this plasmid was grown

604 overnight in LM at 30°C, shaking, with antibiotic and 100 uM IPTG. The next day the culture was

605 back-diluted into IOW containing 100 uM IPTG. Linear transforming DNA (tDNA) marked with

606 an antibiotic resistance cassette was added and cells were incubated statically at 30°C for 4-6

607 hours. LBv2 was then added and the cells were incubated shaking at 30°C for an additional 1-2

608 hours. The reaction was then plated on a LM plate with the appropriate antibiotic (to select for

609 the marker in the selected/marked tDNA) and an LM plate (to determine the viable cell count).

610 The presence of colonies growing on the antibiotic plate containing the transformation reaction,

611 as well as the absence of colonies growing on the antibiotic plate of the control reaction (cells

612 induced for competence with no tDNA added) indicates a successful transformation reaction.

613 Transformation frequency was calculated as the number of antibiotic-resistant colonies divided

614 by viable cells. Following natural transformation, strains containing the correct target mutation

615 were identified via colony PCR with a forward and reverse detection primer.

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616 For MuGENT, this same process was followed, but both a marked linear tDNA and

617 unmarked tDNA was added to the reaction. Following MuGENT, colonies were screened for

618 integration of unselected genome edits via MASC-PCR as described previously (Ankur B Dalia

619 et al., 2014). Briefly, a standard colony PCR reaction was set up, but a primer mix was added.

620 Instead of 2 individual primers, a universal F primer was mixed with each R detection primer in

621 equal concentrations and this mix was added to the PCR reaction. Detection primers are listed

622 in Table S3. A band on the gel indicates the successful deletion of a targeted gene via the

623 unselected tDNA product (Fig. S4). All mutants were further confirmed by PCR amplifying a

624 product off of the mutant gDNA using the F1 and R2 primers, and sending the product to

625 Eurofins Scientific for sequencing.

626

627 Bioluminescence assays and growth curves

628 For the bioluminescence growth assays, overnight cultures were back-diluted 1:10,000

629 in 200 μl LM with selective antibiotics as needed in a black-welled clear-bottom 96-well plate (all

630 adjacent wells left empty to avoid light carryover) and OD600 and bioluminescence were

631 measured every 30 minutes for 20 hours using the BioTek Cytation 3 Plate Reader

632 (temperature set to 30 °C, gain set to 160 for DS40M4 strains, or 135 for BB120 strains). For

633 bioluminescence growth assays with AHLs, the AHLs were added to each well at a final

634 concentration of 10 µM.

635 For endpoint bioluminescence assays, strains were back-diluted to 1:1,000 in 5 ml LM

636 with selective antibiotics as required and incubated at 30°C shaking at 275 RPM for 7 h. 200 μl

637 of the culture was transferred to a black-welled clear-bottom 96-well plate and the OD600 and

638 bioluminescence were measured using the BioTek Cytation 3 Plate Reader (gain set to 160).

639 For growth assays, overnight cultures were back-diluted 1:1,000 in 200 µl LM in a 96-well plate,

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

640 and incubated at 30°C shaking in the BioTek Cytation 3 Plate Reader. OD600 was recorded

641 every 30 minutes in the plate reader for a total of 24 hours.

642 For autoinducer reporter assays, overnight cultures of supernatant strains were back-

643 diluted 1:100 in fresh media and incubated at 30°C shaking at 275 RPM until the OD600 ~2.0.

644 Cultures were centrifuged to pellet cells at 16,200 x g, supernatants were filtered through 0.22-

645 μm filters, and these were added to fresh medium at 20% final concentration. Cells from strains

646 to be assayed in the presence of supernatants were added to these supplemented media at a

647 1:5,000 dilution of the overnight culture and incubated shaking at 30°C at 275 RPM overnight.

648 Bioluminescence and OD600 were measured in black-welled clear-bottom 96-well plates using

649 the BioTek Cytation 3 Plate Reader.

650

651 β-galactosidase reporter assays

652 Pre-induced cells of the ultra-sensitive Agrobacterium tumefaciens reporter strain

653 KYC55 were prepared and β-galactosidase activity was measured as described previously with

654 slight modifications (Zhu et al., 2003; Joelsson and Zhu, 2006). Briefly, a single aliquot of pre-

655 induced KYC55 cells was thawed on ice, and 1 µl/ml of cells were used to inoculate a large

656 volume of sterile ATGN medium without antibiotics. The inoculated ATGN medium was

657 aliquoted into 4 ml cultures containing 30% cell-free supernatant or 30% fresh LM medium

658 supplemented with 10 µM of an exogenous AHL. Cell-free supernatants from Vibrio strains were

659 prepared as described above and, as an added control, exogenous AHL was spiked into one of

660 the cultures immediately before centrifugation to ensure AHL recovery after filtration. Cultures

661 were incubated at 30°C at 275 RPM to an OD600 = 0.75-1.2 and the final density of each culture

662 was recorded using a spectrophotometer. Each culture was diluted 1:40 into Z buffer and cells

663 were lysed by the addition of 5 µl 0.05% SDS and 10 µl chloroform followed by vigorously

664 vortexing for ~15 seconds. 200 µl of cell lysate was transferred to a clear-welled clear-bottom

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

665 96-well plate, and 20 µl of 4 mg/ml ONPG was added to each well. Immediately following the

666 addition of ONPG, the OD420 of each well was recorded at 1-minute intervals using the BioTek

667 Cytation 3 Plate Reader. β-galactosidase activity for each culture was calculated in Modified

668 Miller Units as described previously (Thibodeau et al., 2004).

669

670 Liquid chromatography - mass spectrometry analyses of bacterial supernatants

671 Filtered DS40M4 or BB120 supernatants (100 ml) were extracted with dichloromethane

672 (3 x 30 ml). The combined organic layers were dried (MgSO4), filtered, and concentrated under

673 reduced pressure. The dried extracts were reconstituted in 1 ml of 0.1% aqueous formic acid.

