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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 Vibrio 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: Quorum sensing in V. campbellii DS40M4
25
26 Keywords: quorum sensing, Vibrio, Vibrio campbellii, 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 bioluminescence, 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 Vibrio harveyi) is a fundamental model
41 strain for studying quorum sensing in vibrios. 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 autoinducer 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 bacteria in which
61 individual cells synthesize and respond to signaling molecules called autoinducers. 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 Vibrionaceae.
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 Vibrio cholerae) 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 ocean 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 200 mM NaCl, 23.14 mM MgCl2, and 4.2 mM KCl). When
578 necessary, strains were supplemented with kanamycin (100 μg/ml), spectinomycin
579 (200 μg/ml), gentamicin (100 μg/ml), or trimethoprim (10 μg/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 50 U/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
788
789 Anetzberger, C., Reiger, M., Fekete, A., Schell, U., Stambrau, N., Plener, L., et al. (2012)
790 Autoinducers Act as Biological Timers in Vibrio harveyi. PLoS One 7:.
791 Bagert, J.D., Van Kessel, J.C., Sweredoski, M.J., Feng, L., Hess, S., Bassler, B.L., and Tirrell,
792 D.A. (2016) Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi. Chem
32 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.
793 Sci 7: 1797–1806.
794 Barrasso, K., Watve, S., Simpson, C.A., Geyman, L.J., van Kessel, J.C., and Ng, W.-L. (2020)
795 Dual-function quorum-sensing systems in bacterial pathogens and symbionts. PLOS
796 Pathog 16: e1008934.
797 Bassler, B.L., Greenberg, E.P., and Stevens, A.M. (1997) Cross-species induction of
798 luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179: 4043 LP –
799 4045.
800 Bassler, B.L., Wright, M., Showalter, R.E., and Silverman, M.R. (1993) Intercellular signalling in
801 Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol
802 Microbiol 9: 773–786.
803 Bassler, B.L., Wright, M., and Silverman, M.R. (1994) Sequence and function of LuxO, a
804 negative regulator of luminescence in Vibrio harveyi. Mol Microbiol 12: 403–412.
805 Bridges, A.A. and Bassler, B.L. (2019) The intragenus and interspecies quorum-sensing
806 autoinducers exert distinct control over Vibrio cholerae biofilm formation and dispersal.
807 PLoS Biol 17: 1–28.
808 Chaparian, R.R., Olney, S.G., Hustmyer, C.M., Rowe-Magnus, D.A., and van Kessel, J.C.
809 (2016) Integration host factor and LuxR synergistically bind DNA to coactivate quorum-
810 sensing genes in Vibrio harveyi. Mol Microbiol 101: 823–840.
811 Chaparian, R.R., Tran, M.L.N., Miller Conrad, L.C., Rusch, D.B., and van Kessel, J.C. (2019)
812 Global H-NS counter-silencing by LuxR activates quorum sensing gene expression.
813 Nucleic Acids Res 1–13.
814 Colston, S.M., Hervey, W.J., Horne, W.C., Haygood, M.G., Petersen, B.D., van Kessel, J.C.,
815 and Vora, G.J. (2019) Complete Genome Sequence of Vibrio campbellii DS40M4 .
816 Microbiol Resour Announc 8: 1–2.
817 Croxatto, A., Chalker, V.J., Lauritz, J., Jass, J., Hardman, A., Williams, P., et al. (2002) VanT, a
818 homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm
33 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.
819 production in Vibrio anguillarum. J Bacteriol 184: 1617–1629.
820 Dalia, A.B. (2018) Natural Cotransformation and Multiplex Genome Editing by Natural
821 Transformation (MuGENT) of Vibrio cholerae. In Vibrio Cholerae: Methods and Protocols.
822 Sikora, A.E. (ed). New York, NY: Springer New York, pp. 53–64.
823 Dalia, Ankur B., Lazinski, D.W., and Camilli, A. (2014) Identification of a membrane-bound
824 transcriptional regulator that links chitin and natural competence in Vibrio cholerae. MBio
825 5:.
826 Dalia, Ankur B, McDonough, E., and Camilli, A. (2014) Multiplex genome editing by natural
827 transformation. Proc Natl Acad Sci 111: 8937–8942.
828 Dalia, T.N., Hayes, C.A., Stolyar, S., Marx, C.J., McKinlay, J.B., and Dalia, A.B. (2017) Multiplex
829 Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio
830 natriegens. ACS Synth Biol 6: 1650–1655.
831 Davidson, C.J., White, A.P., and Surette, M.G. (2008) Evolutionary loss of the rdar morphotype
832 in Salmonella as a result of high mutation rates during laboratory passage. ISME J 2: 293–
833 307.
834 Edgar, R.C. (2004) MUSCLE: A multiple sequence alignment method with reduced time and
835 space complexity. BMC Bioinformatics 5: 1–19.
