A three-component logic gate governs quorum sensing-regulated killing of Stenotrophomonas maltophilia by Pseudomonas aeruginosa

17 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint

18 19 Pseudomonas aeruginosa, an opportunistic pathogen, uses a trio of quorum sensing (QS) 20 systems to regulate the production of some virulence factors. Two of these, the las and rhl 21 systems, involve acyl-homoserine lactone signals; the third, called pqs, primarily uses the signal 22 2-heptyl-3-hydroxy-4(1H)-quinolone (“PQS”). We aimed to identify how interbacterial 23 interactions are regulated between P. aeruginosa and Stenotrophomonas maltophilia, which co-24 occur in the airways of people with cystic fibrosis. We explored P. aeruginosa and S. maltophilia 25 interactions using a co-culture model. S. maltophilia in co-culture with P. aeruginosa grows for 26 12 hours and thereafter exhibits a large decline in CFU, demonstrating that P. aeruginosa is 27 killing S. maltophilia. Co-culture of S. maltophilia with P. aeruginosa deficient in las, rhl, or pqs 28 QS resulted in greater S. maltophilia viability than co-culture with the wildtype. This inhibition 29 was not attributable to las and rhl-regulated toxins. Therefore, we interrogated the role of PQS 30 and found that co-culture of S. maltophilia with P. aeruginosa deficient in PQS biosynthesis 31 showed similar CFUs to monoculture. Exogenous PQS did not complement this phenotype, 32 suggesting that another quinolone is the effector. We found that S. maltophilia killing is reduced 33 in competition with a mutant that cannot make the quinolone HQNO. We show that full killing of 34 S. maltophilia by P. aeruginosa requires three components: HQNO, the chaperone PqsE, and 35 intact PQS biosynthesis. Our work identifies quinolone biosynthesis as a driver for interactions 36 between P. aeruginosa and S. maltophilia and, more generally, in modulating interbacterial 37 interactions. 38 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint

39 Bacteria are typically found in microbial communities where they interact with biotic and abiotic 40 factors, as in the soil and gastrointestinal tract of animals (1, 2). Bacteria can interact with other 41 microbes in these spaces, and these interactions can alter their behaviors (3-6). Bacteria can 42 also be found together in infections, as is the case during airways infections in people with the 43 genetic disease cystic fibrosis (CF). In the case of CF, much of the morbidity can be attributed to 44 chronic airway infections with opportunistic pathogens, including Pseudomonas aeruginosa, 45 Staphylococcus aureus, and Stenotrophomonas maltophilia (7-9). One of the prominent 46 pathogens that infects people with CF is the Gram-negative bacterium Pseudomonas 47 aeruginosa (10, 11). P. aeruginosa produces several secreted factors, including those involved 48 in virulence, that have been shown to affect how it interacts with other microbes (12-17). 49 50 Interactions between P. aeruginosa and some other bacteria, such as the Gram-positive 51 bacterium Staphylococcus aureus, have been well-described. For example, P. aeruginosa in 52 coculture with S. aureus secretes virulence factors including the alkylquinolone 2-heptyl-4-53 quinoline N-oxide (HQNO) and the siderophores pyoverdine and pyochelin (18). These 54 virulence factors negatively impact S. aureus metabolism, allowing P. aeruginosa to gain a 55 competitive advantage and increase its growth (19, 20). While this interaction seems to be more 56 beneficial for P. aeruginosa, growth together results in S. aureus having increased resistance to 57 antibiotics, including gentamicin and tetracycline (21, 22). This finding parallels other studies 58 which show that interactions between P. aeruginosa and other microbes can increase P. 59 aeruginosa growth (20, 23), result in increased drug resistance (21, 22), or both; all of these 60 outcomes likely are detrimental to infected host. These studies highlight the importance of 61 understanding interbacterial interactions and how they affect bacterial behavior, such as in the 62 context of human infection. 63 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 64 P. aeruginosa regulates behaviors within the species using an intercellular signaling system 65 known as quorum sensing (QS). Bacteria use QS to measure their cell density and to alter 66 population behaviors in response (24). In P. aeruginosa, QS regulates the production of 67 secreted products like the protease elastase, toxic biosurfactant rhamnolipids, and hydrogen 68 cyanide (25-28). QS also regulates the production of extracellular polysaccharides that are 69 important for forming biofilms (29, 30). Generally speaking, QS consists of a circuit composed 70 of a secreted signal; this signal is sensed by and activates a regulator. P. aeruginosa harbors 71 three complete QS circuits that involve the transcription factors LasR, RhlR, and PqsR (also 72 called MvfR) (31-33). The circuits use different signals. LasR binds to the signal N-3-oxo-73 dodecanoyl-homoserine lactone (3OC12-HSL) produced by the signal