674 Samples were injected onto an Acquity UPLC Peptide HSS T3 column (2.1 x 150 mm, 1.8 µm

675 100 Å) by Waters with an Acquity UPLC Peptide guard column. The UPLC system consisted of

676 an Agilent 1290 II system consisting of a multisampler, binary pump and thermostatted column

677 compartment (set at 40°C). The components were separated by gradient elution at 0.600

678 ml/min. Mobile Phase A consisted of 0.1% formic acid in Optima grade water and Mobile Phase

679 B consisted of 100% acetonitrile. All components of interest eluted within the first 5 minutes.

680 N-3-hydroxybutanoyl-L-homoserine lactone (HAI-1/HC4) was synthesized as previously

681 described (Chaparian et al., 2019). All other AHLs were purchased from Cayman Chemical

682 Company Inc. and resuspended in 100% acetonitrile: N-butyryl-L-homoserine lactone (C4), N-

683 hexanoyl-L-homoserine lactone (C6), N-octanoyl-L-homoserine lactone (C8), N-decanoyl-L-

684 homoserine lactone (C10), N-3-hydroxyoctanoyl-L-homoserine lactone (HC8), N-3-

685 hydroxydecanoyl-L-homoserine lactone (HC10), N-(β-ketocaproyl)-L-homoserine lactone (OC6),

686 N-3-oxo-octanoyl-L-homoserine lactone (OC8), and N-3-oxo-decanoyl-L-homoserine lactone

687 (OC10). The compounds were detected qualitatively via MRM methodology on an AB Sciex

688 QTrap 4000.

689

690 Comparative genomics analyses

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

691 We downloaded 287 publicly available complete Vibrio genome sequences along with

692 their corresponding annotated protein sequences from the Genbank database. To find any

693 missing annotations of genes, we used Prokka version 1.12 (Seemann, 2014), a gene

694 prediction software, by providing the annotated gene sequences as a training set. We searched

695 the individual assemblies for Vibrio core genes using BUSCO version 4.02 (Seppey et al., 2019)

696 against 1,445 vibrionales_odb10 HMMs. Our search returned 214 BUSCOs being marked as

697 complete and non-duplicated in each of the 287 assemblies. A multiple sequence alignment

698 was generated for the protein sequences matching these common core BUSCOs from each of

699 the assemblies using MUSCLE ver. 3.8.31 (Edgar, 2004), which was then converted to

700 nucleotide space by substituting each amino acid with the corresponding codon sequence from

701 the associated CDS sequences. A best scoring maximum likelihood tree was drawn using

702 RAxML version 8.2.12 (Stamatakis, 2014) with parameters (-m GTRCAT -B 0.03).

703 The protein sequences from annotated and predicted genes from the 287 assemblies

704 were clustered using cd-hit ver. v4.8.1-2019-0228 (Li and Godzik, 2006) with parameters: -M 0 -

705 g 1 -s 0.8 -c 0.65 -d 500. All sequences grouped with VIBHAR_02765 from V. campbellii BB120

706 were considered the LuxM homologs while those grouped with VIBHAR_02766 were

707 considered LuxN homologs. To identify distant homologs with less than 65% identity, we

708 scanned the protein sequences from the Vibrio assemblies against HMMs prepared from the

709 LuxM and LuxN clusters using hmmer version 3.2.1 (http://hmmer.org/). In addition to matching

710 scores, we also used the syntenic relationship between the hits to LuxM and LuxN HMMs to

711 identify the corresponding homologs. The homology information was overlaid on the

712 phylogenetic tree using the Interactive Tree of Life version 5 (Letunic and Bork, 2019).

713

714 Extracellular protease activity assay

715 Extracellular protease activity was determined as previously described (Gu et al., 2016).

716 Briefly, all strains were back-diluted 1:1,000 in LM medium and grown at 30°C shaking at 275

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

717 RPM for 9 h. The culture OD600 was measured, and the strains were collected by centrifugation

718 at 3700 x g for 10 min. The supernatants of each strain were filtered through 0.22 µm filters and

719 1 ml of filtered supernatant was mixed with 1 ml phosphate buffer saline (PBS, pH 7.2) and 10

720 mg of hide powder azure (HPA). These mixtures were incubated at 37°C while shaking at 275

721 RPM for 2 h, and the reactions were stopped by adding 1 ml 10% trichloroacetic acid (TCA).

722 Total protease activity was measured at 600 nm on a spectrophotometer and then normalized

723 for each strain by dividing by the culture OD600.

724

725 Biofilm formation assay

726 Biofilm formation was measured as previously described (O’Toole, 2010). Overnight

727 cultures of strains grown in seawater tryptone (SWT) medium were diluted to OD600 = 0.005 in

728 SWT and grown statically in culture tubes or in 96-well microtiter plates in six replicates at 30°C

729 for 24 h. The OD600 of the strains in the plate were measured, and the plates were rinsed with

730 deionized water. 0.1% crystal violet was added to each well, and the plate was subsequently

731 rinsed with deionized water. The crystal violet-stained biofilms were then solubilized with 33%

732 acetic acid and measured at 590 nm using the BioTek Cytation 3 Plate Reader and normalized

733 to the OD600.

734

735 Interbacterial competition assay

736 Interbacterial killing was measured using a competition assay as previously described

737 (Hachani et al., 2013). Predator strains (V. campbellii) were grown in LM medium overnight at

738 30°C, while the prey strain (E. coli S17-1λpir) was grown in LB at 37°C. Predator strains were

739 diluted 1:20 and prey strains were diluted 1:40 and grown until they reached an OD600 between

740 0.6-1.2. Cells were harvested by centrifugation at 3,700 x g for 10 min, and then resuspended

741 in fresh LB to reach an OD600 = 10. Predator and prey strains were mixed at a ratio of 1:1, and 5

742 µl of each mixture was spotted onto a piece of sterile filter paper fitted to an LB plate. The

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

743 competition was carried out at 30°C for 4 h, and then each filter paper was moved to 0.5 ml of

744 LB and vortexed to resuspend the cells. These suspensions were serially diluted and plated on

745 prey-selective plates (LB+trimethoprim10). Plates were incubated at 37°C until colonies were

746 visible, then colonies were counted to determine CFU of the surviving prey.

747

748 RNA extraction, RNA-seq, and quantitative reverse transcriptase real-time PCR (qRT-PCR)

749 Strains were inoculated in 5 ml LM and grown overnight shaking at 30°C at 275 RPM.

750 Each strain was back-diluted 1:1,000 in LM and grown shaking at 30°C at 275 RPM until they

751 reached an OD600 = 0.1, 1.0, or 3.25. Cells were collected by centrifugation at 3,700 RPM at 4°C

752 for 10 min, the supernatant was removed, and the cell pellets were flash frozen in liquid N2 and

753 stored at -80°C. RNA was isolated from pellets using a TRIzol/chloroform extraction protocol as

754 described (Rutherford et al., 2011) and treated with DNase via the DNA-freeTM DNA Removal

755 Kit (Invitrogen).

756 For RNA-seq, cDNA libraries were constructed as described previously (Papenfort et al.,

757 2015) by Vertis Biotechnology AG (Freising, Germany) and sequenced using an Illumina

758 NextSeq 500 machine in single-read mode (75 bp read length). The raw, demultiplexed reads

759 and coverage files have been deposited in the National Center for Biotechnology Information

760 Gene Expression Omnibus with accession code GSE147616.

761 qRT-PCR was used to quantify transcript levels of specific quorum sensing-controlled

762 genes in DS40M4 and was performed using the SensiFast SYBR Hi-ROX One-Step Kit (Bioline)

763 according to the manufacturer’s guidelines. Primers were designed to have the following

764 parameters: amplicon size of 100 bp, primer size of 20-28 nt, and melting temperature of 55-

765 60°C. All reactions were performed using a LightCycler 4800 II (Roche) with 0.4 µM of each

766 primer and 5 ng of template RNA (10 µl total volume). All qRT-PCR experiments were

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

767 normalized to hfq expression and were performed with 4 biological replicates and 2 technical

768 replicates.

