bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Intra-species signaling between aeruginosa genotypes increases

production of quorum sensing controlled virulence factors

Dallas L. Mould, Nico J. Botelho, and Deborah A. Hogan

Geisel School of Medicine at Dartmouth, Hanover, NH 03755

Running title: Mixed genotype co-culture promotes production

*To whom correspondence should be addressed

Department of Microbiology and Immunology,

Geisel School of Medicine at Dartmouth

Rm 208 Vail Building, Hanover, NH 03755

E-mail: [email protected]

Tel: (603) 650-1252 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 Abstract

2 The opportunistic damages hosts through the

3 production of diverse secreted products, many of which are regulated by quorum

4 sensing. The lasR , which encodes a central quorum-sensing regulator, is

5 frequently mutated, and loss of LasR function impairs the activity of downstream

6 regulators RhlR and PqsR. We found that in diverse models, the presence of P.

7 aeruginosa wild type causes LasR loss-of-function strains to hyperproduce RhlR/I-

8 regulated antagonistic factors, and autoinducer production by the wild type is not

9 required for this effect. We uncovered a reciprocal interaction between isogenic wild

10 type and lasR mutant pairs wherein the -scavenging pyochelin,

11 specifically produced by the lasR mutant, induces citrate release and cross-feeding from

12 wild type. Citrate stimulates RhlR signaling and RhlI levels in LasR- but not in LasR+

13 strains, and the interactions occur in diverse media. Co-culture interactions between

14 strains that differ by the function of a single transcription factor may explain worse

15 outcomes associated with mixtures of LasR+ and LasR loss-of-function strains. More

16 broadly, this report illustrates how interactions within a genotypically diverse population,

17 similar to those that frequently develop in natural settings, can promote net virulence

18 factor production. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

19 Introduction

20 Genetic diversity frequently arises and persists within clonally-derived bacterial

21 and fungal populations in chronic infections and healthy microbiomes, and recent data

22 highlight that this heterogeneity can pose challenges to clearance and treatment (1-3).

23 Genotypic and phenotypic complexity has been particularly well-documented in the

24 chronic lung infections associated with the genetic disease cystic fibrosis (CF), and

25 these studies have convincingly demonstrated that within a species, a common set of

26 is under selection across strains and hosts (4-10).

27 P. aeruginosa loss-of-function mutations in lasR (LasR-) are very commonly

28 found in CF isolates, strains from acute infections, and from environmental sources (11-

29 15). Although infection models often show that lasR loss-of-function mutants have

30 reduced virulence compared to strains with functional LasR (LasR+) in several animal

31 models (16, 17), the presence of LasR- strains is correlated with worse disease

32 outcomes in acute and chronic infections (11, 12). There are several possible

33 explanations for this apparent contradiction. Loss of lasR function confers some fitness

34 advantages including altered catabolic profiles (18) and enhanced growth in low oxygen

35 (19, 20). Further, some LasR- strains exhibit rewired regulation of quorum sensing

36 (QS)-controlled exoproducts (21) and LasR- strains can activate QS signaling in

37 response to products from other species (22) or in specific culture conditions (23, 24).

38 LasR- strains are also frequently found among LasR+ P. aeruginosa strains where

39 exoproducts can be shared or signal cross-feeding can occur (13).

40 P. aeruginosa LasR participates in the regulation of QS in conjunction with two

41 transcription factors: RhlR and PqsR (MvfR). Each of these three regulators has one or bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

42 more autoinducer : 3-oxo-C12-homoserine lactone (3OC12HSL) for LasR, C4-

43 homoserine lactone (C4HSL) for RhlR, and hydroxyalkylquinolones (Pseudomonas

44 Quinolone Signal (PQS) and hydroxy-heptyl quinolone (HHQ)) for PqsR (25). In the

45 regulatory networks described in the widely-used P. aeruginosa model strains, LasR is

46 an upstream regulator of RhlR and PqsR signaling, and together these regulators

47 control the expression of a suite of genes associated with virulence including -

48 active small molecule phenazines (26-28), cyanide (29), (30-33), and

49 rhamnolipid surfactants important for surface motility, dispersal, and host

50 damage (34-36).

51 Other traits that are often heterogeneous across P. aeruginosa isolates relate to

52 strategies for iron acquisition. P. aeruginosa procures iron through the use of

53 , including pyochelin (37-39) and pyoverdine (40), from heme or through a

54 direct iron uptake system (41-43). It is common to encounter P. aeruginosa strains with

55 loss of function mutations in genes encoding pyoverdine, the high affinity siderophore,

56 but genes associated with use of the pyochelin siderophore, heme utilization, and the

57 direct iron uptake are generally intact (44-46). Iron limitation can reduce the function of

58 pathways that require abundant iron including the TCA cycle (47), and when iron access

59 is low, Pseudomonas spp. release partially oxidized metabolic intermediates that

60 accumulate at iron requiring steps (48, 49). Many other species are known to release

61 partially-oxidized metabolic intermediates upon iron limitation (48, 50-52).

62 Here, we show that mixtures of P. aeruginosa LasR- and LasR+ strains have

63 enhanced production of QS-controlled factors across medium types, culture conditions,

64 and strain backgrounds, and that this is due to activation of RhlR likely through bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

65 increased RhlI stability in LasR- strains. Our transcriptomic, genetic and biochemical

66 studies led us to uncover a set of interactions in which ∆lasR production of the

67 siderophore pyochelin was necessary for the activation of RhlR in ∆lasR when co-

68 cultured with the LasR+ wild type (WT). Our studies suggested that ∆lasR responds to

69 citrate in co-culture, and we found that pyochelin is sufficient to stimulate citrate release

70 preferentially by WT cells. Citrate increased RhlI protein levels and RhlR activity in

71 ∆lasR cells but not in the wild type. Together, these data highlight a set of complex,

72 small molecule interactions between strains that differ by a single mutation and lead to

73 increased production of exoproducts known to cause host damage.

74

75 RESULTS

76 P. aeruginosa ∆lasR over-produces pyocyanin in co-culture with wild type.

77 We observed that mixtures of P. aeruginosa LasR+ and LasR- strains had high

78 levels of total pyocyanin, a secreted, blue-pigmented phenazine. As shown in spot

79 colony cultures of P. aeruginosa strain PA14 wild type (WT), ∆lasR, and WT / ∆lasR co-

80 cultures, the strain mixture showed increased blue pigmentation (Fig. 1A) and a

81 significant 2-fold induction of pyocyanin relative to either strain alone (Fig. 1B).

82 Phenazine-deficient derivatives, ∆phz (∆phzA1-G1∆phzA2-G2) and ∆lasR∆phz

83 (∆lasR∆phzC1C2) were also included, and as expected, ∆phz and ∆lasR∆phz, showed

84 no blue colony pigmentation (Fig. 1A) and no pyocyanin signal was evident above

85 background levels (Fig. 1B). The higher levels of pyocyanin in WT / ∆lasR co-cultures

86 relative to single-strain cultures was also observed on pH-buffered M63 medium and on

87 artificial sputum medium indicating that the phenomenon occurred in diverse conditions bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

88 (Fig. S1A). Co-cultures of clonally-derived LasR+ and LasR- clinical isolates collected

89 from single respiratory sputum samples from chronically-infected individuals with cystic

90 fibrosis also had increased production of a pyocyanin when LasR+ and LasR- strains

91 were grown together relative to mono-culture levels (Fig. S1B, C).

92 To assess individual strain contributions to increased pyocyanin in WT / ∆lasR

93 co-cultures, we measured pyocyanin levels in co-cultures wherein one strain was

94 replaced with its phenazine-deficient derivative. When ∆lasR was cultured with the

95 phenazine biosynthesis mutant ∆phz (∆phz / ∆lasR), we still observed increased blue

96 pigmentation (Fig. 1A) and total pyocyanin levels were higher relative to monocultures

97 (Fig. 1B). Surprisingly, pyocyanin levels were statistically higher in ∆phz / ∆lasR co-

98 cultures relative to WT / ∆lasR (Fig. 1B). In contrast, WT / ∆lasR∆phz co-cultures did

99 not display the high pyocyanin phenotype, and pyocyanin concentrations in these co-

100 cultures were more similar to those in WT monocultures (Fig. 1A, B). Pyocyanin levels

101 in the WT / ∆lasR∆phz co-cultures were below the limit of detection as there was no

102 difference in pyocyanin levels when compared with ∆phz / ∆lasR∆phz co-cultures or

103 ∆phz and ∆lasR∆phz monocultures. Collectively, these data suggested that WT / ∆lasR

104 co-cultures produced more pyocyanin than either monoculture alone, and that

105 pyocyanin was contributed by the ∆lasR strain.

106 The higher levels of pyocyanin in WT / ∆lasR co-cultures relative to each strain

107 grown alone was not dependent on the initial ratio of WT to ∆lasR (Fig. 1C). We saw

108 increased co-culture colony pigmentation when the initial proportion of the WT was at

109 0.2, 0.3, 0.5, 0.7, and 0.8 of the initial inoculum, with the balance comprised of ∆lasR

110 (Fig. 1C). No increase in pyocyanin was observed at any ratio when WT was co- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

111 cultured with the ∆lasR complemented derivative (∆lasR + lasR) indicating that the

112 phenomenon was dependent on the lasR mutation (Fig. 1C). To assess the relative

113 abundances of WT and ∆lasR in co-culture, we competed each strain against a

114 neutrally-tagged WT strain (PA14 att::lacZ). We found that ∆lasR increased in

115 proportion after 16 h growth in colony regardless of the starting proportion

116 whereas the proportions of tagged and untagged WT remained stable (Fig. 1D). We

117 have previously shown that Anr activity is higher in ∆lasR strains and contributes to

118 ∆lasR competitive fitness against WT P. aeruginosa in colony biofilms (53, 54), but Anr

119 was not required for co-culture pyocyanin production by ∆lasR (Fig. S2).

