P.)aeruginosa cells were routinely cultured in brain heart infusion (BHI) broth at 37)°C with shaking (250)r.p.m.) unless otherwise specified. Pseudomonas isolation agar (PIA) was used for selecting P.)aeruginosa against other microbes. Escherichia coli cells were cultured in lysogeny broth. To prepare plates, 1.5% (w/v) agar was used. Antibiotics were added to the medium as necessary: 60)µg)m2l1 gentamicin, 75)µg)m2l1 Tet or 300)µg)m2l1 carbenicillin for P.)aeruginosa; 10)µg)m2l1 gentamicin, 10)µg)m2l1 Tet or 100)µg)m2l1 carbenicillin for E.)coli. For P.)aeruginosa static or anaerobic cultivation, 50)mM KNO3 was added to BHI broth. The anaerobic condition was maintained in a vinyl anaerobic chamber (Coy Lab Products) with the following atmosphere: 85% N2, 10% CO2 and 5% H2.

Strains, plasmids and primers used in this study are listed in Supplementary Table 3. To create transcriptional lacZ fusions (pPC100–102), desired promoter regions were PCR amplified (Q5, New England Biolabs) using PA14 genomic DNA as the template, and the resulting amplicons containing overlapping regions of about 25)base pairs (bp) were ligated into the mini-CTX-lacZ vector (linearized with EcoRI and KpnI) in Gibson Assembly Master Mix (New England Biolabs). To create sicX complementation plasmids (pPC103–106), the sicX promoter and sicX alleles (containing mutations as described in Supplementary Table 3) were ligated into mini-CTX1 vector (linearized with EcoRI and KpnI) through Gibson assembly. All sicX point mutations (pPC107–124) were constructed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs), using mini-CTX1-sicXTruncate (pPC106) as the template. To create translational lacZ fusions (pPC125–126), the promoter region of ubiU was PCR amplified and then ligated into pSW205 between the EcoRI and BamHI sites. All ubiUVT 52)UTR point mutations (pPC127–145) were constructed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs), using pSW205-Pubi-lacZ (pPC125) as the template. To construct plasmids used for markerless gene deletion (pPC146–147, 149–150) and mutagenesis (pPC148), upstream and downstream regions (about 1,000)bp) flanking the target gene or region were PCR amplified (containing mutations in the overlapping region if needed) and ligated into pEXG2 (linearized with SacI and KpnI) through Gibson assembly. All plasmids were verified by PCR, restriction enzyme digestion and, if necessary, DNA sequencing.

Verified plasmids were then transformed into E.)coli SM10 » pir, followed by conjugation into P.)aeruginosa PA14, PAO1, B1 and their derivatives. The pSW205-derived plasmids were directly electroporated into P.)aeruginosa. Appropriate antibiotics were used for selection as indicated in Supplementary Table 3. To create markerless in-frame deletion or mutagenesis, conjugants with the deletion cassette integrated at the target loci were counter-selected on low-salt lysogeny broth agar plates supplemented with 5% sucrose. The successful deletion was screened by PCR using flanking primers and verified by DNA sequencing if needed.