769

770

771 Acknowledgements

772 We thank Victoria Lydick for excellent technical support, Ryan Chaparian for assistance

773 with sequence alignments, and Konrad Förstner and Malte Siemers for support with the RNA-

774 Seq analysis. We thank Laura Miller Conrad and Minh Tran for synthesis of HAI-1. We would

775 also like to acknowledge the IU Arts and Sciences Undergraduate Research Experience

776 (ASURE) for encouraging and preparing the undergraduate researchers (LG and AL) as well

777 as Charlotte & Jim Griffin for their support of the ASURE program.

778

779 Funding

780 This work was supported by the National Institutes of Health grant R35GM124698 to JVK. KP

781 acknowledges funding by the European Research Council (StG-758212) and DFG (EXC 2051,

782 Project 390713860).

783

784 Conflict of Interest Statement

785 The authors declare that they have no conflicts of interest.

786

787 References

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938 Tu, K.C. and Bassler, B.L. (2007) Multiple small RNAs act additively to integrate sensory

939 information and control quorum sensing in Vibrio harveyi. Genes Dev 21: 221–233.

940 Urbanczyk, H., Ogura, Y., and Hayashi, T. (2013) Taxonomic revision of Harveyi clade bacteria

941 (family Vibrionaceae) based on analysis of whole genome sequences. Int J Syst Evol

942 Microbiol 63: 2742–2751.

943 Wang, L., Zhou, D., Mao, P., Zhang, Y., Hou, J., Hu, Y., et al. (2013) Cell density- and quorum

944 sensing-dependent expression of type VI secretion system 2 in vibrio parahaemolyticus.

945 PLoS One 8: 1–11.

946 Waters, C.M. and Bassler, B.L. (2005) QUORUM SENSING: Cell-to-Cell Communication in

947 Bacteria. Annu Rev Cell Dev Biol 21: 319–346.

948 Waters, C.M., Lu, W., Rabinowitz, J.D., and Bassler, B.L. (2008) Quorum sensing controls

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949 biofilm formation in Vibrio cholerae through modulation of cyclic Di-GMP levels and

950 repression of vpsT. J Bacteriol 190: 2527–2536.

951 White, A.P. and Surette, M.G. (2006) Comparative genetics of the rdar morphotype in

952 Salmonella. J Bacteriol 188: 8395–8406.

953 Yamamoto, S., Izumiya, H., Mitobe, J., Morita, M., Arakawa, E., Ohnishi, M., and Watanabe, H.

954 (2011) Identification of a chitin-induced small RNA that regulates translation of the tfoX

955 gene, encoding a positive regulator of natural competence in vibrio cholerae. J Bacteriol

956 193: 1953–1965.

957 Zhang, Yiquan, Gao, H., Osei-Adjei, G., Zhang, Ying, Yang, W., Yang, H., et al. (2017)

958 Transcriptional regulation of the type VI secretion system 1 genes by quorum sensing and

959 ToxR in Vibrio parahaemolyticus. Front Microbiol 8:.

960 Zheng, J., Shin, O.S., Cameron, D.E., and Mekalanos, J.J. (2010) Quorum sensing and a global

961 regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae.

962 Proc Natl Acad Sci 107: 21128 LP – 21133.

963 Zhu, J., Chai, Y., Zhong, Z., Li, S., and Winans, S.C. (2003) Agrobacterium Bioassay Strain for

964 Ultrasensitive Detection of N-Acylhomoserine Lactone-Type Quorum-Sensing Molecules:

965 Detection of Autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol 69: 6949–

966 6953.

967

968

969

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

970 Table 1. DS40M4 homologs of established BB120 quorum-sensing proteins.

971

Gene name Amino acid Query Cover BB120 locus taga DS40M4 locus tagb identity cqsA 97.96% 100% VIBHAR_06088 DSB67_15960 cqsS 98.83% 100% VIBHAR_06089 DSB67_15965 luxM N/A N/A VIBHAR_02765 N/A luxN 63.23% 99% VIBHAR_02766 DSB67_09895 luxP 97.26% 100% VIBHAR_05351 DSB67_22210 luxQ 98.37% 100% VIBHAR_05352 DSB67_22215 luxS 98.84% 100% VIBHAR_03484 DSB67_12735 luxU 96.49% 100% VIBHAR_02958 DSB67_10605

luxO 98.34% 100% VIBHAR_02959 DSB67_10610 luxR 99.51% 100% VIBHAR_03459 DSB67_12620 aphA 99.44% 100% VIBHAR_00046 DSB67_14070 hqsK 98.29 92% VIBHAR_01913 DSB67_09490 972 a. Accession numbers: CP000789, CP000790, CP000791.

973 b. Accession numbers: CP030788, CP030789, CP030790

974

975 Table 2. BB120 and DS40M4 homologs of V. cholerae quorum-sensing receptor proteins.

976

BB120a DS40M4b Gene Amino acid Query Locus tag Amino acid Query Locus tag name identity to V. Cover identity to V. Cover cholerae cholerae vqmA 64.5% 92% VIBHAR_07094 64.5% 92% DSB67_18870 tdh 91.84% 100% VIBHAR_05001 91.55% 100% DSB67_20895 cqsR 54.76% 98% VIBHAR_01620 55.03% 98% DSB67_05165 vpsS 50.35% 98% VIBHAR_02306 65% 39.37% DSB67_09490 69% 36.50% DSB67_07055 70% 39.49% DSB67_07080 72% 31.11% DSB67_12445 69% 37.06% DSB67_12965 67% 36.16% LuxQ 65% 40.45% DSB67_19025

977 a. Accession numbers: CP000789, CP000790, CP000791.

978 b. Accession numbers: CP030788, CP030789, CP030790.

979

980

981 Table 3. Classes of genes regulated by LuxR in DS40M4.

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Function Number of Genes Transport 8 Metabolism 31 Virulence 4 Protease 3 Gene Expression 4 Membrane 3 Proteins Signaling 2 Oxidoreductases 2 Type VI Secretion 1 Miscellaneous 15 Hypothetical 17 982

41 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

983 Figure legends

984

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

986 DS40M4. The quorum-sensing models in V. campbellii BB120 (A) and DS40M4 (B). Darker

987 colored circuits represent pathways that have been tested in each strain. Lighter colored circuits

988 include putative homologs to other Vibrio systems that have not yet been tested in these strains.