120 Pyocyanin is a product regulated by quorum sensing (QS) through the

121 transcription factors LasR, RhlR, and PqsR (55-57), and because of the cell-density

122 dependent quorum-sensing regulation, it was important to assess the population size in

123 co-cultures relative to monocultures. Total CFUs did not increase in WT / ∆lasR mixed

124 cultures relative to either strain alone (Fig. S1D). Instead we found that WT / ∆lasR co-

125 cultures had fewer CFUs when compared to WT monocultures on LB (Fig. S1D). Taken

126 together, these data suggested altered behavior, rather than cell number, contributed to

127 the increased phenazine profile of LasR- strains.

128

129 P. aeruginosa WT induces ∆lasR RhlR/I-dependent signaling independent of WT

130 produced autoinducer.

131 In the canonical QS pathway, LasR regulates both PqsR and RhlR, and mutants

132 lacking either of these regulators in a WT background have impaired pyocyanin

133 production (58, 59). Both pqsR and rhlR were required for the production of pyocyanin bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

134 by ∆lasR in co-culture with WT (Fig. 2A). To determine if co-culture increased RhlR- or

135 PqsR-dependent signaling in ∆lasR strains upon co-culture with the WT, we fused lacZ

136 to the promoters of rhlI and pqsA, which provide readouts of the activity of each

137 respective regulator (25). To examine the effects of WT on ∆lasR QS, we examined the

138 interactions between WT and ∆lasR in single-cell-derived colonies. To do so,

139 suspensions containing ~50 cells of WT with ~50 cells of either ∆lasR PrhlI–lacZ or

140 ∆lasR PpqsA – lacZ were spread on LB agar with the colorimetric -galactosidase

141 substrate X-gal. Intercolony distances and -galactosidase activity in ∆lasR strains were

142 measured. We found that PrhlI – lacZ activity was inversely correlated with distance to

143 a WT colony, with a significant increase in ∆lasR PrhlI – lacZ activity (Fig. 2B). Pearson

144 correlation analyses showed that 54% of the variability in ∆lasR PrhlI – lacZ activity

145 could be explained by changes in the distance to a WT colony (p-value ≤ 0.0001). The

146 increased activity due to activation of PrhlI – lacZ in ∆lasR was not observed in

147 ∆lasR∆rhlR, and close proximity to another ∆lasR PrhlI – lacZ colony did not affect

148 promoter activity (Fig. 2B, inset). Because RhlR activity is canonically stimulated by

149 C4HSL (which is synthesized by RhlI) and PrhlI – lacZ is stimulated in ∆lasR by

150 proximity to WT, we examined the role of RhlI in the ∆lasR response. We observed that

151 a ∆lasR∆rhlI strain was greatly impaired in the induction of pyocyanin upon co-culture

152 with the WT (Fig. 2A) which suggests activation of RhlR involved C4HSL synthesis

153 within ∆lasR strains. Although PqsR was required in ∆lasR for co-culture pyocyanin

154 production, there was no significant correlation with proximity to WT for ∆lasR PpqsA –

155 lacZ activity (Fig. 2B). Collectively, these data indicated that a diffusible factor produced bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

156 by WT stimulated RhlR-dependent signaling in ∆lasR to induce downstream production

157 of RhlR and PqsR dependent factors.

158 Because C4HSL is readily diffusible and likely produced by WT cells, we tested

159 the hypothesis that C4HSL or other acylhomoserine lactones produced by WT were

160 necessary to induce RhlR-dependent increases in rhlI promoter activity in ∆lasR co-

161 cultured with WT. To test this hypothesis, we co-cultured ∆lasR with ∆rhlI or ∆lasI∆rhlI,

162 which lacks both acylhomoserine lactone synthases. Surprisingly, we found that like WT

163 / ∆lasR co-cultures, ∆rhlI / ∆lasR co-cultures had higher levels of pyocyanin production

164 relative to mono-cultures (Fig. 2C). Likewise, ∆lasI∆rhlI / ∆lasR had higher levels of

165 pyocyanin production relative to mono-cultures though the interaction was delayed by

166 ~24 h hours relative to the WT / ∆lasR co-cultures (Fig. 2C). Consistent with the data

167 above which indicated that pqsA was not induced upon co-culture with the WT, ∆lasR co-

168 cultures of the PQS-deficient strain ∆pqsA had high pyocyanin colony pigmentation

169 relative to monoculture levels after 24 h of extended incubation (Fig. 2C). The

170 dispensability of WT-produced quorum sensing signals implicated a novel signaling

171 interaction in co-culture-dependent activation of ∆lasR RhlR-dependent signaling.

172 To assess whether RhlR activity in ∆lasR strains grown in the presence of WT

173 was sufficient to elicit other RhlR/I-controlled phenotypes in addition to pyocyanin

174 production, we tested whether co-culture with the WT also enhanced other RhlR-

175 regulated processes in ∆lasR. RhlR regulates the production of rhamnolipid surfactants

176 that are important for surface-associated motility known as swarming (60). While the

177 rhamnolipid-defective mutant ∆rhlA, ∆lasR, and the ∆lasR∆rhlR strains were not able to

178 swarm, we observed increased swarming in co-cultures of ∆lasR with ∆rhlA. The bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

179 phenomenon was dependent on RhlR as the ∆lasR∆rhlR / ∆rhlA co-cultures did not

180 swarm (Fig. S3). Altogether, these data suggested broad RhlR-mediated quorum

181 sensing activation in LasR- strains grown in co-culture with LasR+ P. aeruginosa.

182

183 Pyochelin production by ∆lasR is required for co-culture interactions.

184 With evidence indicating that the induction of RhlR activity in ∆lasR in both mixed

185 strain spot colonies and adjacent colonies by WT occurred through a mechanism that

186 does not require acylhomoserine lactone cross-feeding, we sought to gain further

187 insight into the mechanisms that underlie the co-culture interactions. We investigated

188 the transcriptomes of the lasR mutant when co-cultured with either WT or itself via RNA

189 sequencing analysis. We grew ∆lasR colony biofilms on LB physically separated from a

190 lawn of either ∆lasR or WT by two 0.22 µm filters to prevent mixing of genotypes while

191 allowing for the passage of small molecules. RNA was extracted from cells within the

192 ∆lasR colony biofilms grown on the topmost filter for 16 h in order to examine ∆lasR

193 transcriptional profiles (Fig. 3A). One hundred and ninety-nine genes in ∆lasR were

194 higher and 198 genes were lower by a log 2 (fold change) ≥ 1 with an FDR corrected

195 p-value < 0.05 in co-culture with WT compared to ∆lasR alone (Supplemental Table 1).

196 GO term analyses through PantherDB indicated that the upregulated gene set was

197 significantly enriched in two pathways related to siderophore biosynthesis: pyoverdine

198 biosynthetic process and salicylic acid biosynthetic process (an upstream precursor of

199 pyochelin) with ~44 and ~77-fold enrichment, respectively (p-values < 0.005). Twenty-

200 eight out of the 33 genes in the pyochelin and pyoverdine siderophore biosynthesis- and

201 acquisition-related GO pathways were significantly upregulated in ∆lasR upon co- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

202 culture with WT (Fig. 3B). Other genes regulated by the low iron response were also

203 differentially expressed including the has genes involved in heme uptake and antABC

204 genes (Table S1). While we observed stimulation of rhlI promoter activity and increased

205 production of RhlR regulated products, we did not see a pattern indicative of RhlR

206 activation at this early time point (Table S1), and this point is discussed in more detail

207 with respect to additional data shown below.

208 In light of the upregulation of siderophore biosynthesis genes in ∆lasR co-

209 cultured with WT but not with itself, we qualitatively examined production of fluorescent

210 pyoverdine and pyochelin siderophores in monocultures and co-cultures. To determine

211 the contribution of both pyoverdine and pyochelin to , genes required for

212 pyoverdine biosynthesis (∆pvdA), pyochelin biosynthesis (∆pchE) or both pathways

213 (∆pvdA∆pchE) were disrupted in the lasR mutant (Fig. 3C). Increased fluorescence

214 attributable to both pyoverdine and pyochelin in co-culture was due to siderophore

215 production by ∆lasR strains, consistent with the RNA-Seq data, as the increased

216 siderophore production in WT/ ∆lasR co-cultures was lost for co-cultures in which the

217 ∆lasR strains were replaced with ∆lasR∆pvdA, ∆lasR∆pchE, or ∆lasR∆pvdA∆pchE.