synthase LasI; similarly, 74 RhR binds to N-butanoyl-homoserine lactone (C4-HSL) produced by RhlI. PqsR recognizes 2-75 heptyl-3-hydroxy-4(1H)-quinolone (called the Pseudomonas quinolone signal, PQS) which is 76 synthesized by the products of an operon, pqsABCDE and an unlinked gene, pqsH (34-36) 77 (Supplementary Figure 1). These three QS circuits (las, rhl, pqs) control the expression of 78 hundreds of genes, including several that affect virulence, and they are arranged in a hierarchy: 79 LasR activates expression of the genes encoding RhlR and PqsR. 80 81 las and rhl QS are known to regulate interactions with Gram-negative bacteria (15-17), but the 82 role of pqs QS in these interactions is less well-described. The enzymes of PQS biosynthesis 83 together modify chorismic acid into PQS. Each enzyme controls the production of an 84 intermediate in this pathway, and mutants in any gene in the PQS biosynthesis pathway (except 85 PqsE) are unable to produce PQS (Supplementary Figure 1) (37). Signal-bound PqsR 86 positively regulates the expression of pqsABCDE and thus creates a positive feedback loop. 87 PqsE, a product of this operon, is unique: it is required to fully activate rhl QS (38), and in many 88 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint strains dispensible for PQS biosynthesis. Finally, the enzyme PqsL, a product of another gene 89 unlinked to pqsABCDE, creates a branch in the PQS biosynthesis pathway and is required to 90 produce HQNO (Supplementary Figure 1). PQS biosynthesis is unaffected in a pqsL mutant 91 (36, 39). HQNO and other quinolones have been demonstrated to have antibiotic activity 92 against a range of bacteria, including S. aureus (12, 18, 22, 40, 41). 93 94 P. aeruginosa interacts with a multitude of Gram-negative bacteria as a consequence of its 95 ability to occupy a variety of environmental niches such as soils, hospital sink drains, and the 96 animal hosts (42-45). One such bacterium that can share these niches with P. aeruginosa is 97 another Gram-negative bacterium, Stenotrophomonas maltophilia. Interactions between P. 98 aeruginosa and S. maltophilia have been sparsely studied, mostly in the context of infections of 99 people with CF. S. maltophilia is co-isolated with P. aeruginosa at a frequency ranging from 10 100 to 60% in CF airways (46-49). Interactions between P. aeruginosa and S. maltophilia may 101 influence the antimicrobial resistance profile of P. aeruginosa (50). 102 103 We are interested in understanding the interactions of P. aeruginosa and S. maltophilia, to better 104 define the determinants of competition between Gram-negative bacteria, and the genes that 105 modulating these interactions. We established a co-culture system and determined that, in the 106 conditions of our experiments, P. aeruginosa kills S. maltophilia. We assessed the genes 107 affecting this interaction. We found that all inactivation of any of the three QS circuits 108 ameliorated the killing of S. maltophilia. Because pqs QS is regulated by LasR, and its 109 contributions to interactions with Gram-negative bacteria are relatively poorly understood, we 110 explored its role further. We found that the inhibitor HQNO, the chaperone PqsE, and PQS 111 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint biosynthesis are all necessary for mediating competition between P. aeruginosa and S. 112 maltophilia. 113 114

115 P. aeruginosa competes with S. maltophilia. 116 We developed a co-culture model where we grew both the P. aeruginosa strain PAO1 and the S. 117 maltophilia strain K279a in mono- or co-culture in test tubes at 37˚C with shaking with similar 118 growth rates and yields (Supplementary Figure 2). We monitored bacterial viability using 119 colony forming unit (CFU) assays on selective plates: we selected for S. maltophilia using LB 120 agar supplemented with gentamicin and selected for P. aeruginosa using columbia agar 121 supplemented with C-390 and phenanthroline (51). In co-culture, we found that S. maltophilia 122 viability was unchanged as compared to monoculture until around 12 hours where we observed 123 a ~1-log decrease (Figure 1A). At 16 hours, we observed a sharper decline in CFUs. By 20 124 hours, we observed a striking phenotype where S. maltophilia viability decreased by ~7-logs in 125 co-culture with P. aeruginosa compared to monoculture. S. maltophilia viability remains 126 decreased until the end of the experiment at 24 hours. We found that P. aeruginosa viability was 127 mostly unchanged in the same co-culture conditions compared to monoculture (Figure 1B). 128 These findings demonstrated that S. maltophilia and P. aeruginosa interact and that P. 129 aeruginosa kills S. maltophilia by some mechanism. 130 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 131 Figure 1. P. aeruginosa competes with, and kills, S. maltophilia, while P. aeruginosa 132 viability is mostly unaffected. Wild-type P. aeruginosa PAO1 and wild-type S. maltophilia 133 K279a were grown in mono- or co-culture for 24 h. In co-cultures, PAO1 and K279a were mixed 134 1:1 based on OD600. S. maltophilia CFUs are shown and A and P. aeruginosa CFUs are shown 135 in B. Each graph shows viability; x-axis displays time in hours and the y-axis shows CFUs. The 136 experiment was performed in triplicate. Error bars represent the standard deviation. 