The long-chain fatty acid-CoA ligase FadD1 monitorsPseudomonas aeruginosaquorum sensing as a receptor of cis-2-decenoic acid

64 Quorum sensing (QS) is a cell-to-cell communication mechanism utilized by numerous species of 65 bacteria. The first QS signal discovered in gram -negative bacteria is the acyl homoserine lactone 66 (AHL) produced by Vibrio fischeri1-5. In addition to AHL-family signals, there are many other types of 67 QS signals6-16, including the diffusible signal factor (DSF)-family signals. Pseudomonas aeruginosa is 68 a major source of opportunistic infections in both immunocompromised individuals and cystic fibrosis 69 patients and has evolved at least three types of QS systems, e.g., las, pqs and rhl17-19. The las and 70 rhl AHL QS systems use N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-71 l-homoserine lactone (C4 -HSL), respectively, to regulate biological functions, such as biofilm 72 formation, motility and virulence factor production 20-24. The pqs system employs 2-heptyl-3-hydroxy-73 4(1H)-quinolone (PQS), to regulate biological functions and virulence 25-29. These QS systems of P. 74 aeruginosa are hierarchically interrelated, and the las system was verified to monitor both the rhl and 75 pqs systems17, 30. Moreover, it was also revealed that the fatty acid signaling molecule cis-2-decenoic 76 acid ( cis-DA) exhibited important functions in P. aeruginosa 31. The production of cis-DA in P. 77 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint aeruginosa needs an enoyl-CoA synthase encoded by D spI32. The inactivation of D spI leads to a 78 significant reduction in biofilm dispersion33,34. However, it is not yet clear how cis-DA regulates biofilm 79 dispersion, and its receptor and downstream signaling network remain to be investigated. 80 Fatty acyl coenzyme A (CoA) synthetases (FACSs; fatty acid-CoA ligases) are a class of enzymes 81 that activate alkanoic acids to CoA esters 35. FACSs are widely distributed in both prokaryotic and 82 eukaryotic organisms and exhibit broad substrate specificity 36. The long-chain fatty acid-CoA ligase 83 (FadD) is responsible for the activation of endogenous long-chain fatty acids into acyl-CoAs37. RpfB, 84 which has recently been reported as a long -chain fatty acid-CoA ligase of Xanthomonas campestris 85 pv. campestris (Xcc), is involved in the activation of a wide range of fatty acids to their CoA esters in 86 vitro38. Intriguingly, RpfB is required for the turnover of both cis-11-methyl-dodecenoic acid (DSF) and 87 cis-2-dodecenoic acid (BDSF) QS signals in vivo39. The rpfB deletion mutant produced high levels of 88 both DSF and BDSF signals and displayed increased virulence in Xcc40,41. Moreover, RpfB-dependent 89 QS signal turnover was also detected in various bacterial species40. 90 Transcription factors (TFs) are proteins that bind to specific DNA sequences, thereby regulating the 91 transcription of genetic information from DNA to messenger RNA 42. Once bound to DNA, these 92 proteins can promote or block the recruitment of RNA polymerase to specific genes to control 93 transcriptional expression43. Although there is great diversity in the structure and function of DNA -94 binding proteins, some polypeptide motifs enable binding to the major groove of DNA, including helix-95 turn-helix (HTH), helix-loop-helix (HLH), zinc fingers, and leucine zippers44-47. 96 In this study, we demonstrated that FadD1 of P. aeruginosa could convert BDSF to cis-2-dodecenoic 97 acid-CoA and reduce the competitiveness of B. cenocepacia. FadD1 also controls the physiology and 98 virulence of P. aeruginosa by sensing cis-DA, then monitors the QS hierarchy and controls the 99 expression of various target genes as a global transcriptional regulator. These findings indicate that 100 FadD1 is a member of a new class of response regulators of QS signal that controls the important 101 biological functions and virulence of bacterial pathogens, with an additional role in fatty acid oxidation 102 to catalyze the formation of fatty acyl-CoA. 103 104

105 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint FadD1 of P. aeruginosa shows enzyme activity on the BDSF signal 106 BDSF was first found to be involved in the regulation of biofilm formation, motility and virulence of B. 107 cenocepacia9-12,48,49. It was subsequently reported that BDSF from B. cenocepacia interferes with QS 108 systems and the type III secretion system (T3SS) of P. aeruginosa 50. Interestingly, w e found that 109 BDSF could also be degraded when it was added to the culture of P. aeruginosa PAO1. The amount 110 of BDSF in the culture of P. aeruginosa decreased to 2 1.78% after 6 h of incubation ( Figure 1A). 111 Previous work demonstrated that RpfB is a long -chain fatty acid -CoA ligase responsible for BDSF 112 and DSF signal turnover in Xcc38,39. To discover the enzyme that degrades BDSF, we then searched 113 RpfB homologs in the genome of P. aeruginosa PAO1 by using the Basic Local Alignment Search 114 Tool (BLAST) algorithm51. We found six RpfB homologs, FadD1-652; among them, FadD1 showed the 115 highest homology, with 55.35% identity with RpfB. Therefore, FadD1, which contains 562 aa with a 116 calculated molecular weight of 61.67 kDa, was purified to homogeneity using affinity chromatography 117 (Figure 1B). The in vitro enzymatic activity assays showed that when FadD1 was mixed with BDSF, 118 the free BDSF level in the mixture decreased nearly undetectable after 1 h (Figure 1C). These results 119 suggested that FadD1 exhibits enzyme activity on BDSF. Consistently, deletion of fadD1 in P. 120 aeruginosa caused a substantial reduction in BDSF degradation activity (Figure 1D) but did not affect 121 the growth rate of the bacterial cells (Figure S1). 122 We then investigated the in vivo activity of FadD1 in B. cenocepacia H111 by overexpressing the 123 fadD1 gene. Overexpression of fadD1 did not affect the growth rate but markedly reduced the BDSF 124 production of the bacterial cells (Figure S2A, B). As expected, deletion of rpfFBc resulted in complete 125 abolishment of the production of BDSF in B. cenocepacia H111 ( Figure S2B). Interestingly, 126 overexpression of fadD1 in the B. cenocepacia wild-type strain reduced biofilm formation, swarming 127 motility and protease activity, which could be restored by the addition of exogenous BDSF at 5 µ M 128 (Figure S2C-E). Overexpression of fadD1 in the wild -type strain H111 also decreased cytotoxicity 129 (Figure S2F). 130 131 FadD1 boosts the competitive capability of P. aeruginosa against B. cenocepacia 132 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint As BDSF mediates the cross-talk between B. cenocepacia and P. aeruginosa 50 and FadD1 exhibits 133 catalysis activity on BDSF, we investigated whether FadD1 plays a role in the competitive interactions 134 between the two bacterial species. To this end, we cocultured the green fluorescent protein (GFP) 135 fluorescence-labeled P. aeruginosa PAO1 wild-type, Δ fadD1 and complemented strains in the 136 presence of the B. cenocepacia H111 strain labeled with mCherry and measured the BDSF 137 production of B. cenocepacia (Figure 1D) and the mean fluorescence intensity (MFI) of both B. 138 cenocepacia and P. aeruginosa at different time points as indicated ( Figure 1E, F). The results 139 showed that deletion of fadD1 caused a drastic reduction in the degradation capability of P. 140 aeruginosa toward the BDSF signal. The BDSF signal yield of B. cenocepacia cocultured with the P. 141 aeruginosa deletion mutant ΔfadD1 was more than 5 times higher than that when it was cocultured 142 with wild-type PAO1 or the complemented strain Δ fadD1(fadD1) at 9 h postinoculation ( Figure 1D). 