218 While WT and the pyoverdine-deficient derivative ∆lasR∆pvdA (i.e. WT / ∆lasR∆pvdA)

219 showed increased pyocyanin production relative to either monoculture, ∆lasR∆pchE and

220 ∆lasR∆pvdA∆pchE did not support the overproduction of pyocyanin in co-culture with

221 WT (Fig. 3D). The decrease in ∆lasR-derived pyocyanin was not due to decreased

222 fitness as disruption of pvdA and pchE in ∆lasR individually had no effect on the final

223 proportions; in contrast, ∆lasR∆pvdA∆pchE had a significant defect in competitive

224 fitness compared to the ∆lasR parental strain (Fig. S4). These data suggested that bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

225 pyochelin played a role in the co-culture interaction. To test this model, we

226 complemented the ∆lasR∆pchE in co-culture with supernatant extracts from cultures of

227 ∆pvdA which only produced pyochelin, or ∆pvdA∆pchE which produced neither

228 siderophore (Fig. S5A for supernatant absorption spectra). The two supernatants were

229 analyzed using the chrome azurol S (CAS) assay (61) to confirm that chelator activity

230 was present in the ∆pvdA supernatant extracts but not in extracts from ∆pvdA∆pchE

231 cultures (Fig. S5B). The amendment of the medium with PCH-containing extracts, but

232 not siderophore-free extracts, restored co-culture pyocyanin production in ∆pvdA∆pchE

233 / ∆lasR∆pchE co-cultures (Fig. 3E) lending further support for the model that pyochelin

234 was required for co-culture interactions. Consistent with the requirement of the

235 siderophore for the stimulation of pyocyanin in WT / ∆lasR co-cultures, iron

236 supplementation of the LB medium suppressed siderophore production and the

237 stimulation of pyocyanin production (Fig. 3F).

238

239 Evidence for WT - ∆lasR interactions involving citrate secreted by the WT in

240 response to pyochelin.

241 Many of the genes with higher transcript levels in ∆lasR upon co-culture with WT

242 have annotations related to organic acids such as anthranilate and citrate

243 (Supplemental Table 1, Fig. S6A). First, anthranilate was examined as a candidate

244 molecule that was produced by the WT and that induced RhlR-dependent phenotypes

245 in ∆lasR. Co-cultures of ∆lasR with anthranilate synthase mutant ∆phnAB did not alter

246 high phenazine production compared to WT / ∆lasR (Fig. S6B). Additionally,

247 anthranilate supplementation of the medium did not alter ∆lasR phenazine production at bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

248 any concentration tested up to ~15 mM (Fig. S6C). Furthermore, co-cultures of ∆lasR

249 and the PQS deficient strain ∆pqsA, which accumulates anthranilate as an upstream

250 precursor (62), did not show enhanced stimulation of RhlR-dependent phenotypes

251 beyond WT / ∆lasR levels (Fig. 2C).

252 We then tested the ability of citrate to induce RhlR-dependent phenotypes in

253 ∆lasR in light of the observation that twenty percent of the most strongly differentially

254 expressed genes (log 2 (fold change) ≥ 2 with an FDR-corrected p-value < 0.05) were

255 implicated with citrate sensing, transport, catabolism, and anabolism as annotated by

256 UNIPROT and pseudomonas.com (Fig. 4A). Among the genes more highly induced in

257 ∆lasR / WT co-cultures compared to ∆lasR cultured with itself were genes annotated as

258 playing a role in citrate sensing or metabolism, with the most strongly upregulated

259 genes were involved in citrate catabolism and sensing and transport (Fig. 4B).

260 In light of the findings that ∆lasR strains induced low iron responsive genes when

261 grown near the WT but not itself, that ∆lasR pyochelin production was necessary for co-

262 culture interactions that lead to increased pyocyanin and rhlI promoter activity, that

263 citrate sensing and catabolism genes were induced in ∆lasR by the presence of the WT,

264 and the knowledge that numerous microbes, including Pseudomonas putida, secrete

265 citrate and other organic acids when iron limited (49, 50, 63, 64), we measured citrate in

266 the supernatants of WT and ∆lasR LB cultures. Citrate concentrations were significantly

267 higher in WT supernatants (Fig. 4C). Furthermore, amendment of the LB medium with

268 extracts containing 50 µM pyochelin increased extracellular citrate concentrations by

269 about 2-fold in WT cultures compared to control cultures containing extracts lacking

270 pyoverdine and pyochelin, with a much smaller stimulation in ∆lasR cultures (Fig. 4C). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

271 The difference in the effects of pyochelin on citrate levels in culture supernatants for WT

272 and ∆lasR was significant using data from four independent experiments (p<0.05). This

273 suggested WT-produced citrate was possibly involved in WT / ∆lasR co-culture

274 interactions, and that its increased release was enhanced by ∆lasR-produced pyochelin.

275

276 Citrate induces RhlR activity in ∆lasR and RhlI levels in a ClpX-

277 dependent manner.

278 To determine if citrate was sufficient to stimulate RhlR activity in ∆lasR, we

279 analyzed its effects on both rhlI promoter fusion activity and RhlI protein levels. We

280 found that citrate increased rhlI promoter activity in ∆lasR and that its effects were

281 dependent on the presence of RhlR (Fig. 5A). In contrast, the inclusion of citrate in the

282 medium caused a small but significant reduction in WT PrhlI activity compared to LB

283 control (Fig. 5A).

284 To determine if RhlI protein levels were influenced by citrate, we utilized an

285 arabinose-inducible rhlI-HA construct to assess RhlI protein levels and stability of RhlI-

286 HA in the absence and presence of citrate. RhlI-HA was functional as swarming defects

287 of ∆rhlI were complemented upon expression of RhlI-HA but not by the empty vector

288 (Fig. 5B, inset). RhlI-HA protein levels were 3-fold higher in ∆lasR upon citrate

289 supplementation relative to controls (Fig. 5B). Consistent with the absence of an

290 increase in rhlI promoter activity in WT strains (Fig. 5A), RhlI-HA protein levels were not

291 higher with citrate in the ∆lasR complemented strain (∆lasR + lasR) (Fig. 5B). The

292 differential responses to citrate were also observed in LasR- and LasR+ pairs of clinical

293 isolates (CIs). LasR- CIs from acute (strain 388D) and chronic (strains DH2415) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

294 infections had RhlI-HA levels 1.4- and 1.7-fold higher, respectively, in the presence of

295 citrate (Fig. 5C), whereas alterations in RhlI-HA protein levels in LasR+ CIs from acute

296 (550A) or chronic (DH2417) infections was not observed (Fig. 5D). Through this work,

297 we successfully identified citrate as a molecule in co-culture that specifically promoted

298 RhlI protein levels in LasR- strains, but not LasR+ strains, by a mechanism other than

299 transcriptional control. In order to identify transporters that could be involved in the

300 ∆lasR response to citrate, we deleted two organic acid transporters: dctA (65) and

301 PA14_51300 (66) in the ∆lasR background. The dctA gene was deleted in both a ∆lasR

302 and ∆lasR∆rhlR mutant. We found that the ∆lasR∆dctA strain still showed induction of

303 pyocyanin when co-cultured with the WT and that induction was dependent on RhlR

304 (Fig S7A). Similar results were obtained with the ∆lasR∆PA14_51300 (Fig S7B)

305 suggesting that these transporters were not required for the interaction perhaps due to

306 redundant functions of other proteins or the involvement of other import mechanisms.

307 The temporal pattern suggested RhlI protein induction preceded signal

308 amplification via the positive feedback loop of the quorum sensing transcriptional

309 network. This would be consistent with a primary effect on post transcriptional

310 modulation of RhlI-mediated RhlR activity. To begin to unravel the mechanisms by

311 which citrate promoted RhlR/I-dependent signaling and RhlI stability in ∆lasR, we

312 analyzed the role of two proteases previously found to target and degrade RhlI (i.e. Lon

313 and ClpXP) (67). Given knockouts of Lon protease have a less substantial rise in RhlR/I

314 expression in ∆lasR knockouts compared to WT (68), we focused on the role of ClpXP

315 in ∆lasR. We found that citrate induction of RhlI-HA protein levels in ∆lasR relative to

316 the LB control was dependent on the production of ClpX protease (Fig. 5E). More bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

317 specifically, in the absence of ClpX, a protease shown to degrade RhlI (i.e.

318 ∆lasRclpX::TnM), RhlI-HA levels did not increase on LB + citrate relative to LB control,

319 unlike ∆lasR comparator (Fig. 5E). In LB conditions, RhlI-HA levels were 3.20 ± 2.1 fold

320 higher in ∆lasRclpX::TnM compared to ∆lasR, which mirrors the 3-fold induction

321 observed for ∆lasR on LB + citrate. No significant difference in RhlI-HA were observed

322 for ∆lasRclpX::TnM relative to ∆lasR in citrate supplemented conditions (fold change:

323 1.01 ± 0.53). In other words, as previously noted for WT strains, ClpX appeared to

324 degrade RhlI in ∆lasR, and played a role in ∆lasR response to citrate. The distinct

325 responses and mechanisms identified between LasR+ and LasR- strains under iron

326 limitation and exposure to the low-iron associated molecules, citrate and pyochelin,

327 enabled increases in antagonistic factor production beyond monoculture levels as an

328 emergent property of P. aeruginosa intraspecies interactions.

329 In support of the model that induction in RhlR signaling in response to citrate was

330 due to increased RhlI protein, we found that the induction of rhlI promoter activity (Fig.

331 5F) followed the increase in RhlI-HA levels (Fig. 5G) in response to citrate. The

332 stimulation of rhlI promoter activity was greatest for citrate, but modest stimulation was

333 observed for other organic acids including TCA cycle intermediates (succinate, fumarate

334 and malate) and the common fermentation product acetate. In each case, the

335 stimulation of rhlI-promoter activity was accompanied by higher RhlI-HA protein levels in

336 ∆lasR, but not ∆lasR + lasR, aside from succinate (Fig. 5F,G). Together, these data

337 may imply that organic acids, such as citrate, can serve as mediators of co-culture

338 interactions that can activate RhlR activity in LasR- strains.