137 138 Competition between P. aeruginosa and S. maltophilia is mediated by quorum sensing 139 We were next asked what genes were mediating interactions between P. aeruginosa and S. 140 maltophilia. Competition between P. aeruginosa and other bacteria has been demonstrated to 141 be regulated by QS, so we asked if QS played a role in this competition. To answer this 142 question, we grew S. maltophilia K279a in mono- or co-culture with wild-type PAO1, 143 PAO1∆lasR, PAO1∆rhlR, or PAO1∆pqsR and measured S. maltophilia CFUs after 24 hours 144 (Figure 2). In contrast to co-culture with wild-type P. aeruginosa, we found that S. maltophilia 145 exhibited increased viability when co-cultured with mutants in each of the QS transcriptional 146 regulators. S. maltophilia co-cultured with either the lasR or rhlR mutants had a 5-log increase 147 and the pqsR mutant had a 6-log increase in S. maltophilia viability, as compared to wild-type 148 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint PAO1. These data show that S. maltophilia is better able to compete against P. aeruginosa QS 149 mutants, demonstrating that P. aeruginosa QS mediates competition with S. maltophilia. 150 151 Figure 2. Competition between P. aeruginosa and S. maltophilia is mediated by quorum 152 sensing. Wild-type S. maltophilia K279a was grown in mono- or co-culture with wild-type P. 153 aeruginosa PAO1, PAO1∆lasR, PAO1∆lasR, PAO1∆rhlR, and PAO1∆pqsR. The graph shows S. 154 maltophilia viability at 24 hours where the x-axis shows each condition and the y-axis shows S. 155 maltophilia CFUs. The experiment was performed in triplicate. Error bars represent the standard 156 deviation. 157 158 Prior work has shown that several P. aeruginosa LasR- and RhlR-regulated factors are 159 important for mediating competition with other bacteria. These products include phenazines 160 such as pyocyanin, hydrogen cyanide, and the biosurfactants rhamnolipids. We tested whether 161 these QS-regulated factors were driving the competition by co-culturing S. maltophilia with 162 mutants deficient in phenazine biosynthesis (PAO1∆phzA1), hydrogen cyanide production 163 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint (PAO1∆hcnC), or rhamnolipid biosynthesis (PAO1∆rhlB) (Supplementary Figure 3). We found 164 that P. aeruginosa mutants unable to produce hydrogen cyanide or rhamnolipids had minor 165 competitive defects when co-cultured with S. maltophilia. Further, we found that P. aeruginosa 166 mutants unable to produce phenazines exhibited similar competition with S. maltophilia as the 167 wild-type. We concluded that while these factors explained a component of the competition with 168 S. maltophilia, they do not account for large competitive defects we observed with the QS 169 transcriptional regulator mutants. 170 171 Alkylquinolones by themselves do not mediate competition with S. maltophilia 172 LasR activates pqsR expression, and therefore a lasR mutant does not exhibit PqsR activity 173 (34, 52). Having excluded major LasR- and RhlR- regulated factors as mediators of S. 174 maltophilia killing, we reasoned that the competitive defect in the lasR mutant (Figure 2) may be 175 due to a resultant lack of PQS QS. Alkylquinolones that are produced as part of PQS 176 biosynthesis have been shown to mediate interbacterial interactions, such as HQNO-mediated 177 growth inhibition of S. aureus (12, 18, 22). Therefore, we explored the role of PQS QS in 178 mediating interactions between P. aeruginosa and S. maltophilia. 179 180 We first asked whether alkylquinolones could mediate competition with S. maltophilia, like in the 181 case of S. aureus. Some alkylquinolones are commercially available, including the signaling 182 molecules PQS and HHQ and the inhibitor HQNO, so we treated S. maltophilia monocultures 183 with purified PQS, HHQ, or HQNO at concentrations previously determined in stationary phase 184 cultures (36, 40, 53) (Figure 3A). We found no change in S. maltophilia viability when treated 185 with each of the alkylquinolones. Since P. aeruginosa produces all three of the alkylquinolones 186 during growth, we next probed whether the alkylquinolones mediate competition when added in 187 combination. We discovered that even when added in dual or triple combinations, S. maltophilia 188 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint viability was unchanged compared to the untreated control. These data indicate that 189 alkylquinolones are insufficent to effect killing of S. maltophilia by P. aeruginosa. 