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J. Bacteriol. doi:10.1128/JB.00784-15
Copyright 2015, American Society for Microbiology. All Rights Reserved.
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A factor and anti- factor that control swarming motility and biofilm formation
in Pseudomonas aeruginosa
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Abstract
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infections. Here, we provide evidence that sbrR (PA2895), a gene previously identified
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that inhibits the activity of its cognate extra-cytoplasmic function factor SbrI
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interacts directly with SbrI and was sufficient for inhibition of SbrI-dependent gene
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expression. We show that SbrI associates with RNA polymerase in vivo and identify the
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SbrIR regulon. In cells lacking SbrR, the SbrI-dependent expression of muiA was found
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to inhibit swarming motility and promote biofilm formation. Our findings uncover
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SbrR and SbrI as a novel set of regulators of swarming motility and biofilm formation
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in P. aeruginosa that mediate their effects through muiA, a gene not previously known
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IMPORTANCE
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This study characterizes a factor/anti- factor system that reciprocally regulates the
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anti- factor specific for its cognate factor SbrI and identify the SbrIR regulon in P.
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aeruginosa. We find that cells lacking SbrR are severely defective for swarming motility
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and exhibit enhanced biofilm formation. Moreover, we identify muiA (PA1494) as the
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SbrI-dependent gene responsible for mediating these effects. SbrIR have been
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SbrIR may therefore represent a stress-response system that influences the surface
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INTRODUCTION
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic
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human pathogen notorious for being the principal cause of morbidity and mortality in
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cystic fibrosis (CF) patients (1). In patients with CF, chronic pulmonary colonization by
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eventually respiratory failure and death (1). P. aeruginosa is also the fifth leading cause
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of nosocomial infections overall in the US and is the second most common cause of
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infections (CAUTI) (2, 3). In patients with VAP or CAUTI, P. aeruginosa grows as a biofilm
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thought to persist as a biofilm in the CF lung (7). P. aeruginosa biofilms are associated
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with chronic infection and exhibit increased antibiotic resistance and resistance to
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clearance by the immune system (8). Thus, the ability to form biofilms contributes
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virulence factors and is associated with acute infection (9, 17). Several systems are
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known to mediate the transition from motile, swarming cells, to cells growing as
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function (ECF) factor that we refer to here as SbrI (19). As a group, ECF factors are
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factor (20, 21). Upon stimulation by the appropriate extracytoplasmic signal, the ECF
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factor is released from the anti- factor, allowing it to associate with RNA polymerase
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Here we present evidence that SbrI and SbrR are an ECF and anti- factor pair.
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We identify the SbrIR regulon and show that SbrI and SbrR influence biofilm formation
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we show that cells lacking sbrR are unable to engage in swarming motility and exhibit
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observed in these cells. We have named PA2896 and PA2895 SbrI and SbrR,
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sbrR mutant cells. SbrI and SbrR constitute a pair of regulators controlling swarming
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motility and biofilm formation in P. aeruginosa that mediate their effects through
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MuiA.
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Bacterial strains
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E. coli DH5FIQ (Invitrogen) was used as the recipient strain for all plasmid
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constructions. E. coli SM10 pir served as the conjugative donor for transferring
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cultures were routinely grown at 37C in lysogeny broth (LB), or on plates containing
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LB solidified with 1.5% agar unless otherwise noted. When appropriate, gentamicin (30
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g/ml) and carbenicillin (200 g/ml) were used for selection in P. aeruginosa cultures.
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A list of strains and plasmids used in this study are available in Table S1.
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corresponding to a 3 portion of the sbrI gene into pP30-YTAP cut with HindIII and
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NotI; the portion of the PA2896 gene was cloned such that it was in-frame with the
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DNA specifying the TAP-tag. pP30-SbrI-TAP was used to generate PAO1 SbrI-TAP as
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previously described (22). Strain PAO1 RpoS-TAP was constructed in a similar way
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using vector pP30-RpoS-TAP, which contains a portion of the P. aeruginosa rpoS gene
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fused in-frame to DNA specifying the TAP-tag. The PAO1 AceF-TAP strain, which
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expresses AceF-TAP and serves as a control, has been described previously (22).
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end of sbrI is in-frame with the VSV-G tag (23). PAO1 -TAP SbrI-V was constructed in
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Bacterial two-hybrid assays were performed with the E. coli reporter strain KS1
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(25); KS1 harbors on its chromosome the placOR2-62 test promoter driving expression
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of a linked lacZ reporter gene. Plasmids pACcI32 and pBRLN have been described
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previously (26), and were used to create fusions to the C-terminus of cI and the C-
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1-236) fused to residues 2-64 of SbrR from P. aeruginosa via a small linker composed of
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appropriate NotI-BamHI-digested PCR product into pACcI32 that had been digested
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with NotI and BstYI, thus placing expression of the cI-SbrR-NTR fusion protein under
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residues 1-248 of the subunit of E. coli RNA polymerase fused to residues 2-194 of
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SbrI from P. aeruginosa via a small linker composed of three alanine residues. Plasmid
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into pBRLN digested with NotI and BamHI, thus placing the -fusion under the
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control of tandem lpp and IPTG-inducible lacUV5 promoters. Plasmid pBR encodes
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wild type under the control of tandem lpp and IPTG-inducible lacUV5 promoters and
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sbrI, muiA, and PA4495, respectively, fused to the lacZ gene and integrated in single
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copy into the CTX locus in the PAO1 chromosome. The putative sbrI promoter region
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consisted of the 327 bp intergenic region upstream of the sbrI start codon (see
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www.pseudomonas.com) (27). This region was amplified by PCR and cloned into mini-
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intergenic regions of muiA (122 bp) and PA4495 (275 bp) were also PCR amplified and
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copy into the CTX site to create reporter strains PAO1 attB::PsbrI-lacZ , PAO1 attB::PmuiA-
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~ 700 bp in length that flank sbrR in the PAO1 genome by PCR and then splicing the
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the 3 end of sbrI and the 5 end of sbrR, the deletion was designed such that sbrI
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would not be disrupted by the sbrR deletion construct. The deletion was in-frame and
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regions. The resulting PCR product was cloned on a HindIII/XbaI fragment into plasmid
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pEXG2 (29), yielding plasmid pEXG2-sbrR. The PAO1 sbrR and PA14 sbrR deletion
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described (30). Plasmid pEXG2-sbrR was also used to generate the sbrR mutant
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reporter strains in a similar manner. The sbrIR deletion construct was generated by
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amplifying the ~ 700 bp 5 flanking region of sbrI in the PAO1 genome by PCR. This
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PCR product was digested with XbaI and NotI and cloned into pEXG2-sbrR digested
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with XbaI and NotI, such that the 5-flanking sbrI XbaI/NotI fragment replaced the 5
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flanking region used for deleting sbrR, yielding plasmid pEXG2-sbrIR. This plasmid
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was used to create the sbrIR deletion strains as previously described (30). The muiA
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deletion construct was made in a similar fashion to the sbrR deletion construct.
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Flanking regions ~ 700 bp in length on either side of muiA in the PAO1 genome were
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amplified by PCR and spliced together by overlap extension PCR. The deletion was in-
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frame and included the NotI-linker as above. This PCR product was cloned into pEXG2
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to generate pEXG2-muiA. The resulting plasmid was used to delete muiA as described
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(29). Purified proteins were concentrated using Amicon Ultra-4 centrifugal filtration
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units with a 10 kDa molecular weight cut-off (Millipore), separated on 4-12% Bis-Tris
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Western blots
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Purified proteins and cell lysates were separated on 4-12% Bis-Tris NuPAGE
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(Invitrogen) and Western blotting was performed as described previously (22). The
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g/ml), carbenicillin (100 g/ml), chloramphenicol (25 g/ml), and IPTG at the
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representative data set is shown. Values are averages based on one experiment;
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amplified by PCR and cloned into pPSV38 (31). pPSV38-SbrR directs IPTG-inducible
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synthesis of SbrI and confers resistance to gentamicin. The same process was used to
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fragment encoding the first 64 residues of SbrR flanked by HindIII and BamHI sites was
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amplified by PCR and cloned into pPSV38. To make pHERD20T-MuiA, a DNA fragment
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containing the P. aeruginosa PA14 muiA coding sequence flanked by XbaI and PstI was
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amplified by PCR and cloned into pHERD20T (32). pHERD20T-MuiA directs the
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Microarray experiments
Cells of PAO1 (pPSV38), PAO1 sbrR (pPSV38), PAO1 sbrR (pPSV38-SbrR), and
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PAO1 sbrIR (pPSV38) were grown with aeration at 37C in 200 ml LB supplemented
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with gentamicin (30 g/ml). Biological duplicate cultures of each strain were
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(Affymetrix) and GeneSpring GX was used to analyze data for statistically significant
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changes in gene expression. The genes with changes in expression 2-fold (P value of
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0.01) are listed in Table 1. The data discussed in this publication have been deposited
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in NCBI's Gene Expression Omnibus (34) and are accessible through GEO Series
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GSE74917).
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(30 g/ml). Cells were permeabilized with sodium dodecyl sulfate and CHCl3 and
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performed at least twice in biological triplicate. Representative data sets are shown.
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Motility assays
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described (35, 36). Agar plates for assessing swimming motility and swarming motility
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consisted of M8 medium supplemented with glucose, MgSO4, CAA, and 0.3% agar for
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swim plates or 0.5% agar for swarm plates. 0.1% arabinose was added where indicted.
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Swim plates were stab inoculated from colonies grown overnight on LB agar plates.
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Swarm plates were inoculated with 3l of liquid culture grown overnight in LB. Swim
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plates and swarm plates were incubated ~20 hours at 37C. Quantification of swim
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and swarm zones was performed using ImageJ software. Experiments for testing
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experiments normalized to wild type (WT). Subsurface twitching motility was assayed
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as described previously (37). Bacteria were stab inoculated through a layer of LB agar
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(1% agar) to the bottom of the petri dish. After incubation for ~24 hours at 37C, the
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twitching motility was examined by removing the agar and staining the attached cells
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quadruplicate on two separate occasions. Data shown represent the results of those
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inoculate fresh media to a starting OD600 of 0.1. Media was supplemented with
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incubation, plates were washed twice with water and adherent biofilms were stained
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with 150 l of 0.1% Crystal violet for 15 minutes. Following staining, plates were
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washed twice with water and allowed to dry overnight. Stained biofilms were
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solubilized with 200 l of 33% acetic acid and absorbance at 595 nm was read with a
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Tecan Infinite 200 plate reader. Experiments were performed on at least two separate
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RESULTS
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operon together with sbrI (Fig. 1A). A four base pair overlap in the coding sequences of
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sbrR and sbrI suggests strong translational coupling of these genes. While SbrR has no
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ECF factor on the Pseudomonas Genome Database and shares significant sequence
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homology with other ECF factors (27). To determine if SbrI might function as a
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factor, we purified SbrI from cells of P. aeruginosa and asked whether subunits of RNAP
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of PAO1 that synthesized SbrI with a tandem affinity purification (TAP) tag fused to its
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C-terminus (SbrI-TAP) from its native chromosomal location. As a positive control for
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our ability to detect an association between a factor and RNAP, we also constructed
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a second strain that synthesized the stationary phase-specific factor RpoS with a C-
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expected to interact with RNAP), with a TAP-tag fused to its C-terminus (AceF-TAP)
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(22). We then purified SbrI, RpoS, and AceF by TAP and analyzed those proteins that
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co-purified by SDS-PAGE followed by staining with Coomassie blue. Proteins with the
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expected molecular weights for the , , and subunits of RNAP co-purify with both
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RpoS-TAP and SbrI-TAP, but not the negative control AceF-TAP (Fig. 1B). This suggests
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that SbrI associates with RNAP in vivo, consistent with its predicted function as a
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factor. SbrI appears as a doublet in Fig. 1B, suggesting it can exist as a high molecular
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weight and low molecular weight species. It is unclear if this doublet represents SbrI
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processing, the use of an alternative translational start site, or if there is any functional
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difference between these two forms. However, both forms co-purify with -TAP (Fig.
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S1), suggesting that both forms are capable of associating with RNAP.
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factor and its cognate anti- factor. In the absence of their cognate anti- factor
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abundance of the factor. However, in some cases ECF factors exhibit reduced
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activity and abundance in the absence of their cognate anti- factors, which are
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thought to stabilize and protect the factor from degradation (39, 40). Alternatively,
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anti- factors can also promote the proteolysis of their cognate factor (41). A
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comparison of the abundance of SbrI with a C-terminal VSV-G epitope tag (SbrI-V) in
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wild-type (WT) and sbrR mutant cells revealed that SbrI-V (synthesized from its native
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locus) is more abundant in the absence of SbrR (Fig. 1C). This suggests that SbrR
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negatively influences the abundance of SbrI and that SbrR might inhibit the
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expression of sbrI.
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the sbrI promoter. To test this, we integrated a construct with the putative sbrI
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promoter region upstream of a lacZ reporter at the CTX phage attachment site on the
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created an in-frame sbrR deletion in this strain to test the effects of SbrR on
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expression from the sbrI promoter. In cells of the sbrR mutant reporter strain, -
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reporter strain (Fig. 2A, left graph), suggesting that SbrR inhibits transcription from the
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sbrI promoter. This increase was restored to WT levels by complementation with sbrR
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from a plasmid (Fig. 2A, left graph). Cells of the sbrIR double mutant strain exhibited
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reporter strain (Fig. 2A, left graph), suggesting that expression from the sbrI promoter
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regulatory effect of SbrI on its own promoter. Expression of the PsbrI-lacZ reporter was
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higher in cells of the sbrR mutant than in cells of the sbrIR double mutant in which
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sbrI was under the control of a heterologous promoter (Fig. 2A). This difference might
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expression only when sbrI is under the control of its native promoter. Taken together,
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these results suggest that SbrR inhibits SbrI-dependent transcription and that sbrI is
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positively autoregulated, consistent with a model in which SbrI is an ECF factor that
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controls its own expression and SbrR is its cognate anti- factor.
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can be sufficient for anti- factor activity (40, 42-44). SbrR is predicted to contain a
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topology of other ECF anti- factors (Fig. 2C). To determine if the N-terminal region of
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the start of the predicted transmembrane domain to produce SbrR-NTR (residues 1-64)
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(Fig. 2B). Levels of -galactosidase activity in the sbrR reporter strain expressing SbrR-
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NTR are indistinguishable from those expressing full length SbrR (Fig. 2A), which
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suggests SbrR-NTR contains the region of SbrR necessary for inhibiting SbrI activity. It
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further suggests that SbrR-NTR may contain a domain that is capable of interacting
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with SbrI.
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and preventing their association with RNAP (42, 44-46). We have shown that both SbrR
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and SbrR-NTR inhibit SbrI-dependent gene expression (Fig. 2B), and we next sought to
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determine whether SbrR directly interacts with SbrI using a bacterial two-hybrid
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system.
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RNAP can result in transcription activation of a test promoter (25, 26, 47). In the version
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of the assay used here, contact between a protein fused to the subunit of E. coli
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RNAP and another protein (or protein domain) fused to the cI DNA-binding protein
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To test whether SbrR could interact directly with SbrI we created two
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compatible plasmids, one expressing SbrR-NTR (residues 2-64) fused to the C-terminus
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of cI and the other expressing an fusion protein where the C-terminal domain (CTD)
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of has been replaced with full-length SbrI (residues 2-194). We then determined
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whether the resulting cI-SbrR-NTR fusion protein could activate transcription from
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the test promoter in cells that also synthesized the -SbrI fusion protein. Plasmids
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directing the synthesis of the cI-SbrR-NTR and the -SbrI fusion proteins were used to
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transform E. coli strain KS1, which harbors the Plac-OR2-62 test promoter (depicted in
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Fig. 2C) linked to lacZ and integrated in single copy in the E. coli chromosome (26). We
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found the cI-SbrR-NTR fusion protein strongly activated the transcription of the lacZ
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reporter in cells that also synthesize the -SbrI fusion protein, but not in cells that only
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expression of the lacZ reporter in the presence of the -SbrI fusion protein or in the
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presence of WT (Fig. 2D). These results suggest that SbrR and SbrI directly interact,
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consistent with the hypothesis that SbrR is the cognate anti- factor of SbrI.
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Next, we wanted to identify the genes controlled by SbrI and SbrR. Based on
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our above results that show SbrR inhibits the expression of sbrI and that SbrI is
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expression in sbrR mutant cells, sbrIR mutant cells, and WT cells containing an
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empty vector, together with sbrR mutant cells containing a vector that expresses
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sbrR.
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the sbrR mutant (Table 1). The expression of three genes in particular was strongly
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influenced by the deletion of sbrR. Specifically, the expression of muiA (PA1494) was
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103-fold higher in cells of the sbrR mutant than in WT cells, while expression of
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PA4495 and sbrI was 22- and 18-fold higher, respectively, in cells of the sbrR mutant
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when compared to WT (Table 1). The effects of the sbrR deletion on muiA, PA4495
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and sbrI expression could be complemented by providing sbrR in trans from a plasmid
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(Table 1). Furthermore, upregulation of muiA and PA4495 did not occur in cells of the
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sbrIR double mutant, suggesting that the upregulation of these genes observed in
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cells of the sbrR single mutant is dependent upon SbrI (Table 1). Consistent with the
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results of our microarray analyses, the expression of putative muiA promoter- and
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synthesis of SbrR or SbrR-NTR from a plasmid, and was dependent upon SbrI (Fig. 2A).
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Taken together, our findings suggest that transcription from the PmuiA, PPA4495, and PsbrI
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promoters is positively regulated by the ECF factor SbrI and inhibited by SbrIs
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expression in sbrR mutant cells relative to WT cells that did not respond to
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complementation with pPSV38-SbrR, suggesting SbrR may not control the expression
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of these genes (Table 1). Indeed, previous work in our lab may explain the differential
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regulation of three genes (PA1202, PA1203, and PA2432) that fit this profile. We have
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previously shown that expression of PA1202, PA1203, and PA2432 (bexR) is bistable
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and subject to the control of a bistable switch mediated by the transcription regulator
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BexR (48). Therefore, the differential expression of these genes may result from
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bistable BexR activity, rather than changes in expression attributable to SbrI activation.
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PA3876 (narK2) also exhibited higher expression levels in sbrR mutant cells relative to
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expression of narK2 was also elevated in sbrIR double mutant cells relative to WT
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(Table 1), suggesting that the observed changes in narK2 expression are independent
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of SbrI.
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fold in sbrR mutant cells relative to WT (Table 1). MmsA and MmsB are enzymes
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involved in valine metabolism (49). The mmsAB operon is positively regulated by the
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suggest that although SbrR functions principally as a negative regulator of the muiA,
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PA4495 and sbrI genes, SbrR might also exert positive effects on the expression of
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some genes.
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mediated by type-IV pili (50). Swimming motility occurs in low viscosity liquid
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aeruginosa colonies grown overnight expand their borders via surface motility (51).
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When we compared our WT and sbrR mutant strains, we noticed colonies of sbrR
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mutant cells were slightly smaller (data not shown), suggesting these cells might have
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Compared to WT, PAO1 sbrR and PA14 sbrR mutants exhibit reductions in
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twitching motility of 14% and 20%, respectively (Fig. 3A). These findings suggest that
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although sbrR mutants have reduced twitching motility, cells of these mutants
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Next we tested the swimming motility of our strains. PAO1 flgK and PA14
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flgK mutants that do not produce flagella were unable to swim from the point of
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inoculation (data not shown). Compared to WT, PAO1 and PA14 sbrR mutants
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exhibited reductions in swimming motility of 12% and 19%, respectively (Fig. 3B).
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Thus, swimming motility is only slightly reduced in sbrR mutant cells, suggesting that
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functional flagella. Fluorescent staining of WT PAO1 and PAO1 sbrR revealed both WT
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To test whether cells of our sbrR mutant strains exhibited a swarming defect,
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we inoculated cells from overnight cultures onto plates containing a minimal media
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solidified with 0.5% agar. WT PA14 is a robust swarmer, forming colonies with
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dendrites that extended radially from the point of inoculation in an irregular starburst
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(Fig. 3D). In contrast to our WT PA14 strain, cells of our WT PAO1 strain did not swarm
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strain background (data not shown). When we examined our PA14 sbrR mutant strain
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we discovered it does not spread from the point of inoculation and is completely
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defective for swarming motility (Fig. 3C and D). This suggests that SbrR promotes
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The effect of SbrR on swarming motility is dependent upon SbrI and MuiA
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SbrI might be responsible for the motility defect in sbrR mutant cells. To determine if
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the swarming defect in PA14 sbrR mutants was dependent upon sbrI, we constructed
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the double mutant PA14 sbrIR. Swarming motility of cells of the PA14 sbrIR strain
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was restored to WT levels (Fig. 4A), demonstrating that SbrI is necessary for swarming
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sbrR mutants was likely due to the constitutive expression of a gene(s) in the SbrI
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regulon. The three most highly upregulated genes in the cells of the sbrR mutant in
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our microarrays were muiA, PA4495, and sbrI itself (Table 1). MuiA and PA4495 have
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mucoid strains that retain WT MucA (52). Neither MuiA nor PA4495 have any
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significant homology to any previously characterized proteins, but both proteins are
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generated PA14 sbrR muiA double mutants and PA14 sbrR PA4495 double
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mutants. Unlike the cells of a sbrR single mutant, cells of a sbrR muiA mutant did
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not exhibit a swarming motility defect and instead resembled WT PA14 with respect to
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their ability to swarm (Fig. 4A). This finding indicates that MuiA is necessary for
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swarming inhibition in sbrR mutant cells. The PA14 sbrR muiA double mutant
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swarming inhibition (Fig. 4B). In contrast, the PA14 sbrR PA4495 double mutant
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strain exhibited a swarming motility defect similar to that of the PA14 sbrR single
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mutant (Fig. S2), suggesting PA4495 is not necessary for the inhibition of swarming
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motility in sbrR mutant cells. Together, these results suggest that the inhibition of
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expression of MuiA.
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increased (Table 1), and swarming motility is inhibited (Fig. 4A and B). Moreover, MuiA
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is necessary for the inhibition of swarming motility in the sbrR mutant strain (Fig. 4A).
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mutant strain required activation of the entire SbrI regulon. To test this, we
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transformed WT PA14 cells with the same muiA expressing plasmid used to
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complement the PA14 sbrR muiA double mutant (pHERD20T-MuiA) and an empty
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that of the sbrR mutant strain in WT PA14 cells transformed with pMuiA (pHERD20T-
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MuiA), but not in WT PA14 cells transformed with the empty vector control
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(pHERD20T) (Fig. 4B). Thus, ectopic expression of muiA is sufficient for the inhibition of
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swarming motility.
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are inversely co-regulated in P. aeruginosa PA14 (9-12). That is, strains that exhibit
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increased swarming motility produce less biofilm, while strains that produce increased
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relationship, we next asked whether PA14 sbrR mutant cells were altered with
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respect to their ability to form biofilms. Following 8 hours of static growth, cells of the
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non-swarming PA14 sbrR mutant strain formed ~2-fold more biofilm than cells of the
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WT strain (Fig. 4C). This finding suggests that SbrR reduces biofilm formation or
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increase in biofilm formation in the sbrR mutant strain was SbrI-dependent. PA14
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sbrIR formed biofilms at levels similar to WT (Fig. 4C), suggesting that in addition to
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inhibiting swarming motility, increased expression of the SbrI regulon in the SbrR
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Our previous finding that the swarming motility defect of the sbrR mutant
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strain was MuiA-dependent led us to ask whether MuiA was also required for
524
enhanced biofilm formation in the sbrR strain. Cells of a PA14 sbrR muiA mutant
525
strain formed biofilms at levels similar to WT (Fig. 4C), indicating MuiA is necessary for
526
the enhanced biofilm formation observed in the sbrR mutant strain. In addition,
527
biofilm formation in PA14 sbrR muiA mutants could be restored to sbrR mutant
31
528
levels by expressing muiA from a plasmid (Fig. 4D). These findings suggest that
529
enhanced biofilm formation in the sbrR mutant strain may be the result of increased
530
SbrI-dependent expression of muiA. Lastly, while MuiA is required for increased biofilm
531
formation in the sbrR mutant strain, the PA14 muiA mutant strain formed biofilms at
532
WT levels (Fig. 4C). This indicates MuiA is not required for biofilm formation in WT cells
533
534
535
536
537
equivalent to that observed in cells of the sbrR mutant strain (Fig. 4B), suggesting it is
538
sufficient for the inhibition of swarming motility. In light of this observation and the
539
inverse relationship between swarming motility and biofilm formation, we next asked
540
whether ectopic expression of muiA was also sufficient to enhance biofilm formation.
541
In WT PA14 cells transformed with a muiA expression construct, but not those
542
543
comparable to that seen in sbrR mutants (Fig. 4D), demonstrating that ectopic
544
545
findings suggest that in sbrR mutant cells, constitutive activation of SbrI results in
32
546
high levels of muiA expression, which enhances biofilm formation via an unknown
547
mechanism.
33
548
549
DISCUSSION
We have presented evidence that SbrI and SbrR constitute a factor and anti-
550
factor pair with the N-terminal portion of SbrR interacting directly with SbrI. Cells
551
lacking SbrR are defective for swarming motility and exhibit enhanced biofilm
552
553
these cells. SbrR and SbrI represent a novel set of regulators of swarming motility and
554
biofilm formation in P. aeruginosa that mediate their effects through MuiA, a protein
555
556
557
558
559
genes that appear to be negatively controlled by SbrR and are expressed in an SbrI-
560
dependent manner. In particular, the expression of the putative sbrIR operon, muiA,
561
and PA4495 exhibited the largest changes in expression in cells of the sbrR anti-
562
factor mutant relative to WT (Table 1). Using promoter-lacZ fusion reporter strains we
563
564
these genes. We also showed that the increase in transcription that occurs from these
565
promoters in the absence of SbrR is dependent upon SbrI. These findings support the
34
566
idea that SbrR is an anti- factor specific for SbrI and are consistent with those of a
567
568
and PA4495 in response to the overexpression of the periplasmic protease ctpA (19).
569
By aligning the transcription start sites of sbrI, muiA, and PA4495 derived from
570
RNA-seq studies, Seo and Darwin identified putative -10 and -35 promoter elements,
571
with the sequence TAACCCG-N16-CGTCTCA-N6-A (+1) (19). Using the Find Individual
572
573
promoter sequence in the PAO1 and PA14 genomes revealed statistically significant
574
matches to this consensus only in the promoters of sbrI, muiA, and PA4495 (False
575
Discovery Rate q-value 0.01) (data not shown). The sbrI, muiA, and PA4495 promoters
576
may therefore be the only ones that are recognized directly by RNAP containing SbrI.
577
We note that the SbrIR regulon defined here is not unusually small for ECF factors,
578
which frequently control the expression of relatively small sets of genes (20).
579
Taken together, our results suggest a model in which the anti- factor SbrR
580
binds to SbrI and sequesters it at the membrane, preventing it from associating with
581
RNAP (Fig. 5). In response to an unknown extracytoplasmic signal, SbrI is released from
582
SbrR and associates with RNAP, resulting in expression of the SbrI regulon (Fig. 5). The
583
SbrI regulon likely consists of the putative sbrIR operon, muiA, and PA4495. SbrI-
35
584
585
expression of the SbrI regulon. muiA and PA4495 are expressed at high levels and
586
587
and enhanced biofilm formation. Previous work has shown that the expression of sbrI,
588
muiA, and PA4495 becomes elevated following osmotic shock (54), following
589
treatment with the cell wall inhibitory antibiotic D-cycloserine (55), and following
590
ectopic expression of the periplasmic protease CtpA, which leads to disruption of the
591
cell envelope (19). We suggest that cell envelope stress might be sensed by SbrR
592
leading to SbrI activation, expression of muiA, and a transition from motile swarming
593
cells to growth as a biofilm (Fig. 5). It is also possible that CtpA is capable of directly
594
595
596
SbrR and SbrI control swarming motility and biofilm formation in P. aeruginosa
597
598
The muiA gene was the most highly upregulated member of the SbrI regulon in
599
sbrR mutant cells relative to WT (Table 1), and expression from the PmuiA promoter was
600
shown to be SbrI-dependent (Fig. 2A) (19). We have shown that increased SbrI-
601
dependent expression of muiA in sbrR mutant cells inhibits swarming motility and
36
602
enhances biofilm formation, and that ectopic expression of muiA in WT cells has the
603
same effect.
604
605
(14, 15). As demonstrated by our swimming assays, PA14 sbrR mutants are capable of
606
chemotaxis and produce functional flagella (Fig. 3B). On the 0.5% agar plates used to
607
608
609
610
continues to produce surfactants (data not shown). These findings suggest the MuiA-
611
612
613
MuiA has previously been shown to inhibit the mucoid phenotype of certain
614
615
that activate AlgW (52). However, we think it unlikely that MuiA inhibits swarming
616
617
618
alginate production; e.g. MuiA did not detectably influence the production of alginate
37
619
in cells that produce truncated versions of MucA (52). Furthermore, alginate does not
620
621
In addition to MuiA, several other systems have been shown to exert reciprocal
622
control over swarming motility and biofilm formation in P. aeruginosa, including c-di-
623
GMP and the GacS/GacA two-component system (9, 11, 12). We note that the
624
abundance of the muiA and PA4495 transcripts is elevated in cells in which the
625
GacS/GacA system is artificially activated through deletion of retS (9). The GacS/GacA
626
system functions by activating the expression of genes encoding the small RNAs RsmY
627
and RsmZ (57). These small RNAs in turn sequester the RNA-binding protein RsmA that
628
binds many mRNAs directly to influence their translation or abundance (or both) (58).
629
Neither sbrI nor sbrR transcript abundance was altered in cells of a retS mutant relative
630
to WT (9), suggesting that the muiA and PA4495 transcripts may be direct targets of
631
RsmA. Future work will be aimed at identifying the mechanism by which MuiA
632
633
634
635
636
Connections to virulence
A signature-tagged mutagenesis screen identified sbrR (PA2895) as a gene
required during chronic respiratory infection in a rat lung model (18). Interestingly, a
38
637
638
639
640
641
for protease secretion, we do not observe any protease secretion defect in cells of our
642
PAO1 sbrR or PA14 sbrR mutant cells (data not shown). The results presented here
643
suggest the virulence defect of sbrR mutants in the rat lung model of chronic
644
645
646
formation, or both. Recent Tn-Seq analyses indicate that SbrI is required for
647
648
SbrIR may therefore play important regulatory roles during both chronic and acute
649
infections.
39
650
FUNDING INFORMATION
651
This work was funded by grant AI105013 from the NIH (to S.L.D). The funders had no
652
role in study design, data collection and interpretation, or the decision to submit the
653
654
655
ACKNOWLEDGEMENTS
656
657
McFarland for help with microarray data analysis, and Roger Levesque and Ann
658
40
659
FIGURE LEGENDS
660
661
FIG. 1. SbrI interacts with RNAP and is more abundant in sbrR mutants. (A) The
662
putative sbrIR operon. (B) The , , and subunits of RNAP copurify with RpoS-TAP
663
(lane 2) and SbrI-TAP (lane 3), but not with AceF-TAP (lane 1). Purified proteins were
664
separated by SDS-PAGE and stained with Coomassie blue. AceF-CBP, RpoS-CBP, and
665
SbrI-CBP indicate the purified proteins with the calmodulin binding protein (CBP)
666
moiety that remains after cleaving the protein A moiety of the TAP tag during
667
purification. (C) SbrR has a negative effect on SbrI-V protein abundance. Anti-VSV-G
668
Western blot of WT PAO1 (lane 1), PAO1 SbrI-V (lane 2), and PAO1 sbrR SbrI-V (lane 3).
669
670
FIG. 2. SbrR is an anti- factor that directly interacts with SbrI and inhibits its activity.
671
(A) -galactosidase activity of PAO1 PsbrI-lacZ, PAO1 PmuiA-lacZ, and PAO1 PPA4495-lacZ
672
reporter strains. sbrR and sbrIR mutants were generated in each reporter strain and
673
transformed with the indicated plasmids. pPSV38 is an empty vector control. Error bars
674
675
representation of SbrR and the location of its predicted transmembrane (TM) domain.
676
41
677
the N-terminal region of SbrR (SbrR-NTR) fused to cI and SbrI fused to the subunit of
678
RNA polymerase activates transcription from the test promoter driving expression of
679
lacZ. The diagram depicts test promoter PlacOR2-62, which bears the operator OR2
680
centered 62 bp upstream from the transcription start site of the lac core promoter.
681
This test promoter is linked to lacZ and located on the chromosome. (D) Effect of cI-
682
SbrR-NTR on transcription from PlacOR2-62 in the presence of the -SbrI chimera. Cells
683
harboring compatible plasmids directing the synthesis of the indicated proteins were
684
685
galactosidase activity.
686
687
FIG. 3. Swimming, twitching, and swarming motility of WT and sbrR mutant strains.
688
(A) The relative diameter of twitching motility zones for the indicated PAO1 and PA14
689
strains normalized to WT for each strain. *p=0.02, **p<0.001 (B) The relative diameter
690
of swimming motility zones of the indicated PAO1 and PA14 strains normalized to WT
691
for each strain. **p<0.001 (C) The relative swarming motility of the indicated PA14
692
strains. Plates were photographed and the area of motile cells was quantified with
693
694
sbrR strains. Significant differences between strains were determined by t-test. Error
42
695
696
697
FIG. 4. MuiA inhibits swarming motility and enhances biofilm formation. (A) Swarming
698
motility inhibition in sbrR mutants is dependent upon sbrI and muiA. Representative
699
images of swarming plates are shown above the quantification of the area of each
700
701
702
dependent upon sbrI and muiA. (D) Ectopic expression of muiA results in enhanced
703
704
705
706
FIG. 5. A model of the SbrIR regulon. SbrR is an anti- factor that binds to SbrI and
707
inhibits its activity. In response to an unknown signal, SbrI is released from SbrR,
708
709
710
43
711
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55
Table 1
4
PAO1
sbrR
PAO1
sbrR
PAO1
sbrIR
pPSV38 (fold
pPSV38 (fold
change)
change)
PA0086
tagJ1
2.3
PA0089
tssG1
2.2
PA0167
-2.4
PA0171
2.5
PA0839
3.8
PA0971
tolA
3.5
PA0972
tolB
2.6
13.0
14.0
PA1202
PA1203
4.2
3.1
PA1493
cysP
2.8
PA1494
muiA
103.1
-2.2
-2.2
PA2432
bexR
14.2
9.5
PA2895
sbrR
6.0
-2.0
PA2896
sbrI
18.0
-2.4
2.1
2.2
-9.7
-9.3
2.8
6.9
4.9
PA3923
-5.4
PA4495
21.9
PA3179
PA3569
mmsB
mms
PA3570
A
PA3876
narK2
PA5172
arcB
-2.6
PA5315
rpmG
2.7
PA5435
-2.4
PA5445
2.5
5
6
7