339 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

340 Discussion

341 In this study, we described an emergent outcome of co-culturing LasR- and

342 LasR+ strains of P. aeruginosa in which their interactions promoted the increased

343 production of toxic exoproducts including pyocyanin and rhamnolipids (see Fig. 6 for

344 model). We determined that, in co-culture, the iron-binding siderophore pyochelin was

345 largely contributed by ∆lasR, and that exogenous pyochelin induced of citrate

346 significantly more strongly in the WT than in ∆lasR. Citrate increased RhlI protein levels

347 and activated RhlR activity only in ∆lasR, but not WT cells (Fig. 6). Western blot

348 analysis of RhlI-HA expressed from a regulated promoter led us to propose that the

349 increase in RhlR signaling is due to decreased degradation of RhlI by ClpXP, a known

350 negative regulator of RhlI (68, 69). The differences in siderophore production, citrate

351 release, and RhlR/I activation between P. aeruginosa LasR+ and LasR- strains in co-

352 culture environments reflect the pronounced differences between strains that drive the

353 reactivation of quorum sensing and enhanced production of secreted factors. Previous

354 studies have shown that LasR- strains increase their production of phenazines in the

355 presence of other species such as Candida albicans (22) and Staphylococcus aureus

356 (see Fig. 3B in (70)) and future work will determine if pyochelin and citrate also

357 participate in these interspecies interactions. Other microbial interactions have been

358 shown to be influenced by iron availability (71-74). Furthermore, the activation of RhlR

359 activity in ∆lasR strains that can occur in late stationary phase cultures (23, 75) may

360 relate to changes in iron or TCA cycle intermediates. While we found that WT

361 production of the diffusible autoinducers 3OC12HSL, C4HSL and PQS were not

362 required for co-culture stimulation, they clearly contributed to the enhanced activation of bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

363 RhlR regulation which is consistent with intercolony QS interactions that have been

364 demonstrated previously (76).

365 The stimulatory relationship between LasR+ and LasR- strains was remarkably

366 stable as it was observed when strains were mixed within single spot colonies (Fig. 1A)

367 and when strains separated by either filters (Fig. 3) or mm distances on an agar plate

368 (Fig. 2B). The LasR- / LasR+ interactions occurred across distinct media (Fig. S1A),

369 among genetically diverse LasR+ and LasR- clinical isolates (Fig. S1B) and over a wide

370 range of relative proportions of each type (Fig. 1C). The consequences of this

371 intraspecies interaction between genotypes may explain the worse outcomes exhibited

372 by patients in which LasR- strains are detected (12), but future studies with data that

373 include genotypes, mono-culture and co-culture phenotypes, and longitudinal outcome

374 data will be required. RhlR plays other important roles in host interactions (77) which

375 may benefit P. aeruginosa LasR- strains. The observation that rhlR mutants are rare in

376 natural P. aeruginosa isolates and that LasR- strains with active RhlR are virulent (21,

377 78) underscores the relevance of this mechanism and highlights the importance of

378 understanding how microbial interactions activate RhlR.

379 As the study of inter- and intra-species interactions progresses, it is becoming

380 increasingly clear that the environment can dictate the outcome of microbial interactions

381 (79). In fact, even the importance of QS regulation for fitness depends on nutrient

382 sources and conditions (80, 81). As ∆lasR-produced pyochelin was a key component of

383 the interaction, and pyochelin production is repressed under conditions of excess iron

384 availability, it was not surprising that the addition of iron to LB medium suppressed the

385 interaction without significantly altering the final colony CFUs or strain ratios relative to bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

386 LB control (Fig. S4). Siderophore-mediated iron uptake is often required in vivo (39, 82,

387 83) due to iron sequestration by host proteins (84-87), thus the in vivo settings could

388 support these interactions. Interestingly pyoverdine, the higher affinity siderophore, was

389 not required for the co-culture response mirroring findings that genes for the

390 biosynthesis of pyoverdine, but not pyochelin, are commonly disrupted in chronic CF

391 clinical isolates (44-46). In the absence of pyoverdine (i.e. ∆lasR∆pvdA), we observed

392 more pyocyanin in co-culture with WT than ∆lasR (Fig. 3D), and we speculate that this

393 is due to increased pyochelin production by ∆pvdA but future studies will be required to

394 test this model. If this is the case, it would be interesting to analyze the outcomes of

395 interactions over gradients of iron and other nutrients. It was interesting to find that in

396 WT / ∆lasR co-culture, heme-related proteins, hasAP, hasS, and hasD, were among the

397 top eight most upregulated genes by ∆lasR because the presence of lasR mutants and

398 heme utilization are both reported biomarkers of disease progression in CF patients (12,

399 88). Co-culture induced lasR mutant phenotypes may link these two correlative

400 observations.

401 Citrate, a TCA intermediate, is released under iron limitation as a result of

402 “overflow metabolism” (48-50, 52) and is also used by P. aeruginosa and other

403 microbes for iron acquisition due to its iron chelating properties (89). The higher

404 siderophore production by ∆lasR and stimulation of ∆lasR siderophore production in co-

405 culture likely reflects different metabolic strategies between the two strains. Ongoing

406 work will investigate the mechanisms that drive differences in metabolism and iron

407 requirements in order to determine how these differences shape microbial and host

408 interactions. It is likely that Crc-mediated catabolite repression is involved in the bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

409 response to citrate and the control of RhlI levels (67, 69). The existence of a mechanism

410 for the induction of RhlR-mediated QS in response to citrate and other TCA cycle

411 intermediates that are secreted when iron is limiting dovetails with reports of increased

412 expression of the P. aeruginosa quorum sensing regulon in low iron in LasR+ cells (90-

413 92). This coordinate regulation may aid in iron acquisition as quorum sensing-controlled

414 phenazines, such as pyocyanin, reduce poorly soluble Fe3+ to Fe2+ and facilitate its

415 uptake via the Feo system (93). Additionally, rhamnolipids have been employed for iron

416 remediation (94, 95) which suggests their surfactant activity may increase P. aeruginosa

417 substrate iron uptake in part through hydroxyalkylquinolone-dependent mechanisms

418 (96).

419 As the presence of heterogeneous genotypes within single species populations

420 becomes increasingly appreciated, it is important to understand how commonly

421 encountered genotypes interact to influence the apparent behavior of the population.

422 Here, we show that inter-genotype interactions lead to increased RhlR signaling in lasR

423 strains; other work shows co-cultures can also influence the survival of other genotypes

424 (97). It is likely that a wide array of such interactions have yet to be uncovered.

425 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

426

427 Methods

428 Strains and Growth Conditions. Bacterial strains used in this study are listed in Table

429 S2. Bacteria were maintained on LB (lysogeny broth) with 1.5% agar. Yeast strains for

430 cloning were maintained on YPD (yeast peptone dextrose) with 2% agar. Where stated,

431 20 mM of indicated metabolite was added to the medium (liquid or molten agar).

432 Planktonic cultures were grown on roller drums at 37°C for P. aeruginosa.

433

434 Plasmid Construction

435 Plasmid constructs for making in-frame deletions, RhlI-HA expression, and for pqsA

436 promoter fusions were constructed using a Saccharomyces cerevisiae recombination

437 technique described previously (98). The RhlI-HA expression vector with the ampicillin

438 cassette (pMQ70) was constructed by amplifying the rhlI gene with primers that added

439 an HA-tag with sites for cloning into pMQ70 (AmpR). All plasmids were sequenced at

440 the Molecular Biology Core at the Geisel School of Medicine at Dartmouth. In frame-

441 deletions and integrated promoter fusions were introduced into P. aeruginosa by

442 conjugation via S17/lambda pir E. coli. Merodiploids were selected by drug resistance

443 and double recombinants were obtained using sucrose counter-selection and genotype

444 screening by PCR. Both RhlI-HA expression vectors were introduced into P. aeruginosa

445 by electroporation. The ∆lasRclpX::TnM was identified from a collection of transposon

446 mutants in the ∆lasR and verified by sequencing.

447

448 Pyocyanin Quantification bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

449 P. aeruginosa strains were grown in a 96-well plate containing 200 µL LB agar per well

450 by inoculation with 5 µL of overnight cultures adjusted to OD600 = 1. After 16 h

451 incubation at 37 C, two agar plugs (with indicated P. aeruginosa mono- or co-cultures)

452 were added to tubes containing chloroform (500 µL), mixed by vortexing for 30 s and

453 then centrifuged for 2 min at 13,000 RPM. The lower chloroform layer (200 µL) was

454 collected into new tubes, and the chloroform extraction was repeated with an additional

455 500 µL of chloroform. The chloroform extracts (400 µL) were acidified with 0.2 N HCl

456 (500 µL) and vortexed for 30 s. The pink aqueous layer containing pyocyanin was

457 diluted 1:2 in 200 mM Tris-HCl (pH 8.0). Relative pyocyanin was measured by reading

458 absorbance at 310 nm relative to media blank extracts. Values were reported per plug.

459 Each condition had at least eight replicates each for two independent experiments.

460

461 Competition Assays

462 Competition assays were performed to determine the relative fitness of P. aeruginosa

463 mutants. Strains to be competed were grown overnight and adjusted to OD600 = 1.