190 191 192 Figure 3. PQS QS mediates competition, but alkylquinolones alone do not mediate 193 competition. A. Wild-type K279a culture was supplemented with 20 µM 2-heptyl-3-hydroxy-194 4(1H)-quinolone (PQS), 50 µM alkyl-quinolone signals 2-heptyl-4-quinolone (HHQ), or 50 µM 2-195 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint heptyl-4-hydroxyquinoline N-oxide (HQNO) individually or in combination. Cultures were grown 196 for 24 hours. B. Wild-type K279a was grown in mono- or co-culture with wild-type PAO1, 197 PAO1∆pqsR, PAO1∆pqsA, PAO1∆pqsB, PAO1∆pqsC, PAO1∆pqsD, or PAO1∆pqsH. C. Wild-198 type K279a was grown in mono- or co-culture with wild-type PAO1, PAO1∆pqsR, PAO1∆pqsA, 199 PAO1∆pqsB, PAO1∆pqsC, PAO1∆pqsD, or PAO1∆pqsH supplemented with 20 µM PQS. All 200 graphs show S. maltophilia viability at 24 hours where the x-axis shows each condition and the 201 y-axis shows S. maltophilia CFUs. Each experiment was performed in triplicate. Error bars 202 represent the standard deviation. 203 204 Products of PQS QS mediates competition between P. aeruginosa and S. maltophilia 205 The PQS QS pathway regulates the expression of the pqs operon (pqsABCDE and phnAB) that 206 not only produces the compounds PQS, HHQ, HQNO, but also over 20 intermediates required 207 to produce these compounds (36, 54). We next wanted to probe the role of these compounds 208 and test the role of the pqs biosynthesis operon in mediating competition between P. aeruginosa 209 and S. maltophilia. To do so, we took a genetic approach by generating deletion mutants in each 210 of the pqs biosynthesis genes and competed these mutants against S. maltophilia. (Figure 3B). 211 Identical to PAO1∆pqsR, we observed a large competitive defect in each of the pqs biosynthesis 212 genes when competed against S. maltophilia, where growth and viability is only slightly less 213 than in monoculture. These data are consistent with the idea that PQS QS, specifically a 214 product of the PQS biosynthesis operon, mediates competition between P. aeruginosa and S. 215 maltophilia. 216 217 We next wanted to identify if any specific genes in the pqs biosynthesis operon, and possibly 218 their products, mediated competition. Gene products of the pqs biosynthesis operon produce a 219 series of intermediates that ends in PQS. PQS is the major quinolone that binds to and activates 220 PqsR, which in turn upregulate expression of the pqs biosynthesis operon, creating a positive 221 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint feedback loop. Therefore, a mutant in any of the pqs biosynthesis operon genes disrupts the 222 production of PQS, the intermediate alkylquinolone products, and also the positive feedback 223 loop required for QS. To test the role of the specific gene in the pqs biosynthesis operon while 224 also having the feedback loop remain active, we competed individual pqs biosynthesis operon 225 mutants against S. maltophilia and added PQS at the beginning of the experiment (Figure 3C). 226 Adding PQS relieves the defect in pqsABCDE expression but not the production of 227 alkylquinolones in these mutants. We observed that addition of PQS to pqsA, pqsB, pqsC, and 228 pqsD mutants did not change the competition phenotype, indicating that these genes and their 229 products were insufficient to kill S. maltophilia. Interestingly, the pqsH mutant (which is only 230 defective in production of PQS itself) supplemented with exogenous PQS regained the ability to 231 compete with S. maltophilia; we observed a ~4-log decrease in S. maltophilia CFUs when PQS 232 was added compared to when PQS was absent. These data indicate that a product of PQS QS 233 mediated competition between P. aeruginosa and S. maltophilia, although it may not be among 234 the enzymes that synthesize intermediates required for producing PQS. 235 236 HQNO is required for competition between P. aeruginosa and S. maltophilia 237 HQNO is another terminal product of alkylquinolone synthesis that is produced by the enzyme 238 PqsL (Supplemental Figure 1). This is a branch from the PQS synthesis pathway. PqsL diverts 239 some of the 2-ABA away from PQS biosynthesis by converting of 2-ABA to a hydroxylamino 240 derivative of 2-ABA that is subsequently converted to HQNO by the enzymes PqsB and PqsC. 241 As such, we assessed the role of PqsL and its products in mediating competition between P. 242 aeruginosa and S. maltophilia by growing S. maltophilia in co-culture with a PAO1∆pqsL mutant 243 (Figure 4A). The pqsL mutant is only deficient in its ability to produce HQNO but it can still 244 activate PQS QS. This pqsL mutant, unlike a pqsR deletion mutant, still killed S. maltophilia, 245 although not as well as the WT (Figure 4A). Compared to S. maltophilia monoculture, we 246 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint observed a ~3-log decrease in S. maltophilia CFU when co-cultured with the pqsL deletion 247 mutant. 248 249 Because this pqsL mutant is only deficient in HQNO production, the increase in S. maltophilia 250 viability as compared to the WT is likely attributable to HQNO. To test this idea, we added 251 exogenous HQNO to the S. maltophilia - PAO1∆pqsL co-culture. We observed that, with the 252 addition of HQNO, S. maltophilia viability was identical to that seen in co-culture with WT P. 253 aeruginosa. This result indicated that HQNO can complement the competitive defect seen in the 254 pqsL mutant and that HQNO mediates an element of the competition between P. aeruginosa 255 and S. maltophilia. 