836 Freeman, J.A. and Bassler, B.L. (1999a) A genetic analysis of the function of LuxO, a two-
837 component response regulator involved in quorum sensing in Vibrio harveyi. Mol Microbiol
838 31: 665–677.
839 Freeman, J.A. and Bassler, B.L. (1999b) Sequence and function of LuxU: A two-component
840 phosphorelay protein that regulates quorum sensing in Vibrio harveyi. J Bacteriol 181:
841 899–906.
842 Freeman, J.A., Lilley, B.N., and Bassler, B.L. (2000) A genetic analysis of the functions of LuxN:
843 A two-component hybrid sensor kinase that regulates quorum sensing in Vibrio harveyi.
844 Mol Microbiol 35: 139–149.
34 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.
845 Gode-Potratz, C.J. and McCarter, L.L. (2011) Quorum sensing and silencing in Vibrio
846 parahaemolyticus. J Bacteriol 193: 4224–4237.
847 Gu, D., Guo, M., Yang, M., Zhang, Y., Zhou, X., and Wang, Q. (2016) A σE-Mediated
848 Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to
849 Thermal Stress Adaptation in Vibrio alginolyticus. PLoS Pathog 12: 1–31.
850 Hachani, A., Lossi, N.S., and Filloux, A. (2013) A visual assay to monitor T6SS-mediated
851 bacterial competition. J Vis Exp e50103.
852 Haygood, M.G., Holt, P.D., and Butler, A. (1993) Aerobactin production by a planktonic marine
853 Vibrio sp. Limnol Oceanogr 38: 1091–1097.
854 Henares, B.M., Higgins, K.E., and Boon, E.M. (2012) Discovery of a nitric oxide responsive
855 quorum sensing circuit in vibrio harveyi. ACS Chem Biol 7: 1331–1336.
856 Henke, J. and Bassler, B. (2004) Quorum sensing regulates type III secretion in Vibrio harveyi
857 and Vibrio parahaemolyticus. J Bacteriol 186: 3794–3805.
858 Henke, J.M. and Bassler, B.L. (2004) Three Parallel Quorum-Sensing Systems Regulate Gene
859 Expression in <em>Vibrio harveyi</em> J Bacteriol 186: 6902 LP – 6914.
860 Hustmyer, C.M., Simpson, C.A., Olney, S.G., Rusch, D.B., Bochman, M.L., and van Kessel, J.C.
861 (2018) Promoter Boundaries for the luxCDABE and betIBA-proXWV Operons in Vibrio
862 harveyi Defined by the Method Rapid Arbitrary PCR Insertion Libraries (RAIL). J Bacteriol
863 200: e00724-17.
864 Joelsson, A.C. and Zhu, J. (2006) LacZ-Based Detection of Acyl-Homoserine Lactone Quorum-
865 Sensing Signals. Curr Protoc Microbiol 3: 1C.2.1-1C.2.9.
866 Jung, S.A., Chapman, C.A., and Ng, W.L. (2015) Quadruple Quorum-Sensing Inputs Control
867 Vibrio cholerae Virulence and Maintain System Robustness. PLoS Pathog 11: 1–19.
868 Jung, S.A., Hawver, L.A., and Ng, W.L. (2016) Parallel quorum sensing signaling pathways in
869 Vibrio cholerae. Curr Genet 62: 255–260.
870 van Kessel, J.C., Rutherford, S.T., Shao, Y., Utria, A.F., and Bassler, B.L. (2013) Individual and
35 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.
871 combined roles of the master regulators apha and luxr in control of the Vibrio harveyi
872 quorum-sensing regulon. J Bacteriol 195: 436–443.
873 Kim, B.S., Jang, S.Y., Bang, Y., Hwang, J., Koo, Y., Jang, K.K., et al. (2018) QStatin, a
874 Selective Inhibitor of Quorum Sensing in Vibrio Species. MBio 9: e02262-17.
875 Kim, S.M., Park, J.H., Lee, H.S., Kim, W. Bin, Ryu, J.M., Han, H.J., and Choi, S.H. (2013) LuxR
876 Homologue SmcR Is Essential for Vibrio vulnificus Pathogenesis and Biofilm Detachment,
877 and Its Expression is Induced by Host Cells. Infect Immun 81: 3721 LP – 3730.
878 Lenz, D.H., Miller, M.B., Zhu, J., Kulkarnl, R. V., and Bassler, B.L. (2005) CsrA and three
879 redundant small RNAs regulate quorum sensing in Vibrio cholerae. Mol Microbiol 58:
880 1186–1202.
881 Letunic, I. and Bork, P. (2019) Interactive Tree of Life (iTOL) v4: Recent updates and new
882 developments. Nucleic Acids Res 47: 256–259.
883 Li, W. and Godzik, A. (2006) Cd-hit: A fast program for clustering and comparing large sets of
884 protein or nucleotide sequences. Bioinformatics 22: 1658–1659.