464 Competing strains were combined with PA14att::lacZ strain in a 1:1 ratio, unless

465 otherwise stated. Following 15 s vortex, 5 µL of the combined suspension was spotted

466 on LB agar. After 16 h, colony biofilms (and agar) were cored, placed in 1.5 mL tubes

467 with 500 µL LB, and agitated vigorously for 5 min using a Genie Disruptor (Zymo). This

468 suspension was diluted, spread on LB plates supplemented with 150 µg / mL 5-bromo-

469 4-chloro-3-indolyl-D-galactopyranoside (X-Gal) using glass beads, and incubated at 37

470 °C until blue colonies were visible (~24 h). The number of blue and white colonies per bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

471 plate were counted and the final proportions recorded. Each competition was run in

472 triplicate on 3 separate days.

473

474 Colony Proximity Image analysis

475 Glass beads were used to spread 50 µL of a 1:1 mixture of untagged WT and ∆lasR

476 possessing the indicated promoter fusion to lacZ onto LB plates (2% agar)

477 supplemented with 150 µg / mL 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-

478 Gal). After 16 h incubation at 37 °C, plates were placed at 4 °C for an additional 24 h to

479 allow all ∆lasR colonies to develop blue coloration of various intensities. Plates were

480 imaged on glass sheet to reduce glare using Canon EOS Rebel T6i digital camera. To

481 process images, they were first cropped to remove background area surrounding each

482 plate, converted to 8-bit for particle analysis in ImageJ, and the threshold was

483 determined to count WT and ∆lasR CFU’s, separately. Colony parameters were

484 collected for each individual CFU, including x, y coordinates and area for WT and ∆lasR

485 CFU lists. A simple distance calculation was made between every WT and ∆lasR colony

486 using the x,y coordinates and the minimum distance to a WT colony was plotted for

487 each ∆lasR CFU against its area value, representative of the approximate lacZ intensity.

488

489 Swarming Motility Assays

490 Swarm assays were performed as previously described in (99), with a few

491 modifications. Briefly, M8 medium with 0.5 % agar was poured into 60 x 15 mm plates

492 and allowed to dry at room temperature for 4 h prior to inoculation. LB grown cultures

493 (16 h at 37 °C) were diluted to OD600 = 1 in fresh LB, and co-cultures were mixed such bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

494 that ∆rhlA was at 0.7 proportion of the final cell suspension. Each plate was inoculated

495 with 5 µL of the final cell suspensions and incubated upright for 24 h at 37 °C in an

496 incubated chamber followed by 12 – 16 h at room temperature. Each strain was

497 inoculated in four replicates and assessed on at least three separate days.

498

499 RNA Collection

500 A 200 µL aliquot of optical density normalized (OD600 = 1) cultures of PA14 or PA14

501 ∆lasR from three independent overnights were spread onto LB plates with glass beads

502 and briefly allowed to dry. Two isopore 0.2 µm PC membrane filters were stacked on

503 the lawn (37 mm diameter filter directly on lawn then 25 mm diameter filter on top), and

504 three 15 µL spots of normalized (OD600 = 1) ∆lasR cultures were spotted on the top-

505 most filter. After 16 h incubation at 37 C, the top filter was collected and ∆lasR cells

506 were resuspended in 1 mL LB by 5 min of vigorous shaking on the genie disruptor. Cells

507 were pelleted for 10 min at 13,000 RPM and snap-frozen in ethanol and dry ice for RNA

508 extraction. RNA was extracted according to manufacturer’s protocol with the QIAGEN

509 RNeasy Mini kit and DNase treated twice with Invitrogen Turbo DNA-Free kit. DNase-

510 treated samples were prepared for sequencing with ribodepletion and library

511 preparation in accordance with Illumina protocols. Samples were barcoded and

512 multiplexed in a NextSeq run by the Dartmouth Sequencing Core.

513

514 RNA-Seq Processing

515 Reads were processed using CLC Genomics Workbench wherein reads were trimmed

516 and filtered for quality using default parameters. Reads were aligned to the P. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

517 aeruginosa UCPBB_PA14 genome from www.pseudomonas.com. Results were

518 exported from CLC including total counts, CPM and TPM. EdgeR was used to process

519 differential gene expression (100). Generalized linear models with mixed effect data

520 design matrices were used to calculate fold-change, p-value and FDR. Volcano plots

521 and heatmaps using EdgeR output (log fold-change and -log(p-value)) were made in R

522 (ggplot2 and pheatmap respectively) (101-103). GO term pathway enrichment analysis

523 was carried out using PantherDB (104).

524

525 Accession Number

526 Data for our RNA-Seq analysis of P. aeruginosa ∆lasR grown on ∆lasR or WT in co-

527 culture has been uploaded to the GEO repository (https://www.ncbi.nlm.nih.gov/geo/)

528 with the accession number GSE149385.

529

530 Pyochelin extraction, quantification, and validation

531 Pyochelin was extracted based on the methods of Cox et al. (37). Briefly 50 mL cultures

532 of PA14 ∆pvdA and pyochelin biosynthesis deficient strain PA14 ∆pvdA∆pchE (negative

533 control) were grown in Chelex-treated (i.e. media treated for > 2 h with 0.5 g Chelex resin

534 per 10 mL media concentrate, followed by centrifugation and filtration with 0.22 µm filter

535 unit) LB for 16 h. Cultures were pelleted for 15 min at > 5000 RPM, and the supernatant

536 was passed through a 0.22 µm filter unit. The cell-free supernatant was acidified to pH 2

537 with 10 N HCl. For extraction, 5 mL ethyl acetate was added per 50 mL acidified solution

538 in a separatory funnel. The top ethyl acetate layer was concentrated using a speedvac

539 and quantified in 50 / 50 methanol:dH2O in a 1 mm quartz cuvette at 313 nm. Absorbance bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

540 was checked from 200 - 600 nm for expected peak profile. For some extractions, a 5 µL

541 aliquot of ∆pvdA extract in 50:50 methanol:dH2O was viewed under light for

542 expected fluorescence relative to ∆pvdA∆pchE extract that dissipated upon 10 µM FeSO4

543 supplementation. The concentration was determined using the molar extinction

544 coefficient at 313 nm in 50 / 50 methanol:dH2O in a 1 mm quartz cuvette (37). Upon

545 quantification, concentrated extract was lyophilized using rotovap to yellow resin, and

546 used within 2 days of initial extraction by resuspension in LB for supplementation. As

547 validation of biological activity, 10 µL of extract was spotted along with EDTA chelator as

548 control on CAS agar prepared as described in.

549

550 Citrate Quantification

551 Citrate was quantified from cell-free supernatant of 5 mL LB-grown cultures inoculated

552 from single colonies. Cultures were grown in quadruplicate and incubated on a roller

553 drum for 16 h. OD 600 nm was recorded, and cultures were pelleted for 15 min at > 5,000

554 RPM. The supernatant was passed through a 0.2 µm syringe filter unit. Citrate in the

555 filtered supernatant was quantified according to manufacturer’s “manual assay” protocol

556 (Megazyme) in ½ reactions. The extinction coefficient at 340 nm in a quartz cuvette was

557 used to quantify concentration of citrate relative to OD 600 at the end of the 5 min

558 enzymatic reaction. Citrate standard and blank media conditions were included in every

559 assay. The average for 4 replicates for each experiment was reported across 3 - 4

560 independent days.

561

562 Beta galactosidase assays bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

563 Cells with a promoter fusion to lacZ - GFP integrated at the att locus were grown in 5 mL

564 cultures of LB at 37°C for 16 h. The cultures were diluted to a starting OD 600 of 1 and 5

565 µL were spotted onto LB agar plates ± 20 mM pH 7 citrate (or other specified

566 metabolite) in triplicate. After 24 h (or other indicated time) at 37 °C, colony biofilms

567 were cored, resuspended in 500 µL LB by vigorous shaking on the Genie Disrupter for 5

568 min as previously described, and β-Gal activity was measured as described by Miller

569 (105). The average for each experiment was reported across 3-4 independent days.

570

571 Western Blot

572 Strains were grown in LB broth under selection (60 μg / mL gentamycin or 60 μg / mL

573 carbenicillin as appropriate) for 16 h at 37 C on a roller drum, and 5 μL of culture was

574 spotted onto LB plates under selection with 0.2% L-arabinose (v/v) and +/- 20 mM

575 indicated carbon source. Inoculated plates were incubated at 37 C for 16 h. Colony

576 biofilms were resuspended in 325 μL of Laemmli buffer without reducing agent and

577 heated at 100 C for 15 min. Protein was quantified on 1:10 dilution of protein sample

578 according to standard procedure via Thermo scientific BCA Protein Assay Kit. Reducing

579 agent was added and samples were run on a 4 - 15% SDS gradient gel (Bio-Rad) at 60

580 V for 40 min followed by 110 V for 45 min. After SDS page electrophoresis, protein was

581 transferred onto LF-PVDF (Bio-Rad) using the mixed molecular weight option on a turbo

582 blot apparatus (Bio-Rad). After transfer the membrane was dried, rehydrated, and then

583 a total protein stain was run according to manufacturer’s procedure (Li-Cor). Following

584 protein quantification, the membrane was incubated in TBS blocking buffer (Li-Cor) for 1

585 h, and then purified anti-HA mouse monoclonal antibody (Biolegend) in TBS blocking bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

586 buffer (1:2,500 dilution) for 1 hr. Following primary antibody detection, the membrane

587 was washed 4 times in TBST 0.1%. Secondary detection was done by incubation with

588 goat anti-mouse in TBS blocking buffer (1:15000 dilution) for 1 hour in the dark.