256 257 The result that HQNO is important for S. maltophilia killing was seemingly contrary to our prior 258 data: when we added HQNO to S. maltophilia monoculture, we did not observe any changes in 259 viability (Figure 3A), unlike what has been shown for the Gram-positive bacterium S. aureus 260 (12, 18, 22). To investigate this paradox, we asked whether S. maltophilia killing by any of the 261 other mutants of the pqs biosynthesis operon could be complemented with the addition of 262 HQNO (Figure 4B). We found that the addition of HQNO did not change S. maltophilia viability 263 in co-culture with any of these mutants. Because the pqsL mutant is the only mutant in the PQS 264 and HQNO biosynthesis pathway that maintains the ability to activate pqs QS, we reasoned that 265 it was likely that killing by HQNO is dependent on the presence of another PQS QS-regulated 266 factor. 267 268 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 269 Figure 4. Competition between Pa and Sm is partially mediated by HQNO and requires 270 PQS QS. A. Wild-type K279a was grown in mono- or co-culture with wild-type PAO1 or 271 PAO1∆pqsL supplemented with 50 µM HQNO. B. Wild-type K279a was grown in mono- or co-272 culture with wild-type PAO1, PAO1∆pqsA, PAO1∆pqsB, PAO1∆pqsD, or PAO1∆pqsH 273 supplemented with 50 µM HQNO. All graphs show S. maltophilia viability at 24 hours where the 274 x-axis shows each condition and the y-axis shows S. maltophilia CFUs. Each experiment was 275 performed in triplicate. Error bars represent the standard deviation. 276 277 A PqsE-regulated factor potentiates HQNO activity 278 We therefore aimed to pinpoint the identity of this other factor that could potentiate HQNO 279 activity. Our data thus far indicate that pqs QS was important in addition to HQNO activity for 280 killing (Figure 4). We turned our attention to PqsE. PqsE has thioesterase activity and can 281 catalyze the conversion of 2-aminobenzoylacetyl-coenzyme A to 2-aminobenzoylacetate; 282 however, it is disposable for PQS biosynthesis in PAO1 (55, 56). PqsE also links the PQS and 283 rhl QS pathways by serving a chaperone-like function for RhlR (57, 58); there are RhlR 284 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint regulated genes (such as the phenazines) whose expression are exquisitely dependent on 285 PqsE (59-62). 286 287 We assessed the individual role of PqsE and, by connection, RhlR, activity in modulating 288 competition between P. aeruginosa and S. maltophilia (Figure 5A). The pqsE mutant killed S. 289 maltophilia, but not as well as the WT, resulting in a ~3-log decrease in S. maltophilia CFU as 290 compared to monoculture growth. We next tested whether the pqsE mutant could be 291 complemented with the addition of exogenous HQNO (Figure 5A). We did not observe any 292 changes in S. maltophilia viability when HQNO was added to the co-culture with PAO1∆pqsE. 293 These indicate a role for PqsE in competition and suggest that factors regulated by PqsE may 294 be important for potentiating HQNO activity against S. maltophilia. 295 296 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 297 Figure 5. HQNO and PqsE are both required to mediate competition between Pa and Sm. 298 A. Wild-type K279a was grown in mono- or co-culture with wild-type PAO1 or PAO1∆pqsE 299 supplemented with 50 µM HQNO. B. Wild-type K279a was grown in mono- or co-culture with 300 wild-type PAO1 or a PAO1∆pqsA mutant overexpressing pqsE supplemented with 50 µM 301 HQNO. C. Wild-type K279a was grown in mono- or co-culture with wild-type PAO1, 302 PAO1∆pqsE, PAO1∆pqsL, and PAO1∆pqsE∆pqsL supplemented with 50 µM HQNO. All graphs 303 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint show S. maltophilia viability at 24 hours where the x-axis shows each condition and the y-axis 304 shows S. maltophilia CFUs. Each experiment was performed in triplicate. Error bars represent 305 the standard deviation. 306 307 We next asked whether the PqsE alone was necessary for HQNO-mediated killing of S. 308 maltophilia. To answer this question, we created a pqsA knockout mutant that expressed an 309 arabinose-inducible copy of pqsE at a neutral location in the chromosome. We competed this 310 mutant against S. maltophilia with and without HQNO present (Figure 5B). This mutant allowed 311 us to test whether PqsE is important for potentiating HQNO activity in the absence of other 312 alkylquinolones. Surprisingly, we observed no difference in S. maltophilia CFUs when it was co-313 cultured with the pqsA mutant or the pqsA mutant overexpressing pqsE. Further, we observed 314 that the pqsA mutant overexpressing pqsE did not kill S. maltophilia even when HQNO was 315 added. These results indicated that pqsE expression is insufficient to mediate competition with 316 S. maltophilia, whether HQNO is present. These data also are consistent with the idea that 317 some other part of pqs QS, in addition to PqsE expression and HQNO production, is necessary 318 for P. aeruginosa to kill S. maltophilia. 