885 Lin, B., Wang, Z., Malanoski, A.P., O’Grady, E.A., Wimpee, C.F., Vuddhakul, V., et al. (2010)
886 Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-
887 1116 and HY01 as Vibrio campbellii. Environ Microbiol Rep 2: 81–89.
888 Liu, Z., Hsiao, A., Joelsson, A., and Zhu, J. (2006) The transcriptional regulator VqmA increases
889 expression of the quorum-sensing activator HapR in Vibrio cholerae. J Bacteriol 188:
890 2446–2453.
891 McLoon, A.L., Guttenplan, S.B., Kearns, D.B., Kolter, R., and Losick, R. (2011) Tracing the
892 domestication of a biofilm-forming bacterium. J Bacteriol 193: 2027–2034.
893 Meibom, K.L., Blokesch, M., Dolganov, N.A., Wu, C.Y., and Schoolnik, G.K. (2005)
894 Microbiology: Chitin induces natural competence in vibrio cholerae. Science (80- ) 310:
895 1824–1827.
896 Meighen, E.A. (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev 55: 123–
36 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.
897 142.
898 Miyata, S.T., Kitaoka, M., Wieteska, L., Frech, C., Chen, N., and Pukatzki, S. (2010) The Vibrio
899 Cholerae Type VI Secretion System: Evaluating its Role in the Human Disease Cholera.
900 Front Microbiol 1: 117.
901 Mok, K.C., Wingreen, N.S., and Bassler, B.L. (2003) Vibrio harveyi quorum sensing: A
902 coincidence detector for two autoinducers controls gene expression. EMBO J 22: 870–881.
903 Ng, W.L., Perez, L.J., Wei, Y., Kraml, C., Semmelhack, M.F., and Bassler, B.L. (2011) Signal
904 production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems. Mol
905 Microbiol 79: 1407–1417.
906 O’Grady, E.A. and Wimpee, C.F. (2008) Mutations in the lux operon of natural dark mutants in
907 the genus vibrio. Appl Environ Microbiol 74: 61–66.
908 O’Toole, G.A. (2010) Microtiter dish Biofilm formation assay. J Vis Exp 10–11.
909 Papenfort, K., Förstner, K.U., Cong, J.P., Sharma, C.M., and Bassler, B.L. (2015) Differential
910 RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm
911 formation. Proc Natl Acad Sci U S A 112: E766–E775.
912 Papenfort, K., Silpe, J.E., Schramma, K.R., Cong, J.-P., Seyedsayamdost, M.R., and Bassler,
913 B.L. (2017) A Vibrio cholerae autoinducer–receptor pair that controls biofilm formation. Nat
914 Chem Biol 13: 551–557.
915 Rutherford, S.T. and Bassler, B.L. (2012) Bacterial quorum sensing: Its role in virulence and
916 possibilities for its control. Cold Spring Harb Perspect Med 2: 1–25.
917 Rutherford, S.T., van Kessel, J.C., Shao, Y., and Bassler, B.L. (2011) AphA and LuxR/HapR
918 reciprocally control quorum sensing in vibrios. Genes Dev 25: 397–408.
919 Schauder, S., Shokat, K., Surette, M.G., and Bassler, B.L. (2001) The LuxS family of bacterial
920 autoinducers: Biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 41:
921 463–476.
922 Seemann, T. (2014) Prokka: Rapid prokaryotic genome annotation. Bioinformatics 30: 2068–
37 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.
923 2069.
924 Seppey, M., Manni, M., and Zdobnov, E.M. (2019) BUSCO: Assessing Genome Assembly and
925 Annotation Completeness. Methods Mol Biol 1962: 227–245.
926 Shao, Y. and Bassler, B.L. (2014) Quorum regulatory small RNAs repress type VI secretion in
927 Vibrio cholerae. Mol Microbiol 92: 921–930.
928 Simpson, C.A., Podicheti, R., Rusch, D.B., Dalia, A.B., and van Kessel, J.C. (2019) Diversity in
929 Natural Transformation Frequencies and Regulation across Vibrio Species. MBio 10: 1–16.
930 Stamatakis, A. (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of
931 large phylogenies. Bioinformatics 30: 1312–1313.
932 Steensels, J., Gallone, B., Voordeckers, K., and Verstrepen, K.J. (2019) Domestication of
933 Industrial Microbes. Curr Biol 29: R381–R393.
934 Sun, Y., Bernardy, E.E., Hammer, B.K., and Miyashiro, T. (2013) Competence and natural
935 transformation in vibrios. Mol Microbiol 89: 583–595.
936 Thibodeau, S.A., Fang, R., and Joung, J.K. (2004) High-throughput β-galactosidase assay for
937 bacterial cell-based reporter systems. Biotechniques 36: 410–415.
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
38 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.
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