989 (A) In the established BB120 pathway, the autoinducers CAI-1, HAI-1, AI-2, are produced by

990 autoinducer synthases CqsA, LuxM, and LuxS and sensed by receptors CqsS, LuxN,

991 and LuxPQ, respectively. At LCD, autoinducers are at low concentrations, resulting in the

992 receptors acting as kinases. The receptors phosphorylate LuxU (phosphorelay protein), which

993 transfers the phosphate to LuxO. Phosphorylated LuxO activates qrr expression through Sigma-

994 54. The Qrrs together with Hfq bind to the aphA and luxR mRNAs, and AphA is expressed

995 and LuxR production is minimal. The combination of AphA and LuxR protein levels leads to LCD

996 behaviors, such as T3SS and biofilm formation. As autoinducers accumulate at HCD, the

997 receptors bind autoinducers and in this state act as phosphatases. De-

998 phosphorylated LuxO does not activate the qrr genes, thus leading to maximal LuxR production

999 and absence of AphA. This ultimately leads to HCD behaviors such as

1000 bioluminescence, proteolysis, and type VI secretion (T6SS). In the absence of nitric oxide (NO),

1001 HqsK acts as a kinase. In the presence of NO, NO-bound H-NOX inhibits the kinase activity

1002 of HqsK contributing to the de-phosphorylation of LuxO. In V. cholerae, the CqsR receptor

1003 senses an unknown autoinducer from an unidentified synthase. In the V. cholerae DPO-

1004 dependent QS pathway, the autoinducer DPO is produced from threonine catabolism which is

1005 dependent on the Tdh enzyme. Extracellular DPO binds to the receptor VqmA. The VqmA-DPO

1006 complex activates transcription of the VqmR sRNA, which represses genes required for biofilm

1007 formation and toxin production. (B) Our proposed model for quorum-sensing regulation in

1008 V. campbellii DS40M4. The CqsA/CqsS and LuxS/LuxPQ receptor pairs are conserved and act

42 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1009 as described above. The LuxN receptor is active either in the absence of a ligand or in the

1010 presence of an unidentified ligand other than an AHL, but the mechanism by which it functions

1011 is undetermined. We propose that there is an additional regulator outside of the LuxO pathway

1012 that also converges on the Qrrs. At HCD, LuxR controls fewer behaviors than in BB120. Image

1013 created with BioRender.com.

1014

1015 Figure 2. Quorum sensing regulation of bioluminescence in BB120 and DS40M4. (A, B)

1016 Bioluminescence production is shown normalized to cell density (OD600) during a growth curve

1017 for wild-type and mutant strains of BB120 (A) and DS40M4 containing pCS38 (B) (see Table S2

1018 for strain names). For each graph, the data shown are from a single experiment that is

1019 representative of at least three independent biological experiments. In panel A, luxR and luxO

1020 are complemented on plasmids pKM699 and pBB147, respectively, compared to the pLAFR2

1021 empty vector control. In panel B, luxR and luxO are complemented on the chromosome at the

1022 luxB locus.

1023

1024 Figure 3. V. campbellii DS40M4 produces and responds to CAI-1 and AI-2 autoinducers,

1025 but not HAI-1. (A) Diagram of the luxN loci in BB120 and DS40M4 strains. Shaded areas

1026 indicate the percent amino acid identity shared between the strains for each gene. (B) Diagram

1027 of the predicted structure of the DS40M4 LuxN monomer. The conserved C-terminal domain is

1028 colored purple, and the N-terminal region that is not conserved is colored orange. (C)

1029 Bioluminescence production normalized to cell density (OD600) for strains in which supernatants

1030 from the indicated strains were added to BB120 strains. Different letters indicate significant

1031 differences in 2-way analysis of variance (ANOVA) of log-transformed data followed by Tukey’s

1032 multiple comparison’s test (n = 4, p < 0.05). Statistical comparisons were performed comparing

1033 the effects of the three supernatant conditions for each reporter strain; data were not compared

1034 between reporter strains. (D) Superimposed total ion chromatograms for extracts of supernatant

43 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1035 from strains BB120 and DS40M4. (E) Superimposed extracted ion chromatograms of 10 μM

1036 AHLs. Each color corresponds to a different AHL species. (F) β-galactosidase activity as

1037 measured by Modified Miller Units in A. tumefaciens reporter strain KYC55 whole-cell lysate.

1038 KYC55 was grown in cell-free supernatant (sup) from Vibrio strains or supplemented with an

1039 exogenous AHL (10 µM) as indicated. Data shown represent at least 2 independent biological

1040 replicates except BB120 sup with spiked-in HC8 (10 µM) and V. fischeri sup which represent a

1041 single replicate each. (G) Bioluminescence production normalized to cell density (OD600) for

1042 BB120 ΔluxPQM ΔcqsS (TL25) and DS40M4 ΔluxPQ ΔcqsS (cas322) with or without the

1043 supplementation of exogenous AHLs (10 µM) . The data shown in panels G and H are

1044 representative of three independent experiments. (H) Bioluminescence production normalized to

1045 cell density (OD600) for wild-type DS40M4 (cas291), ΔluxO (BDP065), luxO D47E (BDP062),

1046 Δqrr1-5 (cas269), ΔluxPQ ΔcqsS (cas322), and ΔluxPQ ΔcqsS ΔluxN ΔcqsR ΔhqsK (cas399).

1047

1048 Figure 4. DS40M4 LuxN positively regulates biofilm formation and bioluminescence. (A)

1049 Biofilm formation measured by crystal violet staining (OD590) normalized to cell density (OD600).

1050 Culture tubes grown statically and assayed for biofilm formation by crystal violet staining. (B, C)

1051 Biofilm formation measured by crystal violet staining (OD590) normalized to cell density (OD600).

1052 Strains containing complementation plasmids or empty vector were induced with 10 μM IPTG.

1053 (D) Bioluminescence production normalized to cell density (OD600). For panels B, C, and D,

1054 different letters indicate significant differences in 2-way ANOVA of log-transformed data

1055 followed by Tukey’s multiple comparison’s test (n = 3 or n = 4, p < 0.05). See Table S2 for strain

1056 names for all panels.

1057

1058 Figure 5. Conservation of LuxM/LuxN in Vibrio strains. (A) Phylogenetic tree of 287

1059 sequenced Vibrio genomes from Genbank. The assembly number, genus, species, and strain

44 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1060 are indicated for each organism. The red shading indicates the amino acid identity of a protein

1061 in these strains to either LuxM or LuxN in BB120. The presence of pseudogenes for luxN or

1062 luxM, or the presence of multiple LuxN genes is indicated by symbols. (B) Enlarged inset (from

1063 the gray region in panel A) of the V. campbellii region of the tree in panel A. The scale of 0.1

1064 indicated in both panels refers to genetic distance between nodes based on nucleotide identity.