589 Following detection, the membrane was washed 3 times in TBST 0.1% and once in

590 TBS. The membrane was then dried and imaged on the Li-Cor Odyssey CLx imager

591 relative to REVERT total protein stain.

592

593 Acknowledgements

594 Research reported in this publication was supported by grants from the National

595 Institutes of Health to D.A.H. (R01 GM108492 to D.A.H) and HOGAN19G0, and NSF

596 1458359 (D.A.H. and D.L.M). Support for D.L.M came in part from NIH/NIAID

597 T32AI007519 (D.L.M). NIGMS P20GM113132 through the Molecular Interactions and

598 Imaging Core (MIIC). STANTO19R0 from the Cystic Fibrosis Foundation and NIDDK

599 P30-DK117469 (Dartmouth Cystic Fibrosis Research Center). RNA-Seq and

600 NanoString were carried out at Dartmouth Medical School in the Genomics Shared

601 Resource, which was established by equipment grants from the NIH and NSF and is

602 supported in part by a Cancer Center Core Grant (P30CA023108) from the National

603 Cancer Institute. We also thank Georgia Doing for preliminary RNAseq analysis, Pat

604 Occhipinti for swapping the antibiotic marker on the rhlI-HA expression vector, Carla

605 Cugini for strain construction, and Alan Collins for the gradient plate prop.

606

607

608 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

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928 101. Wickham H. 2016. ggplot2: Elegent Graphics for Data Analysis, Springer-Verlag, 929 New York. https://ggplot2.tidyverse.org. 930 102. Team RDC. 2010. R: A language and environment for statistical computing, R 931 Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org. 932 103. Kolde R. 2019. pheatmap: Pretty Heatmaps, https://CRAN.R- 933 project.org/package=pheatmap. 934 104. Mi H, Muruganujan A, Ebert D, Huang X, Thomas PD. 2018. PANTHER version 935 14: more genomes, a new PANTHER GO-slim and improvements in enrichment 936 analysis tools. Nucleic Acids Research 47:D419-D426. 937 105. Miller JH. 1992. A short course in bacterial genetics. Cold Spring Harbor Press. 938 939 Figure legends 940 941 Figure 1. ∆lasR produces pyocyanin in wild type / ∆lasR co-cultures beyond 942 monoculture concentrations. A. Wild type (WT), ∆lasR, and their phenazine-deficient 943 derivatives (∆phz) visualized from the bottom of the 96 well LB agar plate after 16 h 944 growth as mono- (top) and co-cultures (bottom). B. Pyocyanin levels quantified for 945 cultures described in A; ****, p<0.001. C. Pyocyanin production for wild type co-cultures 946 with ∆lasR or ∆lasR complemented with the lasR gene at the native locus (∆lasR + 947 lasR) across several initial (i) proportions on LB for 20 h. D. Final proportions quantified 948 after 16 h growth for WT and ∆lasR co-cultured with a WT tagged with lacZ. 949 Experimental setup as described in C. 950 951 Figure 2. P. aeruginosa WT induces RhlR/I dependent activity in ∆lasR even in the 952 absence of WT AHLs. A. Pyocyanin production by monocultures and WT co-cultures 953 of ∆lasR and ∆lasR derivatives that are deficient PQS or RhlR/I quorum sensing on LB 954 after 24 h growth. B. Promoter activity, quantified by relative pixel intensity of single cell- 955 derived colony forming units (CFU) in co-culture with untagged WT CFU for ∆lasR P 956 pqsA - lacZ (grey) and ∆lasR P rhlI - lacZ (black). Inset shows representative CFUs for 957 RhlR-dependent ∆lasR P rhlI – lacZ activity when in monoculture and co-culture with 958 WT (red circles). C. Co-culture pyocyanin production by ∆lasR still occurs upon co- 959 culture with ∆pqsA, ∆rhlI, and ∆lasI∆rhlI on LB after 24 h. All monocultures are shown at 960 48 h time point for comparison. 961 962 Figure 3. Biosynthesis of the co-culture-induced iron scavenging siderophore 963 pyochelin is required in ∆lasR for pyocyanin over-production when cultured with 964 wild type (WT). A. Scheme for the collection of RNA from ∆lasR colony biofilms grown 965 above a lawn of WT or ∆lasR. B. Volcano plot of ∆lasR expression data with each point 966 representing the log2(∆lasR grown on WT / ∆lasR grown on ∆lasR) expression and – 967 log10(P Value) of a single gene. Genes involved in pyoverdine (blue) and pyochelin 968 (green) iron acquisition systems are indicated. ccmC and ccmF (indicated with arrows) 969 of the pyoverdine GO term are involved in c-type cytochrome biosynthesis, and strains 970 with knockouts of these genes are reported to produce more pyochelin. C. Mono- and 971 co-cultures with ∆lasR strains deficient in pyoverdine (∆pvdA) and/or pyochelin (∆pchE). 972 Colonies are visualized under ultraviolet light (UV) in order to see fluorescent 973 siderophores. D. Pyocyanin production visualized for the colonies shown in panel C. E. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

974 Pyocyanin production by siderophore deficient strains grown in mono- and co-culture on 975 LB with (+PCH) or without (LB) pyochelin-containing extract. Colonies were grown in a 976 12 well plate and imaged after 48 h. F. Wild-type and ∆lasR mixed colony biofilms 977 grown on LB (-) or LB supplemented with either 10 or 100 µM FeSO4 visualized under 978 ambient (top) and UV (bottom) light. 979 980 Figure 4. Citrate release by WT is induced by pyochelin exposure. A. Subset of 981 ∆lasR co-culture expression data (see Fig. 3A for set up) for genes annotated as being 982 involved in citrate sensing, transport, catabolism, anabolism, and those shown to be 983 responsive to citrate. B. Volcano plot of ∆lasR co-culture expression data with genes 984 shown in panel A highlighted in red. C. Citrate concentrations in supernatants from wild 985 type and ∆lasR stationary phase cultures after growth in LB supplemented with extracts 986 containing 50 µM pyochelin (PCH +) or an equal volume of control extracts (PCH -). A 987 representative experiment with four biological replicates is shown; ***, p≤ 0.001 and **** 988 , p≤ 0.0001 by two-tailed t-test of paired ratios. 989 990 Figure 5. Citrate and related compounds induce RhlR-dependent rhlI promoter 991 activity and stabilize RhlI protein in LasR- strains in a ClpX protease dependent 992 manner. A. β-galactosidase activity for ∆lasR, ∆lasR∆rhlR, and wild type harboring 993 att::PrhlI - lacZ on LB ± 20 mM citrate at 24 h. Each point is the average of three 994 biological replicates from 3 - 4 independent experiments. Statistical analyses performed 995 by one-way ANOVA with Dunnet’s multiple hypotheses correction. B. RhlI-HA protein 996 signal normalized to REVERT total protein stain (Licor) on LB ± 20 mM citrate for ∆lasR 997 and lasR complemented at the native locus (∆lasR+lasR). n = 3 biological replicates 998 performed on three independent days. Dunnet’s multiple comparison test with LB 999 control. Inset illustrates that plasmid-borne RhlI-HA, but not the empty vector (EV) can 1000 complement an ∆rhlI mutant for swarming. C. RhlI-HA protein levels on LB and LB 1001 supplemented with 20 mM citrate of LasR LOF (LasR-) clinical isolates (CI) from acute 1002 corneal (388D) and chronic CF infections (DH2415). D. RhlI-HA protein levels on LB ± 1003 20 mM citrate of LasR+ acute corneal CI (550A) of same MLST type as 388D and 1004 LasR+ chronic CF CI (DH2417) from which DH2415 evolved. E. Representative image 1005 and quantification of replicates for the anti-HA antibody analysis of ∆lasR and 1006 ∆lasRclpXTn::M + pRhlI-HA or pEV grown in LB and LB supplemented with 20 mM 1007 citrate. F. RhlR-dependent rhlI promoter activity of ∆lasR P rhlI - lacZ on LB 1008 supplemented with citrate, acetate, succinate, fumarate, and malate at 16 and 24 h 1009 relative to LB control. G. Normalized RhlI-HA protein levels ∆lasR and ∆lasR + lasR at 1010 16 h of growth on citrate, acetate, and succinate. Inset shows representative blot of 1011 RhlI-HA protein under these same conditions. p-values: * (p<0.05), ** (p ≤ 0.05), *** (p≤ 1012 0.001), and **** (p≤ 0.0001). 1013 1014 Figure 6. Model for wild type and ∆lasR co-culture interactions. (1.) ∆lasR 1015 produced pyochelin promotes citrate release in wild type. (2.) Citrate released by wild 1016 type in co-culture stimulates RhlR/I dependent activity (3.) by stabilizing RhlI protein in 1017 ClpXP protease dependent mechanism (4.) to promote the production of antagonistic 1018 factors like pyocyanin and rhamnolipid surfactant. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 1. ∆lasR produces pyocyanin in wild type / ∆lasR co-cultures beyond monoculture concentrations. A. Wild type (WT), ∆lasR, and their phenazine-deficient derivatives (∆phz) visualized from the bottom of the 96 well LB agar plate after 16 h growth as mono- (top) and co-cultures (bottom). B. Pyocyanin levels quantified for cultures described in A; ****, p<0.001. C. Pyocyanin production for wild type co- cultures with ∆lasR or ∆lasR complemented with the lasR gene at the native locus