143 On the other hand, we found that the GFP mean fluorescence intensity (MFI) of the P. aeruginosa 144 ΔfadD1 strain was lower than that of the P. aeruginosa wild-type and complemented strains when 145 they were cocultured with the B. cenocepacia strains (Figure 1E). The mCherry mean fluorescence 146 intensity (MFI) of the B. cenocepacia H111 strain cocultured with the P. aeruginosa ΔfadD1 strain 147 was higher than that of the B. cenocepacia H111 strain cocultured with the P. aeruginosa wild-type 148 and complemented strains (Figure 1F). These results indicated that FadD1 plays an important role in 149 the competition between P. aeruginosa and B. cenocepacia. 150 151 Deletion of FadD1 impairs biological functions and pathogenicity in P. aeruginosa 152 It was determined that FadD1 plays an important role in the interaction between P. aeruginosa and 153 B. cenocepacia. We continued to study whether FadD1plays a role in the regulation of biological 154 functions and pathogenesis in P. aeruginosa. We found that deletion of fadD1 resulted in decreases 155 in biofilm formation, swarming, and pyocyanin production by 30.47%, 36.72% and 62.15%, 156 respectively (Figure 2A-C). Deletion of fadD1 also attenuated cytotoxicity by 69.78% when A549 cells 157 were incubated with the P. aeruginosa strains at 8 h postinoculation (Figure 2D). We tested whether 158 there was a change in the expression level of upstream (PA3298) and downstream (fadD2) genes of 159 the fadD1 mutant gene. The results showed that there was no change in the expression of upstream 160 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint and downstream genes in the fadD1 deletion mutant (Figure S3). To further study the regulatory roles 161 of FadD1 in controlling bacterial physiology, we analyzed and compared the transcriptomes of the 162 wild-type strain and the ∆fadD1 mutant strain by using RNA sequencing (RNA-Seq). Differential gene 163 expression analysis showed that the expression levels of 23 genes were increased and those of 208 164 genes were decreased in the ∆fadD1 mutant strain compared with the expression levels in the wild-165 type strain (|Log2 fold-change| ≥ 1.0) (Table S1 and Figure S4A). These differentially expressed genes 166 are associated with a range of biological functions (Table S1 and Figure S4B). These genes include 167 the las, rhl and pqs QS system genes PA1432 (lasI), PA1430 (lasR), PA3476 (rhlI), PA3477 (rhlR), 168 PA0996 (pqsA), and PA1003 (mvfR), which were shown to be involved in pathogenicity in P. 169 aeruginosa. 170 171 FadD1 monitors the QS systems of P. aeruginosa 172 As FadD1 obviously affects the QS-regulated genes and phenotypes of P. aeruginosa Figure 2A-D, 173 Figure S4 and Table S1), we infer red that FadD1 might affect the QS systems. Analysis of the 174 expression profiles of lacZ under the control of the lasR, lasI, rhlR, rhlI, mvfR and pqsA promoters 175 showed that deletion of fadD1 resulted in reduced expression levels of lasR, lasI, rhlR, rhlI, mvfR and 176 pqsA (Figure 2E-J), which are signal molecule receptor genes and synthase-encoding genes of the 177 las, rhl and pqs QS systems, respectively. RT-qPCR analysis and RNA-seq also showed that the 178 mutation of fadD1 caused a decrease in the expression levels of lasR, lasI, rhlR, rhlI, mvfR and pqsA 179 (Figure 2K). Therefore, we measured and compared the production of 3-oxo-C12-HSL, C4-HSL and 180 PQS in the wild-type, fadD1 mutant, and complemented strains. We found that the production of 3-181 oxo-C12-HSL, C4-HSL and PQS was reduced in the fadD1 mutant strain (Figure 2L-N). These results 182 demonstrated that FadD1 positively regulated the las, rhl and pqs QS systems in P. aeruginosa. 183 184 FadD1 regulates target gene expression by directly binding to the promoter 185 FadD1 obviously affects the QS gene expression levels and QS signal production of P. aeruginosa. 186 Therefore, we performed electrophoretic mobility shift assays (EMSAs) to test whether the 187 transcriptional regulation of the signal molecule synthase-encoding genes and receptor genes of the 188 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint las, rhl and pqs QS systems is achieved by direct binding of FadD1 to their promoters. The 339-bp, 189 321-bp, 270-bp, 330-bp, 306-bp and 324-bp DNA fragments from the lasR, lasI, rhlR, rhlI, mvfR and 190 pqsA promoters were PCR-amplified and used as probes. Surprisingly, as shown in Fig ure. 3A, the 191 lasR promoter DNA fragment formed stable DNA-protein complexes with FadD1 and migrated slower 192 than unbound probes. The amount of labeled probe that bound to FadD1 increased with increasing 193 amounts of FadD1 but decreased in the presence of a 50 -fold greater concentration of unlabeled 194 probe (Figure 3A). However, FadD1 did not bind to the promoters of the other tested genes, i.e., rhlR, 195 mvfR, lasI, rhlI and pqsA (Figure S5 and Figure S6). 196 To identify the DNA motif recognized by FadD1 and the genes directly regulated by FadD1, we 197 performed chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. ChIP-Seq data analysis 198 revealed the potential FadD1 binding site as 5’-AGGACGG-3’ in the lasR gene promoter probe (Figure 199 3B). The results showed that FadD1 could directly bind to multiple target gene promoters to regulate 200 their transcription and expression (Table S2). To further examine the binding sites, three target genes, 201 flgM, katA and PA0692, were chosen for EMSA analysis. The amounts of labeled probes of flgM, katA 202 and PA0692 bound to FadD1 were also increased with increasing amounts of FadD1 ( Figure S7). 203 Then, the potential binding sites 5’ -AGGACGG-3’, 5’ -TGGCCGG-3’, 5’ -AGGAGGG-3’, and 5’ -204 AGGGCGG-3’ were deleted from the promoter regions of lasR, flgM, katA and PA0692, respectively. 205 EMSA analysis showed that no DNA-protein complex was formed when the binding site was deleted 206 from the probes (Figure 3C and Figure S8), indicating that this fragment is essential for the binding of 207 FadD1 to the lasR, flgM, katA and PA0692 promoters. Together, these results demonstrated that 208 FadD1 regulates target gene expression by directly binding to a specific region in the gene promoter. 209 210 The leucine zipper motif of FadD1 is responsible for binding with DNA 211 Since FadD1 can bind to lasR and regulate transcription, and the domain structure analysis of FadD1 212 by using the HMMER (https://www.ebi.ac.uk/Tools/hmmer/) shows that it has an AMP-binding 213 enzyme domain (AMP-B) and an AMP-binding enzyme C-terminal domain (AMP-B_C) (Figure 3D). 214 We then purified the AMP-B domain and the AMP-B_C domain for EMSA ( Figure 3E). The results 215 showed that only the AMP -B domain can bind to the promoter region of lasR (Figure 3F, G). To 216 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint determine the location of the new DNA binding motif in the AMP-B domain, we used the protein motif 217 analysis website (https://www.novopro.cn/tools/motifscan.html). A structure called "leucine zipper" 218 was characterized at 275 to 296 amino acids LTGNHNILITNPRDLPSMLKDL in AMP-B domain 219 (Figure 3D and Figure S9). The leucine zipper consists of a periodic repetition of leucine residues at 220 every seventh position over a distance covering eight helical turns. This motif is found in many 221 eukaryotic transcription factors45. However, in bacterial transcription factor, this motif is only found in 222 the classical DNA-binding HTH domain of the LysR family regulator MetR 46 and a BDSF response 223 regulator DsfR identified recently47. So we speculated that the four leucine residues L275, L282, L289 224 and L296 might be important for the binding of FadD1 to target gene promoters. We then introduced 225 four site -specific substitutions into FadD1 (named FadD1 LM), and the results showed that the 226 simultaneous mutation of L275A, L282A, L289A and L296A abolished the binding of FadD1 to the 227 lasR promoter (Figure 3I). In addition, in trans expression of FadD1 LM did not rescue the defective 228 biofilm, swarming motility and pyocyanin production phenotypes of the Δ fadD1 strain (Figure 3J-L). 