1065

1066 Figure 6. The Qrrs regulate bioluminescence in DS40M4. (A) Bioluminescence production is

1067 shown normalized to cell density (OD600) during a growth curve for wild-type BB120, ΔluxR

1068 (KM669), qrr1+ (KT234), qrr2+ (KT212), qrr3+ (KT225), qrr4+ (KT215), qrr5+ (KT133), and

1069 Δqrr1-5 (KT220). (B) Bioluminescence production is shown normalized to cell density (OD600)

1070 during a growth curve for wild-type DS40M4, ΔluxR (BP64), qrr1+ (cas0266), qrr2+ (cas0265),

1071 qrr3+ (cas0368), qrr4+ (cas0264), qrr5+ (cas0268), and Δqrr1-5 (cas0269). For both panels, a

1072 strain expressing only a single qrr is labeled with a ‘+’ sign. For example, qrr4+ expresses only

1073 qrr4, and the qrr1, qrr2, qrr3, and qrr5 genes are deleted. The data shown for both panels are

1074 from a single experiment that is representative of at least three independent experiments.

1075

1076 Figure 7. Identification of the LuxR regulon in DS40M4. (A, B) Data shown are from qRT-

1077 PCR analyses of transcripts of the exsB and tssC genes in the DS40M4 wild-type, ΔluxR, or

1078 Δqrr1-5 strains. Reactions were normalized to the internal standard (hfq).

1079

1080 Figure 8. Virulence phenotypes in DS40M4 and BB120 differ in quorum-sensing

1081 regulation. For all panels, assays were performed with V. campbellii DS40M4 wild-type, ΔluxO

1082 (cas197), luxO D47E (BDP060), and ΔluxR (cas196). (A) Survival of E. coli in interbacterial

1083 killing assays (n = 9). (B) Exoprotease activity measured by HPA digestion and normalized to

1084 cell density (OD600). (n = 3). (C) Biofilm formation measured by crystal violet staining (OD595)

45 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1085 normalized to cell density (OD600) (n = 6). For all panels, different letters indicate significant

1086 differences in 2-way ANOVA of log-transformed data followed by Tukey’s multiple comparison’s

1087 test (p < 0.05).

1088

46 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1

A Quorum sensing in BB120

Low cell density High cell density

B Quorum sensing in DS40M4

Low cell density High cell density bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2

A BB120 106 WT luxO D47E ) 105 Δqrr1-5 600 104 ΔluxO ΔluxO empty vector 3 10 ΔluxO pluxO 2 (Lux/OD 10 ΔluxR

Bioluminescence ΔluxR empty vector 101 0.1 1 ΔluxR pluxR OD B 600 DS40M4 107

) 106 WT

600 luxO D47E 5 10 Δqrr1-5 104 ΔluxO ΔluxO::luxO 3 (Lux/OD 10 ΔluxR Bioluminescence 102 ΔluxR::luxR 0.1 1 OD600 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3 A B LuxN septation h.p. protein A BB120 trpA symporter luxM luxN

97% 98% 63% 99% 99%

DS40M4 trpA symporter luxN h.p. septation protein A

BB120 C 106 a b b a b b a a b a b c a a a Supernatants D added DS40M4 105 HAI-1 ) None 1000000

600 DS40M4 104 BB120 103 100000 (Lux/OD 102 Bioluminescence Intensity (cps) Intensity

1 10 10000 D∆cqsAcqsA D∆luxSluxS D∆luxMluxM DcqsA∆3 WildWT-type (cps) Intensity DluxS DluxM BB120 reporter strains 1000 0 1 2 3 4 5 RetentionRetention time Time (min) (min) C4 E C6 F ✱✱✱✱ C8 700000 ✱✱✱✱ C10 4000 ✱✱✱ 600000 OC6 ✱✱✱✱ OC8 3000 500000 OC10 HC4(HAI-1) 2000 400000 HAI-1 HC8 HC10 1000

300000 0 Intensity (cps) Intensity

-1000 200000 Modified Miller units Intensity (cps) Intensity C4 C8 HC8 sup 100000 HAI-1

BB120 sup 0 . fischeri DS40M4V sup Media control 0 1 2 3 4 5 Retention time (min) BB120 sup + HC8 G Retention Time (min) H BB120 ΔluxPQM ΔcqsS 107 107 WT ΔluxPQM ΔcqsS + HAI-1 6 ΔluxO ) 6 10 ) 10 DS40M4 luxO D47E 600 600 105 ΔluxPQ ΔcqsS 105 Δqrr1-5 ΔluxPQ ΔcqsS + HAI-1 ΔcqsS ΔluxPQ 104 104 ΔluxPQ ΔcqsS + HC8 ΔcqsS ΔluxPQ 3 ΔluxPQ ΔcqsS + HC10 3 (Lux/OD 10 (Lux/OD 10 ΔluxN ΔcqsR ΔhqsK ΔluxPQ ΔcqsS + C4 Bioluminescence Bioluminescence 102 ΔluxPQ ΔcqsS + C8 102 0.1 1 0.1 1 OD600 OD600 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4 A B 100 a a b c b c ) 10 600

/OD 1 590

0.1 (OD Biofilm Formation 0.01 WT luxO ΔluxN WT luxO ΔluxN D47E D47E BB120 DS40M4 type type luxN luxN - - D47E D47E D D Wild Wild luxO luxO BB120 DS40M4 C DS40M4 biofilm formation D DS40M4 bioluminescence 7 1 a b b a b a b a b a a b b a 10 a b b a a a a b

106 ) 600

600 105 /OD

590 104 (Lux/OD OD 103 Biofilm Formation Bioluminescence

0.1 102

WT luxR luxO luxS luxS luxN luxN Δ cqsA Δ Δ Δ Δ luxR- luxO- cqsA- luxS- Δ type luxS type Δ luxR luxS luxO luxR luxN luxN luxN luxN luxN luxO - cqsA - cqsA cqsA luxS cqsA - - D47E

wild-type D D47E cqsA D D - D D D D D D D luxO D47E D D luxS - luxO D47E luxS- cqsA- D cqsA Δ tac Δ emptytac vector tac Wild Wild tac P luxS Ptac-luxS+ P luxN Ptac-luxN+luxNP Ptac-luxN+ luxS- cqsA- luxN- luxO luxO cqsA Ptac-cqsA+Δ Δ P Δ luxS luxS

cqsA cqsA cqsA Δ Δ D D D vector empty D luxS luxN luxN

Δ luxS D D cqsA tac D +10µM IPTG luxS D luxS cqsA P

Δ D D cqsA type type D - Wild luxS D A V. metschnikovii Tree scale: 0.1

V. cholerae

S V. mimicus

D B Identity to etalo FM

V 1701K

s 1

ASM976306v1 V.cholerae strain 2015V-1118 i 550 CROF niart CROF 550 i

ASM976291v1 V.cholerae strain 2015V-1126 c.