(∆lasR + lasR) across several initial (i) proportions on LB for 20 h. D. Final proportions quantified after 16 h growth for WT and ∆lasR co-cultured with a WT tagged with lacZ. Experimental setup as described in C. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. lasR lasR Figure 2. P. aeruginosa WT induces WT lasR pqsR RhlR/I dependent activity in ∆lasR even in the absence of WT AHLs. A. Pyocyanin production by monocultures

Mono- and WT co-cultures of ∆lasR and ∆lasR WT WT WT WT derivatives that are deficient PQS or lasR lasR lasR RhlR/I quorum sensing on LB after 24 h pqsR growth. B. Promoter activity, quantified by relative pixel intensity of single cell- derived colony forming units (CFU) in co- Co- culture with untagged WT CFU for ∆lasR P pqsA - lacZ (grey) and ∆lasR P rhlI - B. lacZ (black). Inset shows representative lasR P-lacZ CFUs for RhlR-dependent ∆lasR P rhlI – lasR PpqsA-lacZ lacZ activity when in monoculture and ) 2 1500 B . P rhlI - lacZ co-culture with WT (red circles). C. i Mono-culture and ∆lasR co-culture lasR lasRrhlR images for ∆pqsA, ∆rhlI, and ∆lasI∆rhlI on LB after 48 h, rather than 24 h as in

1000 Mono- panel A, due to slower color development. Co- WT 500

Relative Promoter Activity (Pixel Relative Promoter 0 0 10 20 30 Minimum Distance to WT

C. lasR pqsA lasI Mono-

pqsA lasI lasR Co- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. B. pyochelin acquisition acquisition system system 8 pyoverdine acquisition acquisition system system citrate transport/metabolism

6

4 (P Value) (P

10

- log 2

15 0 -4 -2 0 2 4 log 2 (Fold Change) C. D. lasR lasR lasR lasR lasR lasR WT lasR 10 WT lasR

Mono- Mono- log(lasR_lb_wt$PValue) WT WT WT WT WT WT WT lasR WT lasR lasR lasR

lasR5 lasR lasR lasR Co- Co- E. F. FeSO : - + 10 µM + 100 µM + PCH0 LB 4

WT 4 2 lasR 0 2

lasR_lb_wt$logFC lasR lasR

Figure 3. Biosynthesis of the co-culture-induced iron scavenging siderophore pyochelin is required in ∆lasR for pyocyanin over-production when cultured with wild type (WT). A. Scheme for the collection of RNA from ∆lasR colony biofilms grown above a lawn of WT or ∆lasR. B. Volcano plot of ∆lasR expression data with each point representing the log2(∆lasR grown on WT / ∆lasR grown on ∆lasR) expression and –log10(P Value) of a single gene. Genes involved in pyoverdine (blue) and pyochelin (green) iron acquisition systems are indicated. ccmC and ccmF (indicated with arrows) of the pyoverdine GO term are involved in c-type cytochrome biosynthesis, and strains with knockouts of these genes are reported to produce more pyochelin. C. Mono- and co-cultures with ∆lasR strains deficient in pyoverdine (∆pvdA) and/or pyochelin (∆pchE). Colonies are visualized under ultraviolet light (UV) in order to see fluorescent siderophores. D. Pyocyanin production visualized for the colonies shown in panel C. E. Pyocyanin production by siderophore deficient strains grown in mono- and co-culture on LB with (+PCH) or without (LB) pyochelin-containing extract. Colonies were grown in a 12 well plate and imaged after 48 h. F. Wild- type and ∆lasR mixed colony biofilms grown on LB (-) or LB supplemented with either 10 or 100 µM FeSO4 visualized under ambient (top) and UV (bottom) light. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. B. Responsive 1.5 Citrate PA14_28970 Anabolism PA14_20000 1 Related Catabolism citA 00 Genes Sensing & Transport PA14_37990 PA14_72170 -1.5

PA14_72180 lasR.on.WT.3 lasR.on.WT2 lasR.on.WT1 lasR.on.lasR.3 lasR.on.lasR.2 lasR.on.lasR.1 PA14_37980 8

fecA PA14_28970 PA14_28980 PA14_28970

PA14_28980PA14_20000 1 fecA PA14_20000

PA14_37980

lasR.on.lasR.1 lasR.on.lasR.2 lasR.on.lasR.3 lasR.on.WT1 lasR.on.WT2 lasR.on.WT.3 citA PA14_72180 citA 0

PA14_37990

PA14_72170 PA14_37990 6

PA14_72170

PA14_37990 PA14_72170

PA14_72180 citA 0 PA14_72180

fecA PA14_20000 fecA

1 Value) (P

PA14_28980 PA14_28970 4 PA14_28980 PA14_37980 PA14_3798010 lasR.on.lasR.1 lasR.on.lasR.2 lasR.on.lasR.3 lasR.on.WT1 lasR.on.WT2 lasR.on.WT.3 Sensing & Transport Sensing & PA14_52900 1 - log 1 acnAB coaD coaD coaD 2 0 hpcG hpcG hpcG 0 PA14_52880 PA14_10600 PA14_10600 PA14_10600 prpB prpB prpB 0 PA14_52850 -4 -2 0 2 4 PA14_52870 log (Fold Change) aceK aceK aceK 2 prpD prpD prpD C. PA14_53970 500500 PA14_52890 PA14_53980 Catabolism **** idh idh idh PA14_30050 400400 acnB 600 acnB acnB ) acnA

acnA acnA 600 icd Citrate Related Genes icd icd 300 PA14_52910 300 PA14_10570 lasR.on.lasR.1 lasR.on.lasR.2 lasR.on.lasR.3 lasR.on.WT1 lasR.on.WT2 lasR.on.WT.3 lasR.on.WT.3 lasR.on.WT2 lasR.on.WT1 lasR.on.lasR.3 lasR.on.lasR.2 lasR.on.lasR.1 M / OD µ gltA gltA gltA 200200 1 PA14_53920 1 *** 0 prpC prpC prpC 0

prpB Citrate ( prpB Citrate (µM) / OD prpB lasR.on.lasR.1 lasR.on.lasR.2 lasR.on.lasR.3 lasR.on.WT1 lasR.on.WT2 lasR.on.WT.3 Anabolism lasR.on.WT.3 lasR.on.WT2 lasR.on.WT1 lasR.on.lasR.3 lasR.on.lasR.2 lasR.on.lasR.1 100100

gdhB gdhB 1 gdhB 1 00 alkB1 alkB1 alkB1 0 alkB2 alkB2 alkB2 0 PCH:PCH: - + - + lasR.on.lasR.1 lasR.on.lasR.2 lasR.on.lasR.3 lasR.on.WT1 lasR.on.WT2 lasR.on.WT.3 lasR.on.WT.3 lasR.on.WT2 lasR.on.WT1 lasR.on.lasR.3 lasR.on.lasR.2 lasR.on.lasR.1 WT lasR Responsive PA14 lasR Figure 4. Citrate release by WT is induced by pyochelin exposure. A. Subset of ∆lasR co-culture expression data (see Fig. 3A for set up) for genes annotated as being involved in citrate sensing, transport, catabolism, anabolism, and those shown to be responsive to citrate. B. Volcano plot of ∆lasR co-culture expression data with genes shown in panel A highlighted in red. C. Citrate concentrations in supernatants from wild type and ∆lasR stationary phase cultures after growth in LB supplemented with extracts containing 50 µM pyochelin (PCH +) or an equal volume of control extracts (PCH -). A representative experiment with four biological replicates is shown; ***, p≤ 0.001 and **** , p≤ 0.0001 by two- tailed t-test of paired ratios. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. 50005000 P rhlI - lacZ B. -HABi. Swarming ** 2000 40004000 2000 * * 3000 15001500 3000 WT +EV rhlI-HA 20002000 10001000 Miller Units Miller Units nsns 10001000 500500 RhlI-HA Normalized RhlI-HA

ns Normalized RhlI-HA 0 00 Citrate:Citrate - ++ - + - - + CitrateCitrate: - + - - + + lasR lasRrhlR WT lasR lasR lasRrhlR WT lasR lasR+lasR -HA C. D. -HA 15000 nsns 200000

* 150000 150000 10000 * 100000 5000 50000 RhlI-HA Normalized RhlI-HA RhlI-HA Normalized RhlI-HA Normalized RhlI-HA Normalized RhlI-HA NDnd 0 0 CitrateCitrate: - + - + CitrateCitrate: - + - + 388D DH2415 550A DH2417 388D DH2415 550A DH2417 (Acute CI) (ChronicRelativeRelative CI) Miller Miller Units Units (Acute CI) (Chronic CI)

E. Hour: Hour: EV: + - log log - (+ 2 (+ metabolite metabolite- - / LB / LB control) control)

-1 2

-1 Relative normalized RhlI-HA 0 1 2

0 3 1 2 pRhlI-HA: - + + + + Citrate: Citrate 16 Citrate -16 - + - + + Citrate 3.0 ± 1.1

2 **** **** LB * 24 TnM 24 0.83 ± 0.2 REVERT Acetate 16 Acetate 1 16 total lasR lasR protein ( stain ) 24 clpX::TnM 24 F. 0 G. 3 22 lasR PrhlI - lacZ 16 h Succinate Relative RhlI-HA Succinate Gi. lasR + RhlI-HA 16 16 CitrateCitrate (+ metabolite / LB control) 2 -1 LB +C +A +S AcetateAcetate 2 24 log