229 We then used the HDOCK server to automatically predict the interaction between FadD1 and the 230 DNA-binding site 5’-AGGACGG-3’. The binding model of DNA and FadD1 and the detailed contacts 231 of the complex are shown in Figure S10 and Table S3, respectively. The binding sites of FadD1 are 232 in the leucine zipper structure. The results are consistent with the conclusion that the DNA -binding 233 motif of FadD1 is the leucine zipper. 234 235 FadD1 is a receptor of cis-DA 236 Since cis-DA is a DSF -type QS signal in P. aeruginosa, and the FadD1 controls QS-regulated 237 phenotypes as a global transcriptional regulator in P. aeruginosa, we then measured whether there 238 is an interaction between cis-DA and FadD1. To test this hypothesis, we purified FadD1 to performed 239 microscale thermophoresis (MST) analysis to test whether FadD1 binds cis-DA. As shown in Figure 240 4A, FadD1 exhibited binding activity to cis-DA, with an estimated dissociation constant (KD) of 1.06 ± 241 0.14 μM. To further confirm FadD1 is a receptor of cis-DA, we generated the cis-DA deficient mutant 242 ΔdspI, the double deletion mutants ∆dspI∆fadD1 and in trans expressed fadD1 in the Δ dspI and 243 measured the motility activity and pyocyanin of the se strains . The results showed that in trans 244 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint expression of fadD1 rescued the defective motility and pyocyanin phenotypes of the Δ dspI strain 245 (Figure 4B, C). Interestingly, we found that the addition of exogenous cis-DA rescued the impaired 246 motility and pyocyanin of ∆dspI (Figure 4B, C); however, addition of exogenous cis-DA showed no 247 effect on these phenotypes of the mutant ∆fadD1 and ∆dspI∆fadD1 (Figure 4B, C). We then measured 248 the gene expression levels of lasR, lasI, rhlR, rhlI, mvfR and pqsA in the wild -type strain and the 249 ΔdspI strain. The results indicated that deletion of dspI caused a significant decrease in the gene 250 expression levels of las, rhl and pqs QS system genes (Figure 4D-I). Therefore, we measured and 251 compared the production of 3 -oxo-C12-HSL, C4-HSL and PQS in the wild -type, dspI mutant, and 252 complemented strains. The result showed that the production of 3-oxo-C12-HSL, C4-HSL and PQS 253 was reduced in the dspI mutant strain (Figure 4J-L). These results demonstrated that both cis-DA and 254 FadD1 positively regulates the las, rhl and pqs QS systems in P. aeruginosa. 255 256 cis-DA enhances the binding of FadD1 to target gene promoters 257 To determine how the binding of cis-DA to FadD1 might affect the activity of FadD1, we examined the 258 effects of cis-DA on the binding of FadD1 to the lasR promoter by EMSA. As shown in Figure 5A, the 259 binding of FadD1 to the promoter probe of lasR was enhanced when cis-DA was present in the 260 reaction mixtures, and the amount of probe bound to FadD1 increased as the amount of cis-DA 261 increased (Figure 5A). 262 Then, we attempted to identify the cis-DA-binding sites in FadD1. Autodocking analysis revealed 2 263 amino acid residues , Gly488 (G488) and Arg552 (R552), that might be critical for the interaction 264 between FadD1and cis-DA (Figure 5B). To test whether the roles of these acid residues are related 265 to the binding to cis-DA, we then generated two single point mutants ( FadD1G488A and FadD1R552A) 266 (Figure 5C). MST analysis showed that the mutant FadD1G488A and FadD1R552A did not bind to cis-DA 267 (Figure 5D, E). Consistent with this, EMSA analysis showed that mutation of the cis-DA-binding site 268 (Gly488 and Arg552) completely eliminated the effect of cis-DA on FadD1 (Figure 5F, G). In addition, 269 in trans expression of fadD1G488A and fadD1R552A only partially rescued motility and pyocyanin (Figure 270 5H, I), and the addition of exogenous 20 μM cis-DA showed no effect on these phenotypes of the 271 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint mutant ∆dspI∆fadD1(fadD1G488A) and ∆dspI∆fadD1(fadD1R552A) (Figure 5H, I). Thus, we concluded 272 that FadD1 is a specific response regulator of cis-DA in P. aeruginosa. 273 274 Reconstruction of FadD2 with the leucine zipper motif 275 Our study found that the DNA binding motif of FadD1 is a leucine zipper motif. Therefore, we used 276 the protein motif analysis website (https://www.novopro.cn/tools/motifscan.html) to analyze whether 277 the other FadD proteins , FadD2-6, contain a leucine zipper structure. The results showed that the 278 other FadD proteins did not contain a leucine zipper structure, and EMSA analysis showed that FadD2, 279 which shares about 59.82% amino acid identity with FadD1, did not bind to the lasR gene promoter 280 (Figure S11A, B). To further investigate the role of the leucine zipper motif of FadD1, we 281 reconstructed FadD2 by adding the leucine zipper structure of FadD1 to the same part of FadD2 282 (named FadD2LZ) and tested whether the reconstructed FadD2 has regulatory activity similar to that 283 of FadD1. Therefore, FadD2LZ was purified to homogeneity using affinity chromatography ( Figure 284 S11C), and assayed whether it could bind to the lasR promoter. Intriguingly, we found that the lasR 285 promoter DNA fragment formed stable DNA -protein complexes with FadD2LZ and migrated more 286 slowly than unbound probes. The amount of labeled probe that bound to FadD2LZ increased with 287 increasing amounts of FadD2LZ but decreased in the presence of a 50 -fold greater concentration of 288 unlabeled probe ( Figure S11D). Interestingly, addition of exogenous cis-DA also enhanced the 289 binding of FadD2LZ to the lasR promoter probe at a final concentration of 20 µ M (Figure S11E). In 290 addition, in trans expression of the fadD2LZ gene rescued the defective swarming motility and 291 pyocyanin production phenotypes of the Δ fadD1 strain (Figure 11F, G). And we also found that the 292 addition of exogenous cis-DA rescued the impaired motility and pyocyanin of ∆dspI∆fadD1(fadD2LZ) 293 (Figure S11F, G). These results showed that the leucine zipper motif plays a critical role in the 294 regulatory activity of FadD1. 295 296 Analysis of the catalytic motif of FadD1 297 Previous studies reported that fatty acyl-CoA ligase plays a crucial role in catalyzing the formation of 298 fatty acyl-CoA through hydrolysis of pyrophosphate 35,36. To test whether FadD1 can function in the 299 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint uptake and activation steps of fatty acid degradation, we evaluated its enzymatic activity in vitro. The 300

showed that FadD1 had high activity on lauric acid (C12:0), while it displayed moderate activity 301 on BDSF but no activity on cis-DA (Figure S12A). In addition, the previous study found that mutation 302 of Trp433, Thr436 and Arg453 , the substrate binding stie s, results in complete inactivation of fatty 303 acyl-CoA synthetase in E. coli (37). We continued to generated three single point mutants 304 (FadD1W434A, FadD1T437A and FadD1R454A) to explore whether the mutation affects the enzyme activity 305 of FadD1 (Figure S12B). Similarly, it was shown that the FadD1W434A, FadD1T437A and FadD1R454A 306 exerted no enzyme activity on cis-DA, BDSF and lauric acid (C12:0) (Figure S12C-E). To further 307 characterize the relationship between enzyme activity of FadD1 and perception of cis-DA, we chose 308 to test the regulatory activity of the mutant FadD1R454A. We in trans expressed the fadD1R454A in the 309 ∆dspI∆fadD1 mutant strain. Interestingly, it was found that addition of exogenous cis-DA rescued the 310 impaired motility of ∆dspI∆fadD1(fadD1R454A) (Figure S12F). Consistent with this, EMSA analysis 311 showed that mutation of the substrate binding stie (Arg454) would not affect the perception of cis-DA 312 (Figure S12G). 313 314 Homologs of FadD1 are widespread in bacteria 315 To determine whether FadD1 is widely distributed, homologs of FadD1 were sought in the genome 316 database by using the BLAST program. It was indicated that h omologs of FadD1 are distributed 317 across many different bacterial species, including in the genera Pseudomonas, Azotobacter, 318 Azomonas, and Halopseudomonas (Table S4). Furthermore, the homologs of FadD1 in the genera 319 Pseudomonas, Azotobacter and Azomonas contain a leucine zipper structure (Table S4). We then 320 selected P. fluorescens Migula ATCC17518, which contains both a FadD1 Pf protein with a leucine 321 zipper structure and a lasRPf gene, and the FadD1Pf protein was purified by affinity chromatography 322 and tested for its interaction with the lasRPf gene promoter. The EMSA results showed that the 323 FadD1Pf protein from P. fluorescens could also bind to the lasRPf gene promoter ( Figure S13), 324 indicating that the transcriptional regulatory mechanism of FadD1 might be widely conserved in 325 bacteria. 326 327 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint

328 Fatty acid-CoA ligase plays a vital role in fatty acid metabolism, as it can convert fatty acids to fatty 329 acyl-CoA in bacteria37. In this study, we found that the fatty acid-CoA ligase FadD1 of P. aeruginosa 330 not only exhibits high activity for the catalysis of lauric acid (C12:0) to lauric acid-CoA (Figure. S12A), 331 and employs a key role in the bacterial competition with B. cenocepacia by degrading its QS signal 332 BDSF ( Figure 1), but also plays a critical role in the regulation of P. aeruginosa physiology and 333 pathogenesis by acting as the receptor and response regulator of its QS signal cis-DA (Figure 2 and 334 Table S1). We demonstrated that FadD1 can directly bind to the promoter of the target genes, thereby 335 regulate the transcription levels of the target genes (Figure 3A and Figure S7). It was further identified 336 that mutation of the leucine zipper structure of FadD1 abolished the binding of FadD1 to the target 337 gene promoters ( Figure 3I). Interestingly, we found that FadD2 does not contain a leucine zipper 338 structure, and EMSA analysis showed that FadD2 did not bind to the lasR gene promoter ( Figure 339 S11B). However, the reconstructed FadD2 with addition of the leucine zipper structure of FadD1 to 340 the same part of FadD2 (named FadD2LZ) obtained the regulatory activity similar to that of FadD1. It 341 could bind to the lasR gene promoter and restore the fadD1 mutant phenotypes (Figure S11). 342 Furthermore, our findings indicate that FadD1 is widely conserved in many other bacterial species, 343 including Pseudomonas, Azotobacter, and Azomonas species (Table S4), indicating that FadD1 with 344 a leucine zipper structure might be a new family of transcriptional regulatory proteins widely conserved 345 in bacteria. 346 The fatty acid molecule cis-DA was first identified in P. aeruginosa to act as the autoinducer of 347 biofilm dispersion31. It has also been shown to induce biofilm dispersion in a range of Gram-negative 348 and Gram-positive bacteria and in the fungal pathogen Candida albicans31. Although the previous 349 study reported that the two-component sensor/response regulator hybrid DspS (PA4112, 350 PA14_10770) is required and essential for native biofilm dispersion in response to the cell -to-cell 351 signaling molecule cis-DA34, there was no exploration of other functions of cis-DA. In this study, we 352 found that deletion of dspI, which is the synthase of cis-DA, caused an impairment in motility and 353 pyocyanin production (Figure 4B, C). Interestingly, in trans expression of fadD1 rescued the defective 354 motility and pyocyanin phenotypes of the Δ dspI strain ( Figure 4B, C). However, the addition of 355 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint exogenous cis-DA showed no effect on these phenotypes of the mutant ∆fadD1 and ∆dspI∆fadD1 356 (Figure 4B, C). Moreover, cis-DA is shown to bind to FadD1 with high affinity and enhanced the 357 binding of FadD1 to the target gene promoter probes (Figure 4A and Figure 5A). We also identified 358 that two amino acid residues at positions Gly488 (G488) and Arg552 (R552) are critical for the 359 interaction between FadD1 and cis-DA (Figure 5). Mutations at Gly488 and Arg552 abolished the 360 binding between FadD1 and cis-DA (Figure 5D, E). Another interesting observation in this study was 361 that mutation at the substrate binding stie Arg454 (R454) completely abolished the enzyme activity 362 of FadD1 on both BDSF and lauric acid (C12:0) (Figure S12C-E), but could not affect the effect of 363 cis-DA on FadD1 (Figure S12G), suggesting that the cis-DA-binding site is different from the 364 substrate-binding stie in FadD1. Together, FadD1 was identified to be a new response regulator of 365 cis-DA QS signal. 366 BDSF has been identified to be a DSF -family QS signal in B. cenocepacia11. As cis-DA shares a 367 similar chemical structure with that of BDSF, we then continued to explore the interaction between 368 BDSF and FadD1. Although FadD1 showed a degradation activity on BDSF, we found that FadD1 369 tightly binds to BDSF with an estimated dissociation constant (KD) of 1.65 ± 0.21 μM (Figure S14A). 370 Interestingly, the addition of exogenous BDSF also enhanced the binding of FadD1 to the lasR 371 promoter probe at a final concentration of 10 µ M (Figure S14B). To distinguish the role of BDSF to be 372 a “substrate” or “ligand” of FadD1, we then used EMSA analysis and the result showed that mutation 373 of the cis-DA-binding sites abolished the perception of cis-DA (Figure 5), but will not affect the effect 374 of BDSF on FadD1 (Figure S14C). However, EMSA analysis showed that mutation of the substrate-375 binding stie (Arg454) completely abolished the effect of BDSF on the binding of FadD1 to target gene 376 promoter (Figure S14D), while not affect the effect of cis-DA on FadD1 (Figure S12G). These results 377 suggested that BDSF also interferes with the binding of FadD1 to target gene promoters in vitro as a 378 substrate of the canonical enzyme FadD1. 379 In P. aeruginosa, there are at least three QS systems, namely, las, rhl and pqs, which are involved 380 in the regulation of a broad range of genes important for metabolism and virulence 18,19. These QS 381 systems are hierarchically interrelated, and the las system was verified to monitor both the rhl and 382 pqs systems17,30. In this study, we identified that FadD1 affects the QS -regulated phenotypes, 383 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint including the biofilm formation, swarming, and pyocyanin in P. aeruginosa (Figure 2A-C). Surprisingly, 384 we also identified that FadD1 is a cis-DA receptor protein that positively regulates the las, rhl and pqs 385 QS systems in P. aeruginosa (Figure 2 and Figure 6). These results indicated that FadD1 is not only 386 a response regulator of cis-DA but also an important upstream regulator of the QS signaling network 387 in P. aeruginosa. FadD1 regulates numerous target genes and physiological functions by monitoring 388 the QS signaling systems , which broadens our understanding of the QS signal ing network of P. 389 aeruginosa. 390 In summary, our findings suggest that FadD1, which contains a leucine zipper structure in the AMP-391 B domain, is not only an enzyme of fatty oxidation but also a receptor of cis-DA that directly controls 392 target gene expression and controls the QS network in P. aeruginosa. In addition, the BLAST search 393 revealed that homologs of FadD1 containing the leucine zipper structure are highly conserved in 394 many bacteria (Table S4), suggesting that the regulatory mechanism of FadD1 might be present in 395 various bacterial species. Consistently, our recent study revealed that the fatty acyl-CoA ligase DsfR 396 (BCAM2136), which contains a leucine zipper motif, acts as a global transcriptional regulator to control 397 B. cenocepacia virulence by sensing BDSF 47. Different from FadD1, DsfR has a specific BDSF -398 sensing site and showed no enzyme activity on BDSF , while FadD1 only binds with BDSF on the 399 substrate-binding sites . These results indicate that FadD1 and DsfR share some common 400 characteristics and are distinguished from each other. In conclusion, our work presents a unique and 401 widely conserved signal receptor of cis-DA, which might be an important new type of QS signal 402 receptor in bacteria. 403 404