72

370 CROF niarts e niarts CROF 370

ASM993811v1 V.cholerae O1 strain AAS91 1-L

eloh ASM78871v2 V.cholerae strain 2012Env-9

ASM96923v1 V.cholerae strain 1154-74

ASM185442v1 V.cholerae strain Env-390 E

MSA 1

8MSA 102

3

34

6 ASM188751v1 V ASM188763v1 V.cholerae strain M2140 82 n ASM188761v1 V.cholerae strain NCTC9420 ia

069

rt

v

s ASM964613v1 V.cholerae O395 s i1v203132MSA V et alo si eearelohc.V a r BB120 genes 1v75 e

ASM1624v1 V.cholerae O395

V

a ASM976298v1 V.cholerae strain 3569-08 a

relohc.V 1v751953MSA relohc.V ASM2162v1 V.cholerae O395 r

c.

hc.V 1 e ASM2160v1 V.cholerae M66-2 l o

AS o

ohc oirb

el ASM188747v1 V.cholerae strain E506 hc

M168341v1 V.cholerae 2740-80 .choler l e

.V .V

areloh r AS ear

e

ASM82921v1 V.cholerae MS6 DNA ea

1

M188741v1 V.cholerae strain E1320 s

v6

ae strain NCTC5395 t

ASM188765v1 V.cholerae strain E9120 ar ASM188739v1 V.cholerae strain C5 63

ASM188743v1 V.cholerae strain CRC711 i

n ASM188745v1 V.cholerae strain CRC1106 6

2 N16961 v2 V.cholerae O1 biovar El T 7

9 .sp. 2521-89

00

01CB .rts

MS 1 B F R niarts

00 AS 6 ASM207333v2 V.cholerae strain FDAARGOS 223 17 V. furnissii

A M188749v1 V.cholerae strain E1162 ASM976296v1 V.cholerae strain 3523-03 ASM96926v1 V.cholerae strain 10432-62 30 ASM976304v1 V.cholerae strain 3541-04 ASM976289v1 V.cholerae strain 3528-08

ASM976310v1 V.cholerae strain 3566-08

ASM976294v1 V.cholerae strain 3566-06

ASM306388v1 V.cholerae strain Sa5Y 5174 7 8 v2 V.cholerae strain ASM411711v1 V.cholerae strain FORC 076 59617 E01 V.cholerae strain NCTC 30

ASM836962v1 V.cholerae strain RFB05 M966525v1 V.metoecus strain 2011V-1169 ASM221668v1 V

AS M966527v1 V.metoecus strain 08-2459 ASM76541v1 V.cholerae strain 2012EL-2176 ASM976390v1 V.metoecus strain 2011V-1015 ASM274963v1 V.cholerae strain E7946 AS ASM976394v1 V.cholerae C6706 ASM96355v1 V.cholerae O1 biovar El Tor strain FJ147 ASM976386v1 V.metoecus strain 06-2478 ASM155847v2 V.mimicus strain FDAARGOS 112 ASM976396v1 V.mimicus strain 07-2442 V. fluvialis ASM131818v1 V.cholerae O1 str. KW3 ASM294665v1 V.cholerae O1 biovar or str. N16961 ASM976400v1 V.mimicus strain 06-2455 ASM176735v1 V.mimicus strain SCCF01

ASM976402v1 V.mimicus strain F9458 AS ASM371975v1 V.cholerae strain E4 37 M979982v1 V.cholerae isolate CTMA 1441 A19 ASM966519v1 V.mimicus strain 2011V-1073 ASM1130617v1 V.sp. HDW18 ASM976376v1 V.metschnikovii strain 9502-00 ASM16645v2 V.cholerae O1 str. 2010EL-1786 ASM976392v1 V.metschnikovii strain 07-2421 ASM966523v1 V.metschnikovii strain 2012V-1020 ASM976388v1 V.cincinnatiensis strain F8054 V. anguillarum ASM966539v1 V.cincinnatiensis strain 2409-02 48853 H01 V ASM357415v1 V.cholerae V060002El DNA Tor strain HC1037 ASM976348v1 V.cincinnatiensis strain 1398-82 ASM2258v1 V.cholerae MJ-1236 48853 G01 V.cholerae strain 4295STDY6534216 ASM976370v1 V.cincinnatiensis strain 2070-81 .chole ASM636435v1 V.furnissii strain FDAARGOS 777 48853 ASM976322v1 V.furnissii strain 2013V-1001 rae strain 4295STDY6534232 44 A02 V.cholerae strain 4295STDY6534248 ASM976417v1 V.furnissii strain 2012V-1225

ASM976334v1 V.furnissii strain 2420-04

ASM976382v1 V.cholerae strain F9993 ASM966533v1 V.furnissii strain 2419-04 no luxM ASM309769v1 V.cholerae strain A1552 ASM18432v1 V.furnissii NCTC 11218 ASM295337v1 V. uvialis strain FDAARGOS 100 ASM299721v1 V.cholerae strain A1552 multi luxN ASM195295v1 V. uvialis strain 12605 ASM289285v1 V.cholerae strain A1552 ASM976421v1 V. uvialis strain 2015AW-0233 AS pseudo luxN M674v1 V.cholerae O1 biovar El Tor str. N16961 ASM976368v1 V. uvialis strain 2013V-1145 51 AS ASM976419v1 V. uvialis strain F8658 pseudo luxM M25085v1 V.cholerae IEC224 ASM976378v1 V. uvialis strain 2012V-1235 ASM306378v1 V.cholerae strain N16961 luxN ASM976346v1 V. uvialis strain 2013V-1300 luxM ASM155841v2 V. uvialis strain FDAARGOS 104 ASM976318v1 V. uvialis strain 2015AW-0077