319 320 HQNO, PqsE, and the pqs biosynthesis pathway are required for mediating competition 321 between P. aeruginosa and S. maltophilia 322 We hypothesized that PqsE was required in conjunction with pqs QS and HQNO to mediate 323 competition with S. maltophilia. To assess this hypothesis, we made a pqsE and pqsL double 324 knockout mutant that has no PqsE activity or HQNO production, but this mutant can still activate 325 pqs QS (Figure 5C). As we observed in earlier experiments, both the pqsE and pqsL single 326 knockout mutants exhibited partial competitive defects, which could be complemented in the 327 pqsL mutant with exogenous HQNO. We observed that the pqsE and pqsL double knockout 328 mutant had a competitive defect akin to the pqs biosynthesis mutants. These data also show 329 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint that while pqs QS is important, it is insufficient and likely requires both PqsE and PqsL for killing 330 S. maltophilia. 331 332 We further investigated how PqsE and PqsL may be co-implicated in the competion. Although a 333 pqsL mutant does not impair PQS production, a pqsL mutant has been shown to increase 334 production of the signaling molecules HHQ and PQS (63, 64). We wondered whether pqsE 335 expression was affected in this mutant and performed a quantitative real time PCR experiment 336 to monitor pqsE expression in wild-type PAO1, PAO1∆pqsA, PAO1∆pqsB, and PAO1∆pqsL in 337 late-log phase cultures (Figure 6). We found that compared to wild-type PAO1, pqsE expression 338 decreased by 2-fold in the pqsA and pqsB deletion mutants. This decrease likely reflects a lack 339 of PqsR activity and therefore reduced induction of the pqs operon. Indeed, the decrease was 340 ameliorated by addition of exogenous PQS to these mutants, showing that the deletions do not 341 dramatically affect mRNA stability (Supplementary Figure 4). Unexpectedly, we found that the 342 pqsL mutant exhibited a 6-fold increase in pqsE expression compared to the wild-type. In the 343 pqsL mutant, the enhanced expression of pqsE that likely results in increased PqsE activity. 344 This result suggested to us that in the absence of HQNO production, there is likely some 345 competition mediated by the increased pqs QS and PqsE activity, but full competition requires 346 HQNO. 347 348 Finally, we tested whether the pqsE and pqsL mutant could be complemented by the addition of 349 exogenous HQNO (Figure 5C). We found that unlike the pqsL single knockout mutant, the pqsE 350 and pqsL double knockout mutant is no longer complemented by the addition of HQNO. These 351 data indicate that a PqsE-regulated factor is required for HQNO-mediated competition with S. 352 maltophilia. Taken together, these data demonstrate a role for HQNO, PqsE, and PQS QS in 353 mediating competitive interactions between P. aeruginosa and S. maltophilia. 354 355 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 356 Figure 6. A pqsL mutant has increased pqsE expression compared to wild-type PAO1. 357 Quantitative real-time PCR measuring pqsE transcripts at an OD600 of 1.0 in wild-type PAO1, 358 PAO1∆pqsA, PAO1∆pqsB, and PAO1∆pqsL cultures. pqsE expression was normalized to rplU 359 and reported as 2-∆∆CT. 360 361

362 We explored the competition between the Gram-negative bacteria P. aeruginosa and S. 363 maltophilia and defined a suite of P. aeruginosa genes that modulated interactions between 364 these bacteria. Using our co-culture model, we identified that P. aeruginosa kills S. maltophilia. 365 We showed, using a genetic approach, that this killing depends on P. aeruginosa QS. We 366 identified a mechanism where components of the P. aeruginosa quinolone QS circuit mediate 367 interactions with another Gram-negative bacterium, a new discovery. We showed that both the 368 alkylquinolone HQNO and the protein PqsE are each necessary but insufficient for competition 369 between the two bacteria. Our data suggest that all three of pqs QS, HQNO production, and 370 PqsE are required for full killing; removal of any one of these results in significant attenuation 371 (Figure 7). 372 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint 373 374 Figure 7. P. aeruginosa and S. maltophilia interact via competition largely driven by PQS 375 QS and biosynthesis. A model showing that the competition in co-culture is driven by an AND 376 logic gate where the PQS QS-produced inhibitor HQNO, PQS biosynthesis, and PqsE-mediated 377 activity are required to mediate S. maltophilia killing. 378 379 Our work offers a new perspective on interactions between P. aeruginosa and S. maltophilia. 380 Prior studies, in models systems that interrogated growth in a mouse and in biofilms, 381 established that the interaction between these two bacteria can be cooperative (65, 66). S. 382 maltophilia had increased bacterial load in the presence of viable P. aeruginosa; the bacteria 383 together formed intergrated biofilms (65). Further, S. maltophilia reduced P. aeruginosa 384 mobility, but co-culture induced alginate expression in P. aeruginosa and may protect S. 385 maltophilia against tobramycin in a mixed biofilm (66). A recent study found that PQS induced 386 aggregation in S. maltophilia grown in a modified minimal medium, and this aggregation 387 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint conferred protection against P. aeruginosa competition (67). Our experiments established 388 another example that the interactions between these two bacteria can be antagonistic. We 389 showed that P. aeruginosa decreases S. maltophilia bacterial load through factors, both intrinsic 390 and secreted, regulated by pqs QS. These differences highlight the importance of the conditions 391 of experiments on bacterial physiology and interactions; for example, the buffered medium that 392 we use enhances the stability of AHL signals, possibly revealing QS-specific phenotypes. 393 394 QS has been shown to regulate several factors involved in interspecies interactions, but our 395 finding of a role for pqs QS in competition with a Gram-negative bacterium is new. The RhlR-396 regulated products hydrogen cyanide, rhamnolipids, and phenazines in mediating competition 397 with other bacteria. Although our data are consistent with a minor role for these factors in 398 mediating competition, they highlight the importance of the alkylquinolone HQNO. While HQNO 399 has been shown to act on other bacteria like S. aureus, our work shows a unique mechanism in 400 that HQNO by itself is unable to affect S. maltophilia but instead requires other P. aeruginosa 401 factors for its activity. HQNO, PqsE-dependent activity, and pqs biosynthesis forms a three-402 component AND logic gate for S. maltophilia killing. 403 404 PqsE acts as a chaperone for the transcriptional regulator RhlR and is required for expression 405 of a subset of RhlR-regulated genes (59-62). For example, RhlR can positively regulate the 406 expression of rhlAB, genes encoding enzymes that produce rhamnolipids, but require PqsE to 407 positively regulate the expression of the phenazine biosynthesis genes. We showed that HQNO 408 activity is potentiated by expression of PqsE, likely a RhlR-regulated factor (including, possibly, 409 conventional RhlR-regulated toxins like hydrogen cyanide). Future work may explore PqsE-410 dependent RhlR-regulated factors that drive competition of P. aeruginosa with other Gram-411 negative bacteria. Another question that arises from our work is what is the other factor driving 412 competition that requires the PQS biosynthesis pathway? A PAO1∆pqsA mutant overexpressing 413 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint pqsE in the presence of HQNO did not kill S. maltophilia (Figure 5B). This mutant is deficient in 414 the ability to produce a variety of products in the PQS biosynthesis pathway, but we found that 415 the addition of HHQ, HQNO, and PQS are insufficient to restore competition. The branching 416 nature of the PQS biosynthesis pathway makes it difficult to use a genetic approach to 417 idenfication of this product or products, but doing so would likely reveal novel modes of 418 competition mediated by PQS biosynthesis and further our understanding of competitive 419 interactions between bacteria. 420 421 It would be interesting to see how these interactions change by varying growth conditions. For 422 example, the role of pqs QS, in co-culture during biofilm formation is not addressed by our 423 work. Another context would be to replicate the nutritional environment of infection sites, such 424 as by using synthetic cystic fibrosis medium (68). Beyond the context of infection, experiments 425 could examine the impact of QS, HQNO, or PQS biosynthesis in the context of growth in the 426 environment, including in water or the soil, using a variety of clinical and environmental isolates. 427 Studies such as these would further our understanding of interbacterial interactions. 428 429 Our work furthers our understanding on how bacteria use QS to mediate interactions and 430 sociality. We discovered a mechanism for competition between P. aeruginosa and S. 431 maltophilia, where HQNO acts in concert with PqsE and PQS biosynthesis to kill bacteria. 432 These results have implications that may apply to other interactions. For example, what effect 433 do PqsE and HQNO have on other Gram-negative bacteria? Have other gram-negative bacteria 434 evolved countermeasures to negate the effects of HQNO? Is this quinolone-based mechanism 435 of competition used by bacteria other than P. aeruginosa in the clinical or environmental setting? 436 Our work further expands our understanding of QS and sets the stage to study how 437 environmental conditions affect the mechanisms of bacterial competition. 438 439 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint

440 Bacteria and growth conditions. Strains and plasmids used in this study are listed in 441 Supplemental Tables 1 and 2. P. aeruginosa was grown in lysogeny broth (LB) buffered with 442 50 mM 3-(N-morpholino) propanesulfonic acid, pH 7.0 (LB-MOPS). Escherichia coli was grown 443 in LB. Cultures were grown in 18-mm test tubes at a volume of 3 mL in a shaking incubator (250 444 RPM) at 37ºC. For individual colony growth we used LB supplemented with 1.5% agar. Where 445 required, broth cultures of E. coli and P. aeruginosa were supplemented with gentamicin at a 446 concentration of 10 µg per mL (Gm10). E. coli colonies were grown on LB supplemented with 447 1.5% agar and gentamicin at 10 µg per mL. P. aeruginosa colonies were grown on LB 448 supplemented with 1.5% agar and gentamicin at 100 µg per mL (Gm100) for transformations or 449 10 µg per mL for maintaining cultures. 450 451 Construction of P. aeruginosa mutants. In all experiments with the laboratory strain PAO1, 452 we used strain P. aeruginosa PAO1-UW (69). In-frame deletions of pqsA, pqsB, pqsC, pqsD, 453 pqsE, pqsH, and pqsL were generated using two-step allelic exchange as previously described 454 (70). Briefly, constructs for gene deletions were created by using a pEXG2 vector backbone and 455 Gibson assembly to 1000 bp of DNA flanking each side of the gene of interest to facilitate 456 homologous recombination. E. coli S17-1 was transformed with each construct and used to 457 deliver knockout plasmids to P. aeruginosa via conjugation. Merodiploids were selected by 458 plating on Pseudomonas Isolate agar containing Gm100, and deletion mutants were then 459 selected on LB agar containing 10-15% sucrose and no sodium chloride. All deletion mutants 460 were confirmed by PCR and sequencing of genomic DNA. 461 462 Overexpression constructs were created using a pUC18T mini Tn7T integrating plasmid with an 463 arabinose-inducible promoter and Gibson assembly to clone in either pqsE or mexEF-oprN CDS 464 downstream of the promoter (71). The overexpression construct was electrotransformed into P. 465 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint aeruginosa as described elsewhere and selected using LB supplemented with 1.5% agar and 466 gentamicin at 100 µg per mL (Gm100) (72). Proper integration at the attTn7 attachment site was 467 confirmed by PCR and sequencing of genomic DNA. Gentamicin susceptible mutants were 468 made using Flp-mediated excision of gentamicin resistance marker (73). 469 470 Co-culture assay. P. aeruginosa and S. maltophilia cultures were inoculated in biological 471 duplicate from single colonies into 18-mm test tubes containing LB-MOPS and grown overnight 472 37˚C with shaking. Cultures were back diluted to an OD600 of 0.05 and grown until OD600 of 0.1. 473 Exponential phase cultures were used to inoculate 18-mm tubes containing LB-MOPS to an 474 OD600 of 0.025 with a 3 mL final volume. Chemical supplementation of PQS, HHQ, or HQNO 475 was also added at this time, if required. Cultures were incubated at 37˚C with shaking for 24 476 hours. At various time points, 100 µL of culture was sampled and used for colony forming unit 477 (CFU) assays. To select for S. maltophilia, cultures were plated onto LB agar containing 10 478 µg/mL gentamicin. To select for P. aeruginosa, cultures were plated onto cetrimide agar or 479 Columbia agar containing 30 mg/L 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10-480 phenylacridine hydrochloride (C-390) and 30 mg/L phenanthroline (51). 481 482 483 RNA isolation. Wild-type PAO1, PAO1∆pqsA, PAO1∆pqsB, and PAO1∆pqsL cultures were 484 started from single colonies in LB-MOPS in biological duplicate. Cultures were incubated for 18 485 hours at 37˚C with shaking. Cult3ures were back diluted to an OD600 of 0.025 and grown until 486 cultures reached an OD600 of 0.1, and then cultures are diluted back to an OD600 of 0.05. At an 487 OD600 of 1.0, a total of an OD600 of 2.0 was pelleted at 4000 RPM for 5 min. Supernatant was 488 discarded and pellets were resuspended in 1 mL QIAzol containing lysis beads. Samples were 489 lysed by bead-beating at maximum RPM for 1 min with chilling for 5 min. Bead-beating was 490 repeated twice. Chloroform was added, shaken vigorously, and centrifuged for 15 min at 12,000 491 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint x g at 4˚C. 450 µL of the upper phase was combined with 675 µL of 100% ethanol. Samples 492 RNA was extracted using a RNeasy kit with one on-column DNase (cat. No. 79254, Qiagen) 493 treatment, and RNA was eluted using RNase-free water. 494 495 qRT PCR analysis. RNA was extracted as described above. 250 ng total cDNA was generated 496 by reverse transcription using the qScript cDNA synthesis kit. Quantitative real-time PCR was 497 performed using 2.5 ng total cDNA using the PowerTrack SYBR Green Mix. Primers targeting 498 pqsE were used to monitor pqsE expression. Primers amplifying rplU were used as a 499 housekeeping gene. Data analysis was determined using relative gene expression levels using 500 2-∆∆Ct method. 501 502 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint

503 We thank Maureen Thomason and Nicole Smalley for their assistance in cloning. This work was 504 funded in part by grants from Cystic Fibrosis Foundation (FRANDO24F0) to AF and the NIH 505 (R35GM152107) to AAD. 506 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted January 27, 2026. ; https://doi.org/10.64898/2026.01.27.702039doi: bioRxiv preprint

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