of the study 405 * Limitations of the Study: As it reads right now, the section mostly focuses on future directions of the work 406 to answer outstanding open questions. While future directions can belong to this section, it is mostly the 407 place to openly discuss potential caveats of the work (technical, conceptual, or other limitations) with the 408 intent of promoting clarity and transparency. I would like to suggest that you further develop and edit this 409 section in this direction. 410 411 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint Our findings demonstrated that FadD1 is a specific receptor protein of cis-DA QS signal . It was 412 revealed that cis-DA binds to FadD1 to enhance the binding of FadD1 to the promoter DNA of target 413 genes. However, it remains unknown how does the binding to cis-DA induce the regulatory activity of 414 FadD1 on the transcription of target genes. 415 416 STAR METHODS 417 Detailed methods are provided in the online version of this paper and include the following: 418 KEY RESOURCES TABLES 419 RESOURCE AVAILABILITY 420 Lead contact 421

availability 422 EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS 423 Bacterial strains and culture conditions 424 Cell culture 425

DETAILS 426 BSDF extraction 427 Quantification of BDSF and cis-DA 428 Protein expression and purification 429 Enzyme activity assays 430 Construction of in-frame deletion mutants and complementation 431 Bacterial growth analysis 432 Competition assays in the mixed culture 433 P. aeruginosa phenotype assays 434 B. cenocepacia phenotype assays 435 Cytotoxicity assays 436 Construction of reporter strains and measurement of β-galactosidase activity 437 Quantitative reverse transcription PCR assays 438 Quantitative analysis of QS signal production in P. aeruginosa 439 RNA-Seq analysis 440 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint Electrophoretic gel mobility shift assay 441 Chromatin immunoprecipitation sequencing analyses 442 Molecular docking FadD1 443 Microscale thermophoresis assay 444 STATISTICAL ANALYSIS 445 446 SUPPLEMENTAL INFORMATION 447 Supplemental information can be found online at 448 449 ACKNOWLEDGMENTS 450 This work w as financially supported by the National Key Resear ch and Development Program of 451 China (2021YFA0717003 to Y.D.), the National Natural Science Foundation of China (32300033 to 452 S.S.) and the Scientific Research Foundation of Hainan University (KYQD(ZR)-23006 to S.S.). 453 454 AUTHOR CONTRIBUTIONS 455 Y.D. and S.S. conceived the project. S.S., J.L., and Y.D. designed the research. S.S., J.L., B.W., Y.S., 456 H.H., X.S., M.W., and B.C. performed the research. S.S., J.L., G.W., Y.H., L.X., B.G., L.Y., X.W., L-457 H.Z., and Y.D. analyzed the data. S.S. and Y.D. wrote the paper. 458 459 DECLARATION OF INTERESTS 460 The authors declare that they have no conflict of interest. 461 462 INCLUSION AND DIVERSITY 463 We support inclusive, diverse, and equitable conduct of research. 464 465

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Nature 596, 644 583-589. doi: 10.1038/s41586-021-03819-2. 645 646 647 STAR METHODS 648 KEY RESOURCES TABLES 649 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-His Abcam ab9108 Bacterial Bacterial strains see Table S5 N/A Oligonucleotides For deletion see Table S6 N/A For EMSA see Table S6 N/A For in trans expression see Table S6 N/A For recombinant protein see Table S6 N/A For qRT-PCR see Table S6 N/A Recombinant DNA Plasmids see Table S5 N/A Chemicals, peptides, and recombinant proteins Tryptone oxoid LP0042B Yeast extract oxoid PL0021 NaCl Macklin S805275 Agar Macklin A800728 Gentamicin Macklin G810322 Tetracycline Macklin T829835 Kanamycin Macklin K885955 Ampicillin Macklin A830931 2× T5 Super PCR Mix Tsinekg TSE005 ClonExpress II One Step Cloning Kit Vazyme C112-02 X-gal Solarbio X8050 HisTrap affinity columns Smart-lifesciences SA035010 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint TEV protease Beyotime P2307 LightShift Chemiluminescent EMSA Kit Thermo Fisher 20148 Crystal violet Sigma-Aldrich C6158 Monolith NT.115 Protein Labeling Kit Nano Temper MO-L014 Silicon capillaries Nano Temper MO-L022 PBS buffer Macklin 917808 Tris Beyotime ST760 Eastep Super Total RNA Extraction Kit Promega LS1040 cDNA synthesis Yeasen 11141ES60 SYBR qPCR Green Master Mix Yeasen 11201ES08 Formaldehyde Macklin F809902 Glycine Macklin 810676 Complete proteinase inhibitor cocktail Roche 4693116001 SDS Lablead L5751 EDTA Solarbio E8040 Mini-protease inhibitor cocktail Roche 11836170001 LiCl ThermoFisher AM9480 NP40 ThermoFisher 85124 Deoxycholic acid Solarbio D8460 NaHCO3 Macklin S837271 RNase A ThermoFisher EN0531 Proteinase K Macklin 39450-01-6 IPTG Macklin I811719 Glucose Sigma-Aldrich G6152 Sucrose Aladdin S112228 CytoTox 96 Kit Promega G1780 Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM) Sigma-Aldrich D6046 Fetal Bovine Serum Sigma F4135 Trypsin EDTA GIBCO 25200072 DMSO Sigma-Aldrich D5879 BDSF Abcam ab145341 C4-HSL Sigma-Aldrich 67605-85-0 3-oxo-C12-HSL Sigma-Aldrich 168982-69-2 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint cis-DA Sigma-Aldrich 15790-91-7 Lauric acid Sigma-Aldrich 143-07-7 Deposited data Raw and Analyzed RNA -Seq Data This study PRJNA1121197 Raw and Analyzed ChIP -Seq Data This study PRJNA1120965 Software and algorithms Prism GraphPad https://graphpad.com:443/ scientific-software/prism/ AutoDock AutoDock Tools https://autodock.scripps.edu/ Pymol PyMOL 3.0 https://pymol.org/ Protein Plus Protein Plus https://proteins.plus/ HDOCK HDOCK sever http://hdock.phys.hust.edu.cn/ RESOURCE AVAILABILITY 650 Lead contact 651 Further information and requests for resources and reagents should be directed to and will be 652 fulfilled by the lead contact Yinyue Deng ([email protected]). 653 654

availability 655 Constructs and reagents in this study will be made available upon request, but a completed 656

Transfer Agreement may be required if there is potential for commercial application. 657 658 Data and code availability 659 Unprocessed data related to figures in this manuscript are available from the lead contact upon 660 request. 661 This paper does not report original code. 662 Any additional information required to reanalyze the data reported in this work paper is available 663 from the lead contact upon request. 664 665 EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS 666 Bacterial strains and culture conditions 667 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint The bacterial strains and plasmids used in this work are listed in Table S5. P. aeruginosa PAO1, P. 668 fluorescens Migula ATCC17518 and B. cenocepacia H111 strains obtained from the American Type 669 Culture Collection (ATCC) were maintained in Luria-Bertani broth (LB; 10 g/L tryptone, 5 g/L yeast 670 extract, 5 g/L NaCl ) at 37° C. The following antibiotics were added when necessary: ampicillin and 671 kanamycin at 100 µ g mL-1; tetracycline, 10 µ g mL-1; and gentamicin, 10 µ g mL-1. BDSF and cis-DA 672 (HPLC ≥ 99%) were dissolved in methanol at a final concentration of 100 mM, and this solution was 673 added to the medium in the experiments. Bacterial growth was determined by measuring the optical 674 density at 600 nm. 675 676 Cell culture 677 A549 cell were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% 678 fetal bovine serum (FBS) and 1% penicillin‒streptomycin , cells were cultivated in a humidified 679 atmosphere containing 5% CO2 at 37 ° C. 680 681

DETAILS 682 BSDF extraction 683 The extraction of BDSF from culture supernatants was conducted following previously described 684 methods11,53. To quantify BDSF production in the culture of B. cenocepacia strain or P. aeruginosa 685 strain, 100 mL of the supernatant was collected. Its crude ethyl acetate extract was passed through 686 a 0.22 µ m Minisart filter unit and was then condensed to 1 mL for LC-MS analysis. 687 688 Quantification of BDSF and cis-DA 689 Quantification of BDSF and cis-DA was performed by using an LC-MS system, which consisted of an 690 ACQUITY UPLC system and a Waters Q -Tof Premier high -resolution mass spectrometer 53. An 691 ACQUITY UPLC BEH C18 column 1.7 µ m (2.1 × 50 mm) was used for the chromatography analysis 692 of BDSF and cis-DA, which was eluted with a CH 3OH gradient in water at 65 -100% supplemented 693 with 0.01% formic acid at a flow rate of 0.4 mL· min-1 for 10 min. Next, 65% CH 3OH was used for 3 694 min. The entire elution column was introduced into the Q -TOF mass spectrometer according to the 695 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint manufacturer’s instructions. The BDSF and cis-DA levels in the culture supernatant were measured 696 using the peak area in the extracted ion chromatogram. 697 698 Protein expression and purification 699 The coding regions of proteins were amplified with the primers listed in Table S6, and were attached 700 and fused to the expression vectors pET -21a, pET-28a and PDBHT2. The fusion gene constructs 701 were transformed into E. coli strain BL21. Affinity purification of the 6× His fusion proteins was 702 performed using Ni -NTA resin (Smart-lifesciences). The His -tag was removed using TEV protease 703 (Beyotime), and the cleaved fusion protein was analyzed by SDS-PAGE54,55. 704 705 Enzyme activity assays 706 The activity of FadD1 was determined following the methods 56. cis-DA, BDSF or lauric acid (C12:0) 707 were dissolved in TME buffer (Tris-HCl, 60 mM; MgCl2, 10 mM; EDTA, 1 mM; coenzyme A (CoASH), 708 0.5 mM; and Triton X-100, 0.1%; pH 7.5) and added to the mixture at a final concentration of 200 µ M, 709 while the final concentrations of FadD1 and its derivatives were 20 µ M. The reaction mixture was 710 incubated at 37° C, and the reaction was stopped by placing sample tubes in boiling water for 5 , 15, 711 30 and 60 min. cis-DA, BDSF or lauric acid (C12:0) levels were measured by LC-MS spectrometry. 712 713 Construction of in-frame deletion mutants and complementation 714 P. aeruginosa PAO1 was used as the parental strain for the generation of an in-frame deletion mutant 715 of fadD1 and dspI by the following method described previously 14. The primers used to generate 716 upstream and downstream regions flanking fadD1 and dspI are listed in Table S7. For 717 complementation analysis, the coding regions of fadD1 and dspI were amplified by PCR and cloned 718 into the plasmid pBBR1-MCS5, pBBR1-MCS2 and pLAFR3 using the primers listed in Table S6. The 719 resulting constructs were conjugated into the P. aeruginosa PAO1 mutant, B. cenocepacia H111 720 strain by electroporation14,54. 721 722 Bacterial growth analysis 723 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint An overnight bacterial culture in LB medium was inoculated into fresh media to an OD 600 of 0.01. A 724 10 mL cell suspension was grown in 50 mL conical centrifuge tubes at 37° C with shaking at 220 rpm. 725 Bacterial growth was determined by measuring the optical density at 600 nm every 4 hours . LB 726 medium was used as the negative control47. 727 728 Competition assays in the mixed culture 729 The green fluorescent protein expression vector was used and transformed into the P. aeruginosa 730 PAO1 WT, ΔfadD1 and ΔfadD1(fadD1) strains. The mCherry fluorescent protein expression vector 731 was introduced into the B. cenocepacia H111 strain by electroporation. Then, the P. aeruginosa PAO1 732 WT, ΔfadD1 and ΔfadD1(fadD1) strains were cocultured with the B. cenocepacia H111 strain at an 733 initial ratio of 1:4 (vol/vol) at OD 600 = 0.1 at 37° C with shaking at 220 rpm for 12 h. Then, the 734 quantification of BDSF was performed by using an LC -MS system, and the mixed cultures were 735 analyzed by a Spectra Max i3x multifunctional enzyme labeling instrument (Molecular Devices, CA, 736 USA)57. 737 738 P. aeruginosa phenotype assays 739 For the analysis of biofilm formation 58, bacterial cells were grown overnight at 37 °C and inoculated 740 into LB media (150 μL per well) in 96-well polypropylene microtiter plates. Microtiter plates with fitted 741 lids were incubated at 37°C with shaking at 200 rpm for 12 h. The plates were then washed to remove 742 planktonic cells and stained for 20 min with 1% (w eight/vol) crystal violet (Sigma-Aldrich). After 743 washing with water, 200 µ L of 95% (vol/vol) ethanol was added to the wells to release the stain. 744 Biofilm was determined by measuring the absorbance of the resulting solution at 570 nm. 745 Motility was determined on 0.3% semisolid agar (Becton, Dickinson and Company). Bacteria were 746 inoculated into the center of plates containing 0.8% tryptone, 0.5% glucose, and 0.3% agar. The 747 plates were incubated at 30° C for 18 h before the diameter of the colony was measured54. 748 The pyocyanin assay was performed with the method described previously 49. Briefly, bacteria were 749 cultured at 37° C for approximately 12 hours and centrifuged at 13,000 rpm for 1 min, and 1.5 mL 750 supernatants were collected and extracted with double volume chloroform with vigorous shaking at 751 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint room temperature for 30 min. The solvent phase was transferred to a new tube containing 1 mL of 1 752 N HCl (Sigma-Aldrich). The mixture was shaken gently to transfer pyocyanin to the aqueous phase. 753 The quantity of pyocyanin was determined by measurement of absorbance at 520 nm and 754 normalization against the cell density. 755 756 B. cenocepacia phenotype assays 757 Biofilm formation in 96 -well polypropylene microtiter dishes was assayed essentially as described 758 previously by P. aeruginosa. Swarming motility was determined on semisolid agar (0.3%). Bacteria 759 were inoculated into the center of plates containing 0.8% tryptone, 0.5% glucose, and 0.3% agar. The 760 plates were incubated at 30° C for 18 h before the diameter of the colony was measured. 761 Protease assays were performed following a previously described method14. Briefly, bacteria were 762 cultured at 37° C for approximately 12 hours. Cultures were centrifuged at 13,000 rpm for 5 min, and 763 the supernatants were removed and filtered through a 0.22 μm membrane. One hundred microliters 764 of supernatant was incubated at 30° C with an equal volume of azocasein (Macklin) dissolved in 765 proteolytic buffer (5 mg/mL) for 30 min. The reaction was stopped by the addition of 400 μL of 10% 766 (weight/vol) TCA (Macklin) buffer. After incubation for 2 min at room temperature, the mixture was 767 centrifuged at 13,000 rpm for 1 min to remove the remaining azocasein. Supernatants were removed 768 and mixed with 700 μL of 525 mM NaOH (Macklin). The absorbance of the azopeptide supernatant 769 was measured at a wavelength of 442 nm. Protease activity was obtained after normalization of the 770 absorbance against the corresponding cell density. 771 772 Cytotoxicity assays 773 Cytotoxicity was assessed by measuring the release of LDH from human A549 cells54. The A549 cell 774 line was cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) at 37° C with 5% CO2. A549 775 cells were seeded in 96-well plates at a density of 5 × 104 cells per well and grown until they reached 776 ~90% confluence. Culture supernatants were removed, and the cell monolayer was washed once 777 with phosphate-buffered saline (PBS). Fresh bacterial cells cultured to an OD600 of 1.0 were washed 778 and diluted in DMEM. The diluted bacterial cells were then added to the A549 cell monolayers at a 779 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint multiplicity of infection (MOI) of approximately 1000. After incubation for 8 h, the cytotoxicity was 780 determined by measuring the released lactate dehydrogenase (LDH) in the supernatants using a 781 cytotoxicity detection kit (Roche). 782 783 Construction of reporter strains and measurement of β-galactosidase activity 784 The promoters of lasR, lasI, rhlR, rhlI, mvfR and pqsA were amplified using the primer pairs listed in 785 Table S6. The resulting products were inserted upstream of the promoter less lacZ gene in the vector 786 pME2-lacZ. Transconjugants were then selected on LB agar plates supplemented with tetracycline 787 and X-gal (Solarbio). Measurement of β-galactosidase activities was performed following previously 788 described methods14,54,55. 789 790 Quantitative reverse transcription PCR assays 791 Bacterial cells were cultured at 37° C in LB broth to the logarithmic-growth phase (OD600=1.0). Cells 792 were collected by centrifugation, and total RNA was extracted using an Eastep Super Total RNA 793 Extraction Kit (Promega). cDNA synthesis was performed using a HiScript III 1st Strand cDNA 794 Synthesis Kit (Yeasen). Primers for RT-qPCR are listed in Table S6. The RT-qPCR experiments were 795 performed on a QuantStudio™ 7 RT-qPCR System (Thermo Fisher) using SYBR qPCR Green Master 796 Mix (Yeasen). The expression of the target genes was normalized to the expression level of 16S RNA. 797 The relative transcript abundance was calculated by the 2 −ΔΔCt method55. At least three biological 798 replicates were performed per sample. 799 800 Quantitative analysis of QS signal production in P. aeruginosa 801 Bacterial cells were grown in LB medium overnight with agitation at 37° C. One liter of culture 802 supernatant was collected by centrifugation and extracted with an equal volume of ethyl acetate. The 803 crude extract (organic phase) was dried using a rotary evaporator and dissolved in methanol. All of 804 the above samples were kept at 4° C until analysis. Ultrahigh -performance liquid chromatography -805 electrospray ionization tandem mass spectrometry (UHPLC -ESI-MS/MS) was performed in a 806 Shimadzu LC-30A UHPLC system with a Waters C18 column (1.8 µ m, 150 × 2.1 mm) and a Shimadzu 807 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint 8060 QQQ -MS mass spectrometer with an ESI source interface. The mass spectrometer was 808 operated in positive -ion mode. The mobile phase was prepared as 0.1% formic acid/water and 809 acetonitrile57. 810 811 RNA-Seq analysis 812 RNA was isolated from P. aeruginosa strains grown to OD 600=1.0 by using the Eastep Super Total 813 RNA Extraction Kit (Promega, Madison, USA). The concentration was measured using a Qubit® RNA 814 Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). The purity and integrity were 815 checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and the RNA Nano 816 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. 817 Sequencing libraries were generated using the NEBNext® Ultra ™ Directional RNA Library Prep Kit 818 for Illumina® (NEB, USA) and then purified by the AMPure XP system. Finally, library quality was 819 assessed by using the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples 820 was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3 -cBot-HS 821 (Illumina). After cluster generation, the library preparations were sequenced on an Illumina HiSeq 822 platform, and paired -end reads were generated. Trimmed sequence reads were aligned to the P. 823 aeruginosa PAO1 genome sequence using Bowtie2 -2.2.3, and normalized read counts were 824 compared using HTSeq v0.6.1 as described previously 59,60. Then, the FPKM of each gene was 825 calculated based on the 26 lengths of this gene, and the read count was mapped to the gene. 826 827 Electrophoretic gel mobility shift assay 828 Electrophoretic mobility shift assay (EMSA) experiments were performed as described 829 previously53,54,61. The DNA probes used for EMSA were prepared by PCR amplification using the 830 primer pairs listed in Table S6. The purified PCR products were 3 -end-labeled with biotin following 831 the manufacturer’s instructions (Thermo Fisher). The DNA-protein binding reactions were performed 832 according to the manufacturer’s instructions (Thermo Fisher). A 5% polyacrylamide gel was used to 833 separate the DNA-protein complexes. After UV cross-linking, the biotin-labeled probes were detected 834 in the membrane using a Biotin luminescent detection kit (Thermo Fisher). 835 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint 836 Chromatin immunoprecipitation sequencing analyses 837 Chromatin immunoprecipitation sequencing (ChIP-Seq) was performed according to previously 838 published methods with some modifications 62. ChIP -Seq analysis was conducted by the Wuhan 839 IGENEBOOK Biotechnology Co. Ltd. (Wuhan, China). The anti -HIS antibody (ab9108, 1:1000, 840 Abcam) was used for immunoprecipitation, and DNA fragments enriched by ChIP were sequenced 841 on an Illumina HiSeq 2000. After removing sequencing adaptors and low -quality bases, the clean 842 reads were mapped to the P. aeruginosa PAO1 genome using BWA software (version 0.7.15-r1140). 843 The peak caller MACS (version, 2.1.1.20160309; q value, 0.05) was used to localize the potential 844 binding sites of FadD163. 845 846 Molecular docking FadD1 847 The 3D structure of the double-stranded DNA was built in the MOE1 DNA/RNA Builder. Protein-DNA 848 docking in the HDOCK2 server was used for molecular docking simulation of FadD1 and DNA 64,65. 849 The 3D structure of FadD1 (Q9HYU4) predicted by AlphaFold2 was set as the receptor, and the 3D 850 structure of the DNA was set as the ligand 66. The HDOCK server automatically predicts their 851 interaction through a hybrid algorithm of template-based and template-free docking. First, the server 852 performs template -based modeling to identify possible homologous DNA templates. Second, the 853 intrinsic scoring function for protein -RNA interactions is integrated in template -free docking. Finally, 854 the top 100 predicted complex structures are provided to users for download, of which the top 10 855 models will be visualized on the result web page. The intermolecular contacts from the most likely 856 poses were further evaluated. The models of the complex and interface residues were analyzed using 857 MOE. Molecular graphics were generated by PyMOL. 858 AutoDock and PyMOL were used for docking and binding site analysis. The crystal structure of FadD1 859 from the AlphaFold2 was used for docking with cis-DA by AutoDock software. To screen the potential 860 binding sites, a grid box was prepared to wrap the entire macromolecule. The receptor grid was 861 centered at center_x =  -1.038, center_y = 1. 49, center_z =  11.31, size_x =  126, size_y =  126, and 862 size_z = 126. The exhaustiveness was set at 50. The top scoring ligand conformation was chosen , 863 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint and the potential binding sites were visualized in 3D by PyMOL . The potential binding sites were 864 visualized in 2D by Protein Plus (https://proteins.plus/). 865 866 Microscale thermophoresis assay 867 Protein-binding experiments were carried out with a Nano Temper 16 Monolith NT.115 instrument 868 (NanoTemper Technologies; www.nanotemper-technologies.com)55. In brief, the protein was labeled 869 with the L014 Monolith NT.115 Protein Labelling Kit (Nano Temper). The labeled protein and different 870 titers of unlabeled cis-DA were mixed and loaded onto standard treated silicon capillaries (Nano 871 Temper), and fluorescence was measured. The measurements were carried out at 20% LED power 872 and 40% MST power. 873 874 STATISTICAL ANALYSIS 875 No statistical method was used to predetermine the sample size. No data were excluded from the 876 analyses. The data are presented as the means ± standard deviations. Statistical analyses were 877 performed with Prism 8 software (GraphPad). Statistical significance is indicated as follows: *p < 0.05; 878 **p < 0.01; *** p < 0.001; ns = no significance (one-way ANOVA or two -way ANOVA). Biological 879 replicates and numbers of independent experiments were stated in the legends. All experiments 880 presented as representative LC-MS graphs or gels were repeated at least 3 times with similar results. 881 882 FIGURE LEGENDS 883 Figure 1. Effect of fadD1 on the competitive capability of P. aeruginosa against B. cenocepacia. 884 (A) Analysis of the BDSF signal quenching ability of P. aeruginosa PAO1 (n = 3 biological replicates). 885 (B) SDS-PAGE of the purified FadD1 protein. (C) Analysis of the enzymatic quenching activity of 886 FadD1 on BDSF signal (n = 3 biological replicates). (D) The BDSF signal production of the B. 887 cenocepacia H111 strain cocultured with P. aeruginosa PAO1 wild-type, ΔfadD1 and ΔfadD1(fadD1) 888 strains (n = 3 biological replicates). (E) Green mean fluorescence intensity of the P. aeruginosa PAO1 889 wild-type, ΔfadD1 and ΔfadD1(fadD1) strains carrying the green fluorescent protein expression vector 890 (n = 3 biological replicates). (F) Red mean fluorescence intensity of the B. cenocepacia H111 strain 891 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint carrying the mCherry fluorescent protein expression vector (n = 3 biological replicates). The data are 892 the means ± standard deviations of three independent experiments. The P. aeruginosa PAO1 wild-893 type, ΔfadD1 and ΔfadD1(fadD1) strains carried the green fluorescent protein expression vector. The 894 mCherry fluorescent protein expression vector was carried by the B. cenocepacia H111 strain. The 895 P. aeruginosa PAO1 wild-type, Δ fadD1 and Δ fadD1(fadD1) strains were cocultured with the B. 896 cenocepacia H111 strain at a ratio of 1:4 (v/v) at OD 600 = 0.1. The statistical comparisons were 897 performed using two-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001). 898 899 Figure 2. Effects of fadD1 on the QS-regulated phenotypes and QS of P. aeruginosa. Analysis 900 of biofilm formation (A), swarming motility (B), pyocyanin production (C), and virulence (D) in P. 901 aeruginosa PAO1((A), n = 5 biological replicates; (B), (C), (D), n = 3 biological replicates). Effects of 902 fadD1 on the gene expression levels of lasR (E), lasI (F), rhlR (G), rhlI (H), mvfR (I) and pqsA (J) by 903 assessing the β-galactosidase activity of the promoter-lacZ transcriptional fusions and by RT-qPCR 904 (K), which are the signal molecule synthase -encoding genes and receptor genes of the rhl, las and 905 pqs QS systems in P. aeruginosa PAO1, respectively ((E), (F), (G), (H), (I), (J), n = 6 biological 906 replicates; (K), n = 3 biological replicates). The production of 3-oxo-C12-HSL (L), C4-HSL (M) and 907 PQS (N) in the P. aeruginosa PAO1 wild-type, ΔfadD1 and ΔfadD1(fadD1) strains (n = 3 biological 908 replicates). The production of QS signals in the P. aeruginosa PAO1 wild-type strain was arbitrarily 909 defined as 100%. The data are means ± standard deviations of three independent experiments. The 910 statistical comparisons were performed using one-way ANOVA or two-way ANOVA ((A), (B), (C), (D), 911 (L), (M), (M), one-way ANOVA; (E), (F), (G), (H), (I), (J), two-way ANOVA; *p < 0.05; **p < 0.01; ***p 912 < 0.001). 913 914 Figure 3. Analysis of the binding of FadD1 to target gene promoters. (A) EMSA analysis of in 915 vitro binding of FadD1 to the promoter of lasR, in which the biotin-labeled 339-bp lasR promoter DNA 916 probe was used for the protein binding assay. A protein -DNA complex, represented by a band shift, 917 was formed when different concentrations of FadD1 protein were incubated with the probe at room 918 temperature for 30 min. (B) A potential FadD1 binding region was identified by ChIP -seq. The 919 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint conserved sequence is shown in orange. (C) Analysis of the binding between FadD1 and the mutated 920 lasR promoter with deletion of the FadD1 binding sequence AGGACGG. EMSA analysis was 921 performed in vitro. (D) Domain structure analysis of FadD1 (https://www.ebi.ac.uk/Tools/hmmer/). The 922 red box represents the leucine zipper motif ( https://www.novopro.cn/tools/motifscan.html) in AMP-B 923 domain. (E) SDS-PAGE of the purified AMP-B domain and the AMP-B_C domain of the FadD1 protein. 924 EMSA analysis of in vitro binding of the AMP-B domain (F) and the AMP-B_C domain (G) to the 925 promoter of lasR. (H) SDS-PAGE of the purified mutated FadD1 protein (FadD1 LM: L275A L282A 926 L289A L296A). (I) EMSA analysis of the in vitro binding of the mutated FadD1 protein to the promoter 927 of lasR. In trans expression of the mutated fadD1 could not restore biofilm formation (J), swarming 928 motility (K), and pyocyanin production (L) in the P. aeruginosa ΔfadD1 strain ((J), n = 6 biological 929 replicates; (K), (L), n = 3 biological replicates). The EMSA experiments were performed three times, 930 and representative images from one experiment are shown . The data are the means ± standard 931 deviations of three independent experiments. The statistical comparisons were performed using one-932 way ANOVA (**p < 0.01; ***p < 0.001; ns = no significance). 933 934 Figure 4. Effects of FadD1 on cis-DA-regulated phenotypes of P. aeruginosa. (A) MST analysis 935 of the binding of cis-DA to the FadD1. “Fnorm (‰)” indicates the fluorescence time trace changes in 936 the MST response. Complementation of the dspI mutant with fadD1. In trans expression of fadD1 937 complemented swarming motility (B) and pyocyanin production (C) in the dspI-deficient mutant. 938 Effects of dspI on the gene expression levels of lasR (D), lasI (E), rhlR (F), rhlI (G), mvfR (H), pqsA 939 (I) and the production of 3 -oxo-C12-HSL (J), C4-HSL (K) and PQS (L) of the P. aeruginosa PAO1 940 strains. The production of QS signals in the P. aeruginosa PAO1 wild-type strain was arbitrarily 941 defined as 100%. n = 3 biological replicates. The data are means ± standard deviations of three 942 independent experiments. The statistical comparisons were performed using one-way ANOVA (*p < 943 0.05; **p < 0.01; ***p < 0.001). 944 945 Figure 5. Effects of cis-DA on the binding of FadD1 to the promoters of the target genes. (A) 946 EMSA analysis of the in vitro binding of FadD1 to the promoter of lasR with the addition of different 947 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint amounts of cis-DA. (B) The potential residues (which are shown as stick models and are labeled) of 948 FadD1 involved in cis-DA binding analyzed by AutoDocking software. The hydrogen bonds were 949 represented by yellow dashed lines. (C) SDS‒PAGE gel electrophoresis of purified FadD1 variants. 950 MST analysis of the binding of BDSF to the (D) FadD1G488A and (E) FadD1R552A. EMSA analysis of 951 the in vitro binding of (F) FadD1G488A and (G) FadD1R552A to the promoters of lasR with the addition of 952 different amounts of cis-DA. The EMSA experiments were performed three times, and representative 953 images from one experiment are shown. Analysis of the perception of cis-DA by FadD1G488A and 954 FadD1R552A, cis-DA-regulated phenotypes: (H) swarming motility and (I) pyocyanin production in P. 955 aeruginosa (n = 3 biological replicates) . The data are means ± standard deviations of three 956 independent experiments. The statistical comparisons were performed using one-way ANOVA (***p 957 < 0.001; ns = no significance). 958 959 Figure 6. Schematic representation of cis-DA-regulated QS signaling in P. aeruginosa. DspI 960 synthesized cis-DA signal molecule. FadD1, the signal receptor of cis-DA, perceives cis-DA signal 961 and regulates the transcription of lasR, which positively regulates the las, rhl and pqs QS systems in 962 P. aeruginosa. DspS is also the sensor of cis-DA involved in biofilm dispersion in response to cis-DA. 963 The individual QS circuits are highly interconnected. 964 965 966 .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licensemade available 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 The copyright holder for this preprintthis version posted June 15, 2024. ; https://doi.org/10.1101/2024.06.08.598097doi: bioRxiv preprint