ASM976300v1 V. uvialis strain 2014AW-0434

ASM1106428v1 V.sp. ZWAL4003 58 ASM219651v1 V.gazogenes strain ATCC 43942 ASM154793v1 V.tritonius strain AM2 ASM988381v1 V.parahaemolyticus strain 20140624012-1 ASM371687v1 V.sp. HBUAS61001 ASM225754v1 V.qinghaiensis strain Q67 ASM988383v1 V.parahaemolyticus strain 20140722001-1 ASM339957v2 V.anguillarum strain J360 ASM988385v1 V.parahaemolyticus strain 20140829008-1 ASM228754v1 V.anguillarum strain VIB43 65 ASM221200v1 V.anguillarum strain S3 4/9 ASM976340v1 V.parahaemolyticus strain AM51552 ASM166050v1 V.anguillarum strain 90-11-286 ASM976354v1 V.parahaemolyticus strain 2013V-1244 ASM221198v1 V.anguillarum strain JLL237 ASM359558v1 V.anguillarum strain MHK3 ASM56849v1 V.parahaemolyticus UCM-V493 ASM221202v1 V.anguillarum strain CNEVA NB11008 ASM976342v1 V.parahaemolyticus strain AM46865 ASM231033v1 V.anguillarum strain VIB12 ASM21767v1 V.anguillarum 775 ASM976360v1 V.parahaemolyticus strain 2013V-1136 72 ASM229126v1 V.anguillarum strain ATCC-68554 ASM187958v1 V.parahaemolyticus strain FORC 022 ASM303120v1 V.anguillarum strain 425 ASM46297v1 V.anguillarum M3 ASM19609v1 V.parahaemolyticus RIMD 2210633 ASM216379v1 V.anguillarum strain mvav6203 ASM207377v2 V.parahaemolyticus strain FDAARGOS 191 ASM221150v1 V.anguillarum strain 87-9-116 ASM400651v1 V.parahaemolyticus strain VPD14 Vibrio anguillarum NB10 serovar O1 V.anguillarum strain NB10 79 ASM1058738v1 V.astriarenae strain HN897 ASM361271v1 V.parahaemolyticus strain FORC 071 ASM221434v1 V.mediterranei strain QT6D1

ASM43042v1 V.parahaemolyticus O1 K33 str. CDC K4557 ASM372693v1 V.mediterranei strain 117-T6 ASM170083v1 V.parahaemolyticus strain CHN25 ASM936335v1 V.sp. THAF190c ASM402254v1 V.chagasii strain ECSMB14107 ASM175860v1 V.parahaemolyticus strain FORC 023 ASM514490v1 V.cyclitrophicus strain ECSMB14105 86 ASM976302v1 V.parahaemolyticus strain AM43962 ASM334529v1 V.splendidus strain Vibrio sp ASM220972v2 V.parahaemolyticus strain MAVP-Q ASM9146v1 V.tasmaniensis LGP32 ASM211952v1 V.alginolyticus ASM976356v1 V.parahaemolyticus strain 2013V-1181 strain K08M4 Vibrio tapetis CECT4600 V.tapetis subsp. tapetis isolate Vibrio tapetis CECT4600

ASM168217v1 V.parahaemolyticus strain MAVP-Q ASM80127v1 V.nigripulchritudo HGAP V.parahaemolyticus strain MAVP-R ASM167727v1 V.breoganii strain FF50 93 ASM960176v1 V .sp. SM1977 ASM361269v1 V.parahaemolyticus strain FORC 072 ASM221802v2 V ASM32840v1 V.parahaemolyticus BB22OP .casei strain DSM 22364 ASM221804v2 V.rumoiensis strain FERM P-14531

ASM21577 ASM973432v1 V.parahaemolyticus strain 19-021-D1 3v2 V.aphrogenes strai ASM168780v1 V.scophthalmi strain VS-05 n CA-1004 ASM43040v1 V.parahaemolyticus O1 Kuk str. FDA R31 ASM168546v1 V.scophthalmi strain VS-12 100 V. parahaemolyticus ASM993820v1 V.panuliri strain JCM 19500 ASM869362v1 V.parahaemolyticus strain FDAARGOS 667 ASM993822v1 V.ponticus strain DSM 16217 ASM976362v1 V.parahaemolyticus strain 2013V-1174 ASM993818v1 V.taketomensis strain C4III291 ASM130477v1 V.parahaemolyticus strain FORC 006 ASM993816v1 V.taketomensis strain C4III282 ASM77210v1 V.tubiashii ATCC 19109 ASM155849v2 V.parahaemolyticus strain FDAARGOS 115 ASM936341v1 V.sp. ASM188705v1 V.parahaemolyticus strain FORC 018 807 ASM169361v1 V.coralliilyticus strain 58 THAF100 ASM124431v1 V.parahaemolyticus strain FORC 008 ASM207399v ASM199636v2 V.parahaemolyticus strain MVP1 ASM339137v1 V ASM112127 1 V.coralliilyticus strain SNUTY-1 ASM163603v1 V.parahaemolyticus strain FORC 014 ASM76353v2 V.coralliilyticus.coralliilyticus strain strain OCN014 RE22 ASM369152v1 V.parahaemolyticus strain 160 ASM77206v1 V.coralliilyticus0v1 V strain RE98 .coralliilyticus OCN008 ASM970819v1 V.sp. THAF191c ASM936345v1 V.sp. THAF64 ASM966549v1 V.parahaemolyticus strain 2012AW-0154 ASM967686v1 V.sp. THAF191d ASM976344v1 V.parahaemolyticus strain 2012AW-0353 ASM976380v1 V AARGOS 51 ASM966521v1 V.navarrensis strain 08-2462 ASM308573v1 V.parahaemolyticus strain 20130629002S01 ASM97637 .cidicii strain 2756-81 ASM976411v1 V ASM988389v1 V.parahaemolyticus strain 20151116002-3 2v1 V AS ASM976350v1 V.parahaemolyticus strain 2014V-1125 M304712v1 V .navar ASM976409v1 V.vulnicus strain 06-2410 .vulnicus strarensisin 07-2strai444 ASM220491v1 V.vulnicus strain FORC 037 ASM976316v1 V.parahaemolyticus strain 2015AW-0174 .vulnicus Env1 ASM222426v1 V n 2462-79 ASM976352v1 V.parahaemolyticus strain 2014V-1066 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version postedASM964901v1 V.parahaemolyticus May 10329 10, 2021. The copyright holder for this preprint (which ASM869368v1 V.vulnicus strain FDAARGOS 663 ASM286372v1 V ASM118818v2 V.parahaemolyticus strain FD ASM221513v1 V.vulnicus strain .vulnicusCECT 4999 NBRC 15645 = ATCC 27562 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ASM155851v2 V.vulnicus strain FDAARGOS 119 ASM165377v1 V.vulnicus strain FORC.vulnicus 016 strain FORC 054 ASM976364v1 V.parahaemolyticus strain 2013V-1146 ASM143343v1 V.vulnicus strain FORC 009

AS M974v1 V.vulnicus YJ016 ASM74666v1 V.vulnicus strain 93U204 ASM869374v1ASM976407v1 V.parahaemolyticus V.parahaemolyticus strain FDAARGOS strain 2010V-1106 662 ASM285045v1 V.vulnicus strain VV2014DJH ASM304708v1 V.parahaemolyticus strain S107-1 ASM431964v1 V.vulnicus strain FORC 077

ASM311937v1 V.parahaemolyticus isolate R13 ASM3976v1 V.vulnicus CMCP6 ASM307689v1 V.parahaemolyticus strain R14 ASM966547v1 V.vulnicus strain 2142-77 AS

ASM167524v1M18658v1 V.vulnicus V.vulnicus strain FORC MO6- 01724/O

AS ASM288761v1M354487v1 V.jasicida V.alfacsensis 090810c strain CAIM 1831 ASM143341v1 V.parahaemolyticus strain FORC 004 ASM290665v1 V.hyugaensis strain 090810a ASM718283v1 V.rotiferianus AM7 DNA ASM221439v1 V.rotiferianus strain B64D1 ASM190843v2 V.harveyi isolate QT520 ASM335188v1 V.parahaemolyticus strain PB1937 ASM918474v1 V.harveyi strain WXL538 ASM285029v1 V.harveyi strain 345 ASM988387v1 V.parahaemolyticusASM419451v1 V.parahaemolyticus strain 20160303005-1 strain D3112 ASM966531v1 V.harveyi strain 2011V-1 ASM155843v2 V.harveyi strain FDAARGOS 107 ASM77011v2 V.harveyi strain ATCC 33843 392 MAV rain 2439-01 ASM288765v1 V.owensii strain 051011B ASM369154v1 V.owensii strain V180403 ASM202175v1 V.owensii strain XSBZ03 ASM369150v1 V.owensii strain 1700302 ASM214910v1 V.alginolyticus isolate K04M3 ASM131057v2 V.owensii strain SH14 ASM651779v1 V.parahaemolyticus strain Vb0624 ASM290645v1 V.campbellii strain BoB-90 ASM369148v1 V.campbellii strain 170502 ASM214265v1 V.campbellii strain LA16-V1 ASM196932v1 V.campbellii strain LMB29 ASM976405v1 V.parahaemolyticusASM214908v1 strain 2012AW-0224V.alginolyticus strain K08M3 ASM214005v1 V.campbellii strain 20130629003S01

47361 GUCC niarts snegeirtan.V 1v800861MSA snegeirtan.V niarts GUCC 47361 4M04SD niarts iillebpmac.V 1v852133MSA iillebpmac.V niarts 4M04SD T915 MIAC 02952 CCTA niarts 13651 CRBN = 915 MIAC iillebpmac.V 1v573612MSA ASM211954v1 V.alginolyticusiillebpmac.V strain K04M5MIAC 915 = CRBN 13651 niarts CCTA 02952 MIAC T915 ASM214906v1 V.alginolyticus strain K09K1

1MSA ASM361303v1 V.alginolyticus strain K04M1

ASM361306v1 V.alginolyticus strain K05K4 4464MSA ASM211956v1 V.alginolyticus strain K06K5 ASM211958v1 V.alginolyticus strain K10K4

ASM211950v2 V.alginolyticus strain K01M1

ASM976374v1 V.alginolyticus st

p m ac.V 1v746092 M S A ASM2482v1 V.antiquarius V. vulnificus ASM167974v1 V.alginolyticus strain ZJ-T ASM24138v1 V.sp. EJY3 ASM976308v1 V.alginolyticus strain 2014V-1011 ASM344377v1 V.sp. dhg 164 ASM523936v1 V.neocaledonicus strain CGJ02-2 V. coralliilyticus ASM976384v1 V.alginolyticus strain 2010V-1102 ASM1180143v1 V.alginolyticus strain FA2

C C T A iille b p m a c.V 1 v 0 7 7

ASM1180145v1 V.diabolicus strain FA3

ASM295397v1 V.alginolyticus strain FDAARGOS 114

ASM966543v1 V.alginolyticus strain 2013V-1302

3 5 - B o B n ia rt s iille b ASM35417v2 V.alginolyticusASM295351v1 NBRC 15630 V.alginolyticus = ATCC 17749 strain FDAARGOSASM146973v1 108 V.alginolyticus strain ATCC 33787 6111-AAB

ASM295353v1 V.alginolyticus strain FDAARGOS 110 6 1 1 1- A A B C C T A iille b p m a c.V 1 v 3

ASM295339v1 V.diabolicus strain FDAARGOS 105 ASM295335v1 V.diabolicus strain FDAARGOS 99 ASM295333v1 V.diabolicus strain FDAARGOS 96 ASM168006v1 V.natriegens strain CCUG 16373

ASM168004v1 V.natriegens strain CCUG 16371

ASM145625v1 V.natriegens NBRC 15636 =ATCC 14048 = DSM 759

ASM168002v1 V.natriegens NBRC 15636 =ATCC 14048 = DSM 759 strain ATCC 14048

V. alginolyticus

V. natriegens V. owensii V. harveyi V. campbellii

B Tree scale: 0.1

V. campbellii BoB-90 V. campbellii 170502 V. campbellii LA16-V1 V. campbellii LMB29 V. campbellii 20130629003S01 V. campbellii BoB-53 V. campbellii ATCC BAA-1116 V. campbellii ATCC BAA-1116 V. campbellii CAIM 519/NBRC 15631/ATCC 25920 V. campbellii DS40M4 V. campbellii bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6

A 107 BB120 WT

6 ) 10 ∆qrr1-5

600 qrr1+ 5 10 qrr2+ 104 qrr3+

3 qrr4+ (Lux/OD 10

Bioluminescence qrr5+ 102 0.1 1 ΔluxR OD600 B 107 DS40M4 WT 106 ) Δqrr1-5

5 600 10 qrr1+

104 qrr2+

103 qrr3+

(Lux/OD 102 qrr4+ Bioluminescence 101 qrr5+ 0.1 1 ΔluxR OD600 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 7 A exsB BWT tssC WildWT-type 1000 WT ΔluxR DΔluxRluxR 1 ΔluxR qrr -100 DΔqrr1qrr-15-5 aphA

exsB Δ 1 5

1 tssC Δqrr1-5 10 0.1

1 Relative Relative transcript levels Relative transcript levels 0.1 transcript levels 0.01 0.1 0.1 1.0 3.25 0.1 1.0 3.25 0.1 1.0 3.25 OD OD600 600 OD600 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.019307; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 8 100 A a a

b a E. coli b a a a 10 % Survival 1

WT WT luxO luxR luxO luxR Δ Δ Δ Δ

luxO D47E luxO D47E BB120 DS40M4 B 0.3

a a

0.2

c a a 0.1

(HPA/OD600) b b b

Relative Protease Activity Relative Protease 0.0

WT WT luxO luxR luxO luxR Δ Δ Δ Δ

luxO D47E luxO D47E BB120 DS40M4

C c b 10 b a ) 600 b /OD 1 590 c

(OD a Biofilm Formation a

0.1

WT WT luxO luxR luxO luxR Δ Δ Δ Δ

luxO D47E luxO D47E BB120 DS40M4