Blastn searches were carried out on pseudomonas.com and the National Center for Biotechnology Information website to identify orthologues of sicX and ubiUVT across the Pseudomonas genus and other organisms. The sicX sequence (for Fig. 1c and Extended Data Fig. 1), sicX with its neighbouring genes (Fig. 1d) and ubiUVT with its upstream intergenic region (for Extended Data Fig. 4) from the P.)aeruginosa PA14 strain were used as queries. Orthologues identified from complete bacterial genomes with E values of <1)×)1024, percentage identity >90% and query coverage values of >90% were retained for further analysis. Sequences of sicX and ubiUVT orthologues were aligned using MUSCLE43. To analyse the sequence conservation of sicX and ubiUVT orthologues, we first aligned sicX and ubiUVT orthologues using MUSCLE. Next, alignment ends were trimmed and gaps were removed, and percentages of orthologue sequences that are identical to sicX and ubiUVT queries were calculated at each nucleotide position in R. To create Extended Data Fig. 4b,c, the alignments were visualized in Jalview44 using the default nucleotide colour scheme. ³-galactosidase assay P.)aeruginosa cells were grown overnight in BHI broth supplemented with 50)mM KNO3. The overnight culture was diluted to an optical density at 600)nm (OD600nm) of about 0.05 in fresh BHI broth (with 50)mM KNO3) and then grown to the log phase (OD600nm around 0.5). Log-phase cells were diluted to OD600nm 0.2 and transferred to the anaerobic chamber for 3-h shaking (at 100)r.p.m.) cultivation at37)°C. A 100)µl volume of the anaerobic culture was used to conduct the ³-galactos idase assay (as described in ref.)45). Aerobic log-phase cultures were used as comparisons. For static cultivation, log-phase cultures were diluted to OD600nm 0.05 and incubated in a 96-well plate (500)µl in each 1-ml-deep well) statically at 37)°C for 3)h. The static cultures were then used for the ³-galactosidase assay.

For making Extended Data Fig. 3a,b, strains harbouring ubiUVT-lacZ translational fusions were spotted (3 µl of OD600nm 0.1 cultures) onto 0.2-µm Nuclepore track-etched polycarbonate membranes (Whatman) that were placed (rough side facing upwards) on BHI agar plates containing 200)µg)m2l1 X-gal, 50)mM KNO3 and 100)µg)m2l1 carbenicillin. The plates were then incubated in the anaerobic chamber (protected from light) at 37)°C for 20)h before imaging. ³-galactosidase activities were estimated as described previously46. Briefly, images of colony biofilms were converted to 8)bit in ImageJ. Thresholding was carried out to define colony objects for measurements. Mean pixel intensity was measured for each colony and subtracted with the mean pixel intensity of a colony of WT PA14.

P.)aeruginosa cells were grown aerobically and anaerobically for total RNA extraction. To prepare aerobic cultures, log-phase cells were diluted to OD600nm 0.01 in BHI (with 50)mM KNO3) and incubated at 37)°C with shaking for 3)h. To prepare anaerobic cultures, log-phase cells were diluted to OD600nm 0.1 in BHI (with 50)mM KNO3) and incubated in the anaerobic chamber for 4)h with shaking at 37)°C. For total RNA extraction, P.)aeruginosa cultures were first stored in RNAlater at 4)°C overnight. Next, cells were pelleted and resuspended in RNase-free TE buffer containing 1)mg)m2l1 lysozyme. Samples were incubated at 37)°C for 30)min to enzymatically lyse cells. A 1)ml volume of RNA-Bee was then added, and cells were further mechanically lysed by bead beating three times for 30)s. A 200)µl volume of chloroform was added to the samples, and the aqueous and organic phases were separated by centrifugation at 13,000g for 30 min at 4)°C. The aqueous phase that contained RNA was mixed with 0.5)ml isopropanol and20)µg linear acrylamide. After overnight incubation at 280)°C, RNA was pelleted and washed with 75% ethanol. Air-dried pellets were resuspended in 100)µl of RNase-free water. The RNA concentration for each sample was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Approximately 1)µg of total RNA was separated on a 15% polyacrylamide gel electrophoresis–urea gel and transferred to Hybond-N+ membranes (GE Healthcare). The membranes were then crosslinked with an ultraviolet crosslinker (UVP) and probed in hybridization solution (Takara) with the indicated DNA oligonucleotides (listed in Supplementary Table 3) that were radiolabelled with [³ -32P]ATP (Perkin Elmer) by T4 polynucleotide kinase (New England Biolabs). Probed membranes were exposed to a phosphor screen and visualized using a Typhoon Biomolecular Imager (GE Healthcare). When necessary, membranes were stripped by incubating them with boiled 0.1% SDS with agitation for 15)min three times. The sizes of RNA were estimated with the RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific) as well as the migration of xylene cyanol (about 28)nt) and bromophenol blue (about 8)nt). MSnbase v2.13 (ref.)48) was used for processing proteomics data. ProColony biofilm assay tein differential abundances were evaluated using the Limma package Bacterial cultures in BHI (with 50)mM KNO3) were grown to the log v3.44.3 (ref.)49). Differences in protein abundances were statistically phase (OD600nm around 0.5) and then diluted to OD600nm 0.02 with determined using Student9st-test with variances moderated by the fresh medium. Next, 10)µl diluted cultures were spotted onto 0.2-µm use of Limma9s empirical Bayes method.P values were adjusted for Nuclepore track-etched polycarbonate membranes (Whatman) that multiple testing by the Benjamini–Hochberg method50. Proteins were were placed (smooth side facing upwards) on BHI agar plates (with considered to have increased or decreased in abundance only when 50)mM KNO3). The plates were then incubated in the anaerobic cham- their log2[fold change] value was greater than 1 or less than 21, respecber at 37)°C for 16)h. Membranes containingP.)aeruginosa biofilms tively, and when their P value was <0.01. were transferred to the BeadBug prefilled tubes that contained 1)ml For anaerobic growth condition, two biological replicates for PA14 phosphate-buffered saline (PBS). Biofilms were mechanically disrupted and three biological replicates for — sicX were collected as described by bead beating for 45)s. CFUs were determined by spreading the diluted above (in the section entitled ³-galactosidase assay). Briefly, the pro cell suspensions on PIA plates. teins in each sample were reduced, alkylated and digested with trypsin according to the filter-aided sample preparation protocol51. The pepQuantitative proteomics and data analysis tides were labelled with TMTs, separated by high-pH reversed-phase P.)aeruginosa PA14 and — sicX cells were collected under both static chromatography as described previously52. They were pooled into and anaerobic growth conditions as described above (in the section 12 fractions as described previously53. Each fraction was analysed by entitled ³-galactosidase assay). nano-LC–MS/MS,andpeptideswereidentifiedaspreviouslydescribed54

For static growth condition, three biological replicates for each strain with the following modifications. Reversed-phase chromatography was were collected. Cell pellets were resuspended in lysis buffer (100)mM carried out using an in-house packed column ( 40 )cmlong)×)75)µm inner Tris-HCl, 50)mM NaCl, 10% (v/v) glycerol, 1)mM Tris(2-carboxyethyl) diameter)×)360)µm outer diameter, Dr. Maisch GmbH ReproSil-Pur phosphine) (TCEP), pH 7.5) with cOmplete Mini protease inhibitor 120 C18-AQ 1.9-µm beads) and a 120-min gradient. The Raw files were cocktail (Roche). Following three rounds of sonication ( 3 )×)10)s) on ice, searched using the Mascot algorithm (v2.5.1) against a protein database supernatants were clarified by sedimentation (21,130g, 15)min, 4)°C). constructed by combining the sequences of P.)aeruginosa UCBPP-PA14 Aliquots (100)µg) of each sample were reduced withTCEP, alkylated and a contaminant database (cRAP, downloaded 21 November 2016 with iodoacetamide and labelled with tandem mass tags (TMTs). TMT from http://www.thegpm.org) through Proteome Discoverer v2.1. labelling was carried out according to the protocol specified by the Only peptide spectral matches with expectation value of less than 0.01 manufacturer (Thermo Fisher). (8high confidence9) were used for our analyses as described above.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) experiments were carried out using a Dionex Ultimate 3000 rapid Lipid extractions and ubiquinone (UQ9) quantification separation LC (RSLC) nano-ultraperformance LC system (Thermo To prepare cells for lipid extractions, we first diluted log-phase cultures Fisher Scientific) and a Lumos Orbitrap mass spectrometer (Thermo (OD600nm 0.5; grown aerobically) into the anaerobic BHI broth (supFisher Scientific). Separation of peptides was carried out by C18 plemented with 50)mM KNO3) to a calculated OD600nm 0.05, and after reversed-phase chromatography at a flow rate of 300)nl)min21 using a 6-h anaerobic incubation with shaking, cells were collected, washed Thermo Scientific reversed-phase nano EASY-spray column (Thermo once with PBS and flash-frozen in liquid nitrogen. Cell numbers were Scientific PepMap C18; 2-mm particle size, 100-Å pore size, 75-mm i.d. estimated with OD600nm. by 50-cm length). Solvent A was water/0.1% formic acid, and solvent Frozen samples were thawed on ice. A 100)µl volume of ice-cold isoB was 80% (v/v) acetonitrile/20% water/0.1% formic acid. The linear propanol with 10)nM coenzyme Q10-d9 (deuterated UQ10 as internal gradient used was 2% to 40% solvent B over 93)min (the total LC run standard; Sigma-Aldrich) was added to 109 cells. With the addition time was 120)min, including a high-organic-wash step and column of glass beads, samples were vortexed briefly and homogenized by re-equilibration). TissueLyzer for 5)min twice, followed by centrifugation at 21,100g

The eluted peptides were sprayed into the mass spectrometer by for 5)min. After centrifugation, supernatant was diluted tenfold and means of an EASY-Spray source (Thermo Fisher Scientific). All m/z transferred to 4)°C. Ultraperformance LC–MS was carried out using values representing eluting peptide ions were measured in an Orbit- an UltiMate 3000 fitted with an Accucore C30 column (2.1)×)150)mm, rap mass analyser (set at a resolution of 120,000) and were scanned 2.6)µm particle size; Thermo Fisher) and coupled to an Orbitrap ID-X at between m/z 380 and 1,500)Da. Data-dependent MS/MS scans (top mass spectrometry system. speed) were used to automatically isolate and fragment precursor ions The chromatographic method for sample analysis involved elution by collision-induced dissociation (normalized collision energy value, with acetonitrile/water (60:40, v/v) with 10)mM ammonium formate 35%) analysed in the linear ion trap. Singly charged ions and ions with and 0.1% formic acid (mobile phase A) and isopropanol/acetonitrile unassigned charge states were excluded from selection for MS/MS, (90:10, v/v) with 10)mM ammonium formate and 0.1% formic acid and a dynamic exclusion window of 70)s was used. The top 10 most (mobile phase B) at 0.4)ml)min21 flow rate using the following gradiabundant fragment ions from each MS/MS event were then selected for ent programme: 0)min 20% B; 1)min 60% B; 5)min 70% B; 5.5)min 85% B; a further stage of fragmentation by synchronous precursor selection 8)min 90% B; 8.2)min 100% B hold to 10.5)min, then10.7)min 20% B hold and MS/MS/MS47 in the high-energy-collision cell using high-energy to 12)min. The column temperature was set to 50)°C, and the injection collisional dissociation (normalized collision energy value of 65%). volume was 5)µl.

The m/z values and relative abundances of each reporter ion and of all The targeted molecules UQ9 and deuterated UQ10 were fragfragments (mass range, 100 to 500)Da) in each MS3 step were meas- mented by high-energy collisional dissociation at 20 collision energy ured in the Orbitrap analyser, which was set at a resolution of 60,000. in positive mode. MS/MS transitions for UQ9 and deuterated UQ10 This was carried out in cycles of 10 MS3 events, after which the Lumos are 812.66/197.08 and 889.77/206.18. Standard curves for UQ9 and instrument reverted to scanning the m/z ratios of the intact peptide deuterated UQ10 were generated. The amounts of UQ9 and deuterated ions and the cycle continued. UQ10 in the samples were calculated on the basis of the standard curve.

Proteome Discoverer v2.1 (Thermo Fisher Scientific) and Mascot (Matrix Science) v2.6 were used to process raw data files. Data were Planktonic growth and competition assays aligned to the sequences of P.)aeruginosa UCBPP-PA14 (with com- For generating growth curves, bacterial cells in BHI (with 50)mM KNO3) mon repository of adventitious proteins (cRAP) v1.0). The R package were first grown aerobically to the log phase (OD600nm around 0.5) and then diluted to OD600nm 0.01 with either aerobic or anaerobic BHI (with 50)mM KNO3) broth. Aerobic and anaerobic growth of P.)aeruginosa at 37)°C (with shaking) were monitored by measuring the OD600nm.

For long-term competition experiments, PA14, — sicX, TetR (resistance provided by mini-CTX1) PA14 and TetR — sicX were grown to the log phase (OD600nm around 0.5) in BHI (with 50)mM KNO3) before diluting to OD600nm 0.4 with fresh medium. PA14 and TetR — sicX were mixed at 1:1 ratio, and 100)µl of cell mixtures was transferred to 4)ml anaerobic BHI (with 50)mM KNO3), followed with shaking incubation at 37)°C under anaerobic conditions. After 24)h, 20)µl of the culture was transferred to another tube containing 4)ml anaerobic BHI (with 50)mM KNO3). The culture was then consecutively passaged daily in a similar fashion. The competition between TetR PA14 and — sicX was conducted similarly to that described above. CFU values of TetR and TetS cells were estimated by plating cultures (on each day) on PIA as well as PIA containing 75)µg)m2l1 Tet.

RNA secondary structures were predicted using mfold26 with default parameters. IntaRNA 2.0 was used to predict base-pairing interactions between SicX sRNA and ubiUVT mRNA25 with default settings, and the minimum number of base pairs in the seed region was set as 7.

Mouse chronic wound infections were carried out with female Swiss Webster mice (8–10 weeks old) essentially as described previously55 with a few modifications. Briefly, dorsal full-thickness skin excision was carried out in which a small circular wound (with a diameter of 1.5)cm) was surgically administered. After excision, the wound was immediately covered with a semipermeable polyurethane dressing. A total of 104 CFUs of P.)aeruginosa cells were injected onto the wound bed underneath the dressing to establish infection. As bandages are required to reduce the contractile healing and minimize wound contamination with other bacteria, they were closely monitored and replaced when necessary throughout the infection course.

For assessing the bacterial burdens of WT and — sicX infection, wound tissues and spleens were collected at 10 days post-infection or at an early time point if the animal was found moribund (moribund mice that were not identified immediately were excluded from the CFU analysis). Tissues were homogenized in PBS for 45)s in BeadBug tubes with 2.8-mm steel beads (Sigma-Aldrich) using a Mini-Beadbeater-16 (BioSpec Products). To enumerate CFUs, homogenized samples were serially diluted and plated on PIA plates. For studying the dissemination outcomes of WT, — sicX, — ubiUVT and — sicX-ubiC infection (Fig. 3e), at 10 days post-infection, distal organs including spleens, livers and gallbladders were collected for assessing the presence or absence of P.)aeruginosa cells. For studying the macroscale organization of P.)aeruginosa infection, the wound was excised at 4 days post-infection, and a biopsy punch of 10)mm diameter (Acu-Punch, Thermo Fisher Scientific) was used to separate the edge (about 0.98)cm2) and core (about 0.77)cm2) regions. The CFUs were then normalized by the surface area for comparison purposes.

In vivo biofilm dispersal was carried out with female Swiss Webster mice (8–10 weeks old) essentially as described previously31 with a few modifications. Briefly, the mouse surgical excision wound was administered as demonstrated above. A total of 104 CFUs of P.)aeruginosa cells (PAO1) were injected onto the wound bed underneath the dressing to establish infection. At 48)h post-infection (after the formation of mature biofilms in wounds), the established wound infections were treated through topical application of GHs or cis-DA to induce systemic dissemination. Specifically, GHs are composed of bacterial -³amylase (from Bacillus subtilis; MP Biomedicals) and fungal cellulase (from Aspergillus niger; MP Biomedicals). A 10% (w/v) -³amylase and cellul ase (in a 1:1 combination) solution was prepared by dissolving powdered enzymes in PBS. cis-DA (Cayman Chemical Company) was diluted in PBS to a final concentration of 500)nM. GH andcis-DA solutions were prepared immediately before use. Wound beds were irrigated with a GH or cis-DA solution in three separate topical infusions with 30)min of dwell time for each. Topical infusions containing dispersed cells (n)=)2 animals for each treatment) were aspirated andimmediately placed in RNAlater (Thermo Fisher Scientific). In separate animals, P.)aeruginosa-infected wound tissues (n)=)3 animals) were carefully excised from the surrounding uninfected tissue and the underneath muscle layer at 48)h post-infection (without GH orcis-DA treatment) as a comparison. Infected wound tissues were immediately placed in RNAlater (Thermo Fisher Scientific).

Mouse pneumonia infection was carried out with female BALB/c mice (6–8 weeks old) essentially as described previously56 with a few modifications. Briefly, mice were anaesthetized by intraperitoneal injection of 0.2)ml of a cocktail of ketamine ( 25 )mg)m2l1) and xylazine ( 12 )mg)m2l1). For infection with PA14-derived strains, mice were intranasally instilled with approximately 107 CFUs (a sublethal dose) P.)aeruginosa cells (in 25)µl PBS). Mice were euthanized at 48)h post-infection. Whole lungs and spleens were collected aseptically, weighed and homogenized in 1)ml of PBS. Tissue homogenates were serially diluted and plated on PIA for CFU enumeration. For infection with PAO1-derived strains, 5)×)107 CFUs (a lethal dose) of P.)aeruginosa cells were used as inocula, and tissues were collected at 24)h post-infection.

Mouse subcutaneous skin infection (also known as the mouse abscess model) was carried out with female Swiss Webster mice (8 weeks old) essentially as described previously55 with a few modifications. Briefly, the mouse inner thigh was shaved and any remaining hair was removed with a depilatory agent (Nair). A 100)µl volume ofapproximately 1)×)107 CFUs of P.)aeruginosa cells (PA14 or — sicX) were subcutaneously injected to the inner thigh. Mice were euthanized at 16)h post-infection. Spleens were collected aseptically and homogenized in 1)ml PBS. Tissue homogenates were serially diluted and plated on PIA for CFU enumeration.

RNA extractions were carried out essentially as described previously14. For in vitro static cultivation of PA14, cells were first grown as described above (in the section entitled ³-galactos idase assay) and immediately mixed with five volumes of RNAlater. After storage at 4)°C overnight, cells were pelleted, resuspended innuclease-free TE buffer (Acros Organics) containing 1)mg)m2l1 lysozyme, and transferred to bead-beating tubes containing a mixture of large and small beads (2-mm zirconia and 0.1-mm zirconia/silica, respectively). For in vivo biofilm dispersal experiments (as described above), infected tissues were removed from RNAlater, mixed with TE buffer, and transferred to bead-beating tubes. In vitro as well as tissue samples in TE were briefly disrupted by homogenization using a Mini-Beadbeater-16. Samples were then incubated at 37)°C for 30)min to enzymatcially lyse cells. Next, 1)ml RNA-Bee was added to 300-µl sample homogenate, and samples were further mechanically lysed by bead beating three times for 30)s.

A 200)µl volume of chloroform per 1)ml RNA-Bee wasadded, and the tubes were shaken vigorously for 30)s and then incubated on ice for 5)min. The samples were then centrifuged at 13,000g for 30)min at 4)°C to separate the aqueous and organic phases. The aqueous phase was transferred to a new microcentrifuge tube to which 0.5)ml isopropanol (per 1)ml RNA-Bee) and 20)µg linear acrylamide were added.

After overnight incubation at 280)°C, samples were thawed on ice and centrifuged at 13,000g for 30)min at 4)°C. Pellets were washed twice with 1)ml of 75% ethanol, air dried for 5)min and resuspended in 100)µl of RNase-free water. The RNA concentration for each sample was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Statistics and reproducibility rRNA was depleted using a MICROBExpress Kit (Thermo Fisher Sci- All experiments were carried out independently at least three times entific) for the in vitro samples and a QIAseq FastSelect Kit (QIAGEN) (unless otherwise stated in the figure legends) with similar observafor mouse-derived samples, followed by ethanol precipitation. The tions. The exact n values for animal studies as well as in vitro experidepleted RNA was fragmented for 2)min with the NEBNext Magnesium ments are provided in the figures and legends. Statistical analyses RNA fragmentation module kit, and cDNA libraries were prepared were conducted using Prism GraphPad 9 and are specified in the figure using the NEBNext multiplex small RNA library prep kit (New England legends. For northern blotting, each experiment was conducted at least Biolabs) according to the manufacturer9s instructions. Libraries were twice independently (by using freshly extracted RNA samples) with sequenced at the Molecular Evolution Core at the Georgia Institute of similar observations, and representative images are shown. Technology on an Illumina NextSeq500 using 75-bp single-end runs.

In addition to the above-mentioned RNA-seq libraries prepared in this study, 202 publicly available P.)aeruginosa RNA-seq datasets were analysed, including 28 P.)aeruginosa transcriptomes derived from human samples, 28 from infection models and 146 from a range of well-defined in vitro conditions (listed in Supplementary Table 1). The quality of the raw sequencing reads was first confirmed with FastQC v0.11.7 (ref.)57). Next, RNA-seq reads were trimmed on the 32 endsto remove the Illumina adaptor (AGA TCG GAA GAG CAC ACG TCT GAA CTC CAG TCA C) using Cutadapt 3.0 (ref.)58) with a minimum read length threshold of 22 bases. Trimmed reads were then mapped to the P.)aeruginosa PA14 reference genome (available for download from pseudomonas.com) using Bowtie2 v2.4.2 with default parameters for end-to-end alignment59. As PA1414 (sicX) was originally annotated as a protein-coding gene, we first assigned reads to protein-coding genes with featureCounts Subread v2.0.1 (ref.)60; results shown in Fig. 1a). For the remaining analyses (Figs. 1b, 2a and 4b–d and Extended Data Figs. 2b and 10), reads assigned to both protein-coding genes as well as 199 sRNAs15 were tallied.

Differential expression was determined with DESeq2 (Figs. 1a and 4b–d; ref.)61). To compare sicX expression levels in different samples (Fig. 1a and Extended Data Fig. 5), raw reads were normalized using the varianceStabilizingTransformation() function in the DESeq2 package. To estimate the relative transcript abundance of various features (protein-coding genes, sRNAs, sicX and/or rsmYZ), we calculated TPM. First, raw counts were normalized by feature (gene) length to generate reads per kilobase (RPK). Next, all RPK values were counted up and divided by 1,000,000 to determine the scaling factor. Finally, RPK of each gene was divided by the scaling factor. The resulting TPM values were used for making Figs. 1b and 2a and Extended Data Figs. 2b and 10. To examine the alignment pattern of sicX reads, samtools v1.13 was used to measure the read depth encompassing the sicX locus at each nucleotide position62. The read depth was then normalized using the estimateSizeFactors() function in DESeq2 before plotting.

All mouse procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were treated humanely and housed in temperature- and humidity-controlled facilities ( 18–22 )°C; 40–50%) with 12)h light–dark cycle s.The protocol for the mouse chronic wound infection was approved by the Institutional Animal Care and Use Committee (IACUC) of Texas Tech University Health Sciences Center (protocol number 07044) and by the IACUC of Georgia Institute of Technology (protocol number A100127). The protocol for the mouse subcutaneous infection model was approved by the IACUC of Georgia Institute of Technology (protocol number A100127). For the mouse pneumonia infection model, all experimental procedures were conducted according to the guidelines of the Emory University IACUC, under approved protocol number DAR-201700441.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

A maximum-likelihood phylogenetic tree was constructed in PhyML 43. tEhdrgoaurg,hRp.Cut..MNUuScCleLicE:AmciudlstiRpeles.s3e2q,u1e7n9c2e31a7li9g7n(m20e0n4tw). ith high accuracy and high 3.0 (ref.)63) using 365 16S rRNA sequences representing diverse Pseu- 44. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview version domonas isolates. For better visualization of the tree, we included 24a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, only approximately 50 16S sequences from the P.)aeruginosa strains. 45. 1P1e8a9r3so11n9,1J.(2P0.e0t9 a)l.. Structure of the autoinducer required for expression of Pseudomonas The 16S sequences were aligned with MUSCLE and used as an input in aeruginosa virulence genes. Proc. Natl Acad. Sci. USA 91, 1973201 (1994). PhyML 3.0. The best-fitting evolutionary models were predicted with 46. Mould, D. L., Botelho, N. J. & Hogan, D. A. Intraspecies signaling between common the corrected Akaike information criterion. The tree was visualized vvairruialenntsceoffaPcsteourds.oMmBoinoa1s1,aee0ru1g8i6n5o-s2a0in(2c0re2a0s)e.s production of quorum-sensing-controlled and annotated in iTOL64. 47. McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, Functional enrichment analysis 48. 7G1a5t0to3,7L15.&8 L(2il0le1y4,)K.. S. MSnbase-an R/Bioconductor package for isobaric tagged mass Gene Ontology enrichment analysis was conducted using Galaxy v2.0.1. spectrometry data visualization, processing and quantitation. Bioinformatics 28, 2883289 Differentially expressed genes in response to GH or cis-DA treatment (2012). were analysed using the Gene Ontology file (obtained from http:// 49. BSimocyothn,dGu.cKto.irn(eBdiosinGfeonrmtleamticasna,nRd., CCoarmeyp,uVt.aJt.i,oHnualbBeiro,lWog.,yIrSizoalrurtyi,oRn.sAu.s&inDguRdaonitd,S.) geneontology.org/docs/download-ontology) and the P.)aeruginosa 3973420 (Springer, 2005).

PAO1 Gene Ontology term annotations (obtained from https://pseu- 50. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful domonas.com/goterms/list). A Benjamini–Hochberg test with P-value 51. aWpipsnroieawcshktio,Jm.Ru.,ltZipoluegtmesatinn,gA..J,.NRa.gSatarat.j,SNo.c&. BM5a7n,n2,8M9.3U3n0i0ve(r1s9a9l5s)a.mple preparation cutoff)<)0.05 was carried out. method for proteome analysis. Nat. Methods 6, 3593362 (2009).

Acknowledgements We thank D.)Cornforth and other members of the laboratory of M.W. for helpful discussion of the manuscript and assistance with the animal experiments. We thank G.)Welch and H.)Hui for help with the animal expermients. We thank S.)Brown, S.)Diggle and C.)Baskett for critical reading of the manuscript.The proteomic and metabolomic studies were supported by the Georgia Institute of Technology Systems Mass Spectrometry Core Facility as well as the Cambridge Centre for Proteomics. This study was supported by National Institutes of Health grants R21AI154220 (to M.W.), R21AI137462 (to K.P.R.) and R21AI147178 (to C.S.S.), Cystic Fibrosis Foundation grants WHITEL20A0 and WHITEL22G0 (to M.W.) and Cystic Fibrosis Trust Foundation SRC017 (to M.W.). P.C. and S.K.D. are supported by Cystic Fibrosis Postdoctoral Fellowships CAO20F0 and DOLAN20F0, respectively.

Author contributions P.C. and M.W. conceived and designed the study, analysed data and wrote the manuscript; D.F., D.A.M., P.C., W.K.R. and F.L.D. contributed to various aspects of animal experiments; S.K.D. prepared samples and carried out data analyses for proteomics; K.H.S. carried out northern blotting and analyses; P.C., M.W. and A.R. constructed bacterial strains and carried out reporter assays; P.C. carried out all other experiments and data analysis; C.S.S., J.B.G. and K.P.R. provided technical guidance and analysed data.

Competing interests The authors declare no competing interests.

Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41586-023-06111-7.

Correspondence and requests for materials should be addressed to Marvin Whiteley.

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