24 **** SuccinateSuccinate

Fumarate Fumarate 1 Fumarate -2

1 24 24 Malate 1 lasR lasR + lasR 24 24 0 Relative Miller Units Malate Malate Relative RhlI-HA 2

00 Relative RhlI-HA 2 log

Relative Miller Units Citrate (+ metabolite / LB control) (+ metabolite / LB control) 2 log 2 -1-1 Acetate log log Succinate -1-1 -2-2 Hours:Hour: 1616 2424 1616 242416 16 24 2424 2424 24 lasRlasR lasR + lasR Citrate Acetate Succinate Fumarate Malate bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 5. Citrate and related compounds induce RhlR-dependent rhlI promoter activity and stabilize RhlI protein in LasR- strains in a ClpX protease dependent manner. A. β- galactosidase activity for ∆lasR, ∆lasR∆rhlR, and wild type harboring att::PrhlI - lacZ on LB ± 20 mM citrate at 24 h. Each point is the average of three biological replicates from 3 - 4 independent experiments. Statistical analyses performed by one-way ANOVA with Dunnet’s multiple hypotheses correction. B. RhlI-HA protein signal normalized to REVERT total protein stain (Licor) on LB ± 20 mM citrate for ∆lasR and lasR complemented at the native locus (∆lasR+lasR). n = 3 biological replicates performed on three independent days. Dunnet’s multiple comparison test with LB control. Inset illustrates that plasmid-borne RhlI-HA, but not the empty vector (EV) can complement an ∆rhlI mutant for swarming. C. RhlI-HA protein levels on LB and LB supplemented with 20 mM citrate of LasR LOF (LasR-) clinical isolates (CI) from acute corneal (388D) and chronic CF infections (DH2415). D. RhlI-HA protein levels on LB ± 20 mM citrate of LasR+ acute corneal CI (550A) of same MLST type as 388D and LasR+ chronic CF CI (DH2417) from which DH2415 evolved. E. Representative image and quantification of replicates for the anti-HA antibody analysis of ∆lasR and ∆lasRclpXTn::M + pRhlI-HA or pEV grown in LB and LB supplemented with 20 mM citrate. F. RhlR-dependent rhlI promoter activity of ∆lasR P rhlI - lacZ on LB supplemented with citrate, acetate, succinate, fumarate, and malate at 16 and 24 h relative to LB control. G. Normalized RhlI-HA protein levels ∆lasR and ∆lasR + lasR at 16 h of growth on citrate, acetate, and succinate. Inset shows representative blot of RhlI- HA protein under these same conditions. p-values: * (p<0.05), ** (p ≤ 0.05), *** (p≤ 0.001), and **** (p≤ 0.0001). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 6. Model for wild type and ∆lasR co-culture interactions. (1.) ∆lasR produced pyochelin promotes citrate release in wild type. (2.) Citrate released by wild type in co-culture stimulates RhlR/I dependent activity (3.) by stabilizing RhlI protein in ClpXP protease dependent mechanism (4.) to promote the production of antagonistic factors like pyocyanin toxin and rhamnolipid surfactant. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1.5

A. C. WT ) **** ****

WT lasR 310 lasR 1.01.0 **** ****

) / plug **

LB 310 ****

0.50.5 Pyocyanin (OD ASM Pyocyanin (OD 0.0 M63 pH 6.8 0.2% glucose + CAA 0.0

WTWT lasRlasR lasRlasR

WT /

DH2417DH2417DH2415 DH2415(LasR+) (LasR+) (LasR-) (LasR-)DH1133DH1133DH1132 (LasR+)DH1132 (LasR+) (LasR-) (LasR-) DH2417DH2417 / / DH2415DH2415 DH1133DH1133 / /DH1132 DH1132 B. D. 2.02.0 WT WT lasR lasR 1.51.5 *** ** 1.01.0 ns

DH2417 DH2415 DH2417 (LasR+) (LasR-) DH2415 0.50.5 Total CFUs relative to LasR+ Total CFUs relative to LasR+ Total 0.00.0

WT WT lasRlasR lasRlasR DH2417DH2415 DH1133DH1132 WT / DH2417DH2415 (LasR+) (LasR-)DH1133DH1132 (LasR+)DH1133/D1132 (LasR-) DH2417DH2417/DH2415 / DH2415 DH1133 / DH1132

Figure S1. Increased pyocyanin production of LasR+ and LasR- strain co-cultures relative to either strain alone is stable across media and strains. A. Representative images from PA14 wild type (WT) and ∆lasR mono- and co-culture pyocyanin production on rich media (LB and Artificial Sputum Media-ASM) and buffered minimal medium (M63 pH 6.8 0.2% glucose 0.2% CAA). B. Pyocyanin production of mono- and co-cultures of strain PA14 WT and ∆lasR and clonally-derived clinical isolates DH2417 (LasR+) and DH2415 (LasR-). C. Pyocyanin concentrations of LasR+ and LasR- strains grown in mono- and co-culture on 96 well LB agar plugs for 16 h. Strain PA14 WT and ∆lasR, and previously characterized clinical isolate pairs DH2417 (LasR+) and DH2415 (LasR-) and DH1133 (LasR+) and DH1132 (LasR-), from two distinct CF patients, are also shown. Data are from two independent experiments with four biological replicates each. **** represents a significant difference from the co-culture, p<0.0001. D. Colony forming units (CFUs) in 16 h colony biofilms grown on LB. Each strain set is presented relative to WT or the LasR+ strain. PA14 WT and ∆lasR mono- and co-cultures data are the average from four independent experiments with at least three biological replicates. No significant (ns) differences found by ANOVA with Tukey multiple hypotheses correction for clinical isolate CFU counts. ** p-value < 0.005, *** p-value < 0.0005. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. WT lasR

WT lasR

Figure S2. ∆lasR shows Anr independent increases in pyocyanin in co- culture with LasR+ P. aeruginosa. Pyocyanin production of WT, ∆anr, and ∆lasR∆anr in mono- and co-culture colony biofilms on LB after 24 h. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure S3. ∆lasR shows enhanced production of RhlR-regulated rhamnolipid surfactant in co-culture with LasR+ strain. RhlR- regulated swarming motility on soft agar of WT, rhamnolipid surfactant biosynthesis mutant (∆rhlA), ∆lasR, and ∆lasR∆rhlR in mono- (top) and co-culture (bottom) after 36 h. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure S4. Fitness of ∆lasR lacking one or both major siderophore biosynthesis pathways. Final proportion of untagged colony forming units quantified after 16 h competition with att::lacZ wild type. Dotted line indicates

0.5 initial proportion (Pi). Final proportions (Pf) for ∆lasR, wild type (WT), and ∆lasR complemented with the lasR gene at the native locus (∆lasR + lasR) on LB (white background), for ∆lasR on LB supplemented with 10 µM FeSO4 (grey background), and siderophore deficient ∆lasR derivatives. Statistical analyses performed by one-way ANOVA for comparison to ∆lasR on LB with Sidak multiple hypotheses correction: a-b p-value < 0.0006, a-c p-value < 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. 1.0 Extracts pvdA pvdApchE

0.5 Absorbance

0.0 300 400 500 600 Wavelength (nm)

B. Extracts

EDTA pvdA pvdA chelator (PCH) pchE (NEG) CAS + extract

Figure S5. Pyochelin-containing extracts are biologically active. A. Absorbance spectra (230 to 600 nm) of extracts from supernatants of pyochelin- producing, ∆pvdA (solid line), and pyochelin-deficient, ∆pvdA∆pchE (dotted line)

strains in a 50/50 MeOH/dH2O solution. Grey vertical lines indicate reported peaks for purified, iron-free pyochelin at 248 and 313 nm. B. Indicated extracts spotted on chrome azurol S (CAS) agar with ethylenediaminetetraacetic acid (EDTA) metal chelator as positive control wherein change from blue to yellow indicates iron chelating capacity. CAS activity for the extracts from ∆pvdA (+PCH) and ∆pvdA∆pchE (NEG) are shown. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. Antranilate Metabolism B. WT phnAB lasR 8

6 Mono- WT phnAB phnAB 4 lasR

(P-Value) lasR lasR

10 rhlR

- log 2 Co-

0 -4 -2 0 2 4

log2(Fold Change) C. 14.5 mM 0 AA

lasR

Figure S6. Anthranilate (AA) did not stimulate ∆lasR pyocyanin production. A. Volcano plot indicating anthranilate metabolism gene expression (orange) in ∆lasR grown in co-culture with WT. B. RhlR dependent pyocyanin production of ∆lasR and anthranilate synthase mutant (∆phnAB) mono- and co-cultures on LB after 18 h. C. Colony morphology and pyocyanin production are not different upon anthranilate supplementation across a gradient of concentrations. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.29.068346; this version posted April 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A. lasR WT lasR lasR Mono- WT WT WT lasR lasR lasR Co-

B. lasR WT lasR PA14 _51300 Mono- WT WT lasR lasR PA14 _51300 Co-

Figure S7. Potential di- and tri-carboxylic acid transporters dctA and PA14_51300 were not required for ∆lasR pyocyanin production. A. Major succinate, fumarate, and malate transporter dctA was dispensable in ∆lasR background for RhlR-dependent pyocyanin production in co- culture with WT. B. Broad TCA cycle intermediate transporter PA14_51300 was not required in ∆lasR for pyocyanin production in co- culture with WT.