Antisense RNA that affects Rhodopseudomonas palustris quorum-sensing signal receptor expression

Abstract

Quorum sensing in the bacterium Rhodopseudomonas palustris involves the RpaI signal synthase, which produces p-coumaroyl-homoserine lactone (pC-HSL) and RpaR, which is a pC-HSL–dependent transcriptional activator. There is also an antisense rpaR transcript (asrpaR) of unknown function. Recent RNAseq studies have revealed that bacterial antisense RNAs are abundant, but little is known about the function of these molecules. Because asrpaR expression is quorum sensing dependent, we sought to characterize its production and function. We show that asrpaR is approximately 300–600 bases and is produced in response to pC-HSL and RpaR. There is an RpaR-binding site centered 51.5 bp from the mapped asrpaR transcript start site. We show that asrpaR overexpression reduces RpaR levels, rpaI expression, and pC-HSL production. We also generated an asrpaR mutant, which shows elevated RpaR levels, and elevated rpaI expression. Thus, asrpaR inhibits rpaR translation, and this inhibition results in suppression of RpaR-dependent rpaI expression and, thus, pC-HSL production. The R. palustris asrpaR represents an antisense RNA for which an activity can be measured and for which a distinct regulatory circuit related to a function is elucidated. It also represents yet another subtle regulatory layer for acyl-homoserine lactone quorum-sensing signal-responsive transcription factors.

We have been interested in the Rhodopseudomonas palustris quorum-sensing system because although many Proteobacteria use acyl-homoserine lactone (AHL) quorum sensing signals, R. palustris is unusual in that it uses an aryl-HSL, p-coumaroyl-HSL (pC-HSL) rather than a fatty AHL. The R. palustris quorum sensing circuit is also unusual in that pC-HSL is not made entirely from endogenously produced substrates. Rather, its synthesis requires exogenous p-coumarate (1). The R. palustris signal synthesis gene, rpaI, codes for a member of the large LuxI family of AHL synthases, and the adjacent signal receptor gene, rpaR, codes for a member of the LuxR family of transcription factors. Although rpaI and rpaR are not cotranscribed, they are adjacent and show the same orientation (Fig. 1A). The rpaR translational stop codon is 86 bp upstream of the rpaI transcriptional start site. The rpaI gene is positively autoregulated by its product pC-HSL and RpaR, which binds to an inverted repeat element called the RpaR-box centered 48.5 bp from the rpaI transcript start site (2).

Fig. 1.

The R. palustris rpaR-rpaI region and expression of asrpaR and rpaR mRNA. (A) Map of the rpaR-rpaI region. The intergenic region is 86 bp and contains an inverted repeat RpaR-binding site (white box) (2). Numbers refer to nucleotide coordinates in the R. palustris CGA009 genome (32). The asrpaR transcript is depicted as a two-toned arrow. The black shading indicates the shortest product detected (304 bases), and the gray shading indicates the longest product detected (604 bases) (Table S1). (B) Northern-blot analysis of RNA from the wild-type CGA009 (WT) and the rpaR-deletion mutant CGA850 (R⁻) grown in the presence and absence of 250 nM pC-HSL. Blots were probed for products specific to asrpaR (antisense) or rpaR mRNA (sense) as described in Materials and Methods. The locations of size markers (bases) are indicated on the left.

We recently performed an RNAseq analysis to identify quorum-sensing-dependent transcripts in R. palustris and discovered that there is an rpaR antisense RNA (asRNA) produced in response to pC-HSL and RpaR. This asRNA, which we call antisense rpaR transcript (asrpaR), is among the transcripts showing the greatest pC-HSL dependence (2). We have pursued an investigation of asrpaR for the following reasons: There are extensive regulatory layers on LuxR homologs in different bacteria (3–9), and the implication is that precise fine-tuning of LuxR levels is a critical element of quorum sensing. The pC-HSL-RpaR dependence of asrpaR transcription leads us to speculate that because this RNA is complementary to rpaR message, it could exert a regulatory effect on quorum sensing. Regulation by an antisense RNA would represent yet another element that can fine-tune quorum sensing signal receptor activity. Furthermore, next-generation sequencing technologies have revealed that asRNAs are quite prevalent in bacteria. Although there is an extensive literature on regulatory functions of trans-encoded small-regulatory RNAs in bacteria, relatively little is known about cis-encoded asRNAs (10, 11). There has been speculation that the preponderance of asRNAs detected by next-generation sequencing by and large represents nothing more than insignificant mispriming events (11). There are, however, a few examples of bona fide asRNAs with known regulatory activities (12, 13).

Results

Characterization of asrpaR.

Our published RNAseq analysis of R. palustris quorum-sensing-controlled genes revealed a pC-HSL-activated antisense rpaR transcript or transcripts that appeared to overlap most of the length of the rpaR mRNA. The results indicated there was an asRNA or asRNAs extending from somewhere around 50 bases upstream of the rpaR stop codon to 604 bases upstream of the stop codon (2). A unique feature of the R. palustris pC-HSL quorum sensing system is that the signal is produced only if p-coumarate is added to the growth medium. Thus, the pC-HSL-dependent transcriptome can be identified by comparing wild-type cells grown with either p-coumarate or the pC-HSL signal itself to cells grown without either of these molecules (1, 2). Expression of rpaR is not affected by pC-HSL but the antisense rpaR, which we term asrpaR, shows >20-fold pC-HSL induction (2). To determine whether pC-HSL induced synthesis of a series of small rpaR antisense molecules or whether longer antisense molecules were produced, we performed a Northern blot analysis with RNA harvested from wild-type and rpaR-mutant R. palustris cells by using strand-specific RNA probes corresponding to 207 bp of the 3′ end of rpaR (Materials and Methods). We detected asRNA between 300 and approximately 600 bases from cells grown with pC-HSL (Fig. 1B). As expected, we detected transcripts (400–800 bases) when probing for rpaR mRNA from wild-type cells grown with or without pC-HSL. As a control, we show that there is no detectable rpaR message in the rpaR deletion mutant CGA850 (2) (Fig. 1B).

We mapped the asrpaR transcript start site to a guanine positioned 41 bases from the rpaR stop codon by primer extension and S1 nuclease protection analyses with RNA isolated from cells grown with or without p-coumarate, or RNA isolated from the rpaR deletion mutant (Fig. 2 A and B and Fig. S1). We used a 3′-RACE analysis to detect asrpaR 3′ ends. We sequenced 11 different clones and found asrpaR sizes to vary from 305 to 468 bp (Table S1), in agreement with the sizes of the most abundant species detected by Northern analysis (Fig. 1B). It is not clear whether there may be several different transcripts synthesized or whether the size range results from some degradation of a single long element, but it is clear that pC-HSL induces asRNAs of considerable length. We also observed a range in the sizes of rpaR mRNA. The rpaR ORF is 732 bp, corresponding well with the upper end of rpaR transcript sizes observed in the Northern blot analysis (Fig. 1 B).

Fig. 2.

The asrpaR transcript start site and the dependence of asrpaR expression on the intergenic RpaR box. (A) Primer extension analysis of asrpaR from the R. palustris wild type (CGA009) grown with or without p-coumarate and the RpaR mutant (CGA850) grown with p-coumarate. (B) Sequence of the rpaR-rpaI intergenic region. Transcription start sites for asrpaR (coordinate 349392) and rpaI (coordinate 349293) are indicated by +1 arrows. The RpaR-binding site (RpaR box) is indicated by the gray box (coordinates 349350–349331), the rpaI start codon (coordinates 349264–349262) is underlined, and the rpaR stop codon is bolded (coordinates 349352–349350). Arrows indicate the 5′-most position of the asrpaR-lacZ fusions for pHH102 (thick arrow) and pHH103 (thin arrow). The Ω designates the location of the Ω-cassette insertion in strain CGA854 as described in Materials and Methods. (C) Transcription of asrpaR requires both RpaR and the RpaR-binding site. Wild type (WT) or the RpaR⁻ mutant containing pHH102 (intact RpaR-box) or pHH103 (RpaR-box deletion) was assessed for asrpaR-lacZ activity. Cells were grown with the following additions: none (white bars), 250 nM pC-HSL (gray bars), or 0.5 mM p-coumarate (black bars). Data are the means of three biological replicates, and the error bars indicate ranges.

The asrpaR transcript start site is located 51.5 bp from the RpaR box required for pC-HSL induction of rpaI (Fig. 2 A and B). Because this spacing is similar to that between the rpaI transcript start and the RpaR box (48.5 bp), we reasoned that the RpaR box might be involved in pC-HSL induction of asrpaR. To test this idea, we constructed two asrpaR-promoter-lacZ plasmids: One contained the asrpaR transcriptional start fused to lacZ and extended 81 bp upstream of the asrpaR start site (pHH102; Fig. 2B, thick arrow), and the other extended 44 bp from the asrpaR transcript start and, thus, contained only 3 bp of the RpaR-box (pHH103; Fig. 2B, thin arrow). Expression of lacZ in R. palustris containing the complete RpaR-box plasmid was induced by pC-HSL, but pC-HSL did not induce lacZ in R. palustris carrying the RpaR-box-deletion construct (Fig. 2C). As a control, we used the rpaR deletion mutant to show that pC-HSL induction of lacZ in cells containing the complete RpaR-box-asrpaR-lacZ construct required RpaR (Fig. 2C). Thus, we conclude that induction of both rpaI and asrpaR depends on a single RpaR box in the rpaR-rpaI intergenic region and also depends on RpaR and pC-HSL. It should be pointed out here that a number of genes are activated by RpaR and pC-HSL, but electromobility shift assays show that among these genes, only the rpaI operon is a direct target of RpaR (2). By using the rpaI RpaR-binding site, asrpaR is also directly regulated by RpaR.

Evidence That asrpaR Is Not Translated.

Although uncommon, there is precedence for overlapping (or cryptic) protein-encoding antisense genes in bacteria (14). We examined the asrpaR DNA sequence for potential ORFs by using ORFfinder (www.ncbi.nlm.nih.gov/gorf/gorf.html) and identified a single ORF encoding a polypeptide >50 aa residues (480 bp in length). We tested the idea that asrpaR codes for a polypeptide in several ways, and all of the evidence is consistent with the conclusion that it does not. First, a BLAST analysis showed these peptides do not match any known sequences. Second, we have a large database of peptide fragments from R. palustris grown in the presence of p-coumarate (15), and we did not find peptides corresponding to the ORF. Third, we created an asrpaR-lacZ translational fusion plasmid. β-Galactosidase activity in wild-type R. palustris containing this plasmid was negligible and unaffected by growth in the presence of p-coumarate. Fourth, we constructed an asrpaR plasmid containing a hexaHis-tag–coding sequence at the 3′ end of the predicted 480-bp ORF. We could not detect the putative His-tagged protein by Western blotting with anti-His tag antiserum. Fifth, and most convincing, we constructed an R. palustris asrpaR mutant (CGA854), with a stop codon substituted for the glutamate-codon at position 11 in the ORF. This substitution was designed so that it did not alter the RpaR protein sequence. In experiments described below, we show measurable activity of asrpaR, and the activity is not affected in the stop codon substitution mutant strain.

Overexpression of asrpaR Reduces RpaR Polypeptide Levels, rpaI Transcription, and pC-HSL Production.

Because the RNAseq (2) and Northern blot analyses (Fig. 1B) indicated that pC-HSL did not influence rpaR mRNA levels, but activated asrpaR expression, we asked whether asrpaR might interfere with rpaR translation. Levels of RpaR in R. palustris are low, and for technical reasons, we could not detect native RpaR or even a hexaHis-RpaR encoded on an expression plasmid by Western blotting. We thus sought evidence that asrpaR overexpression would affect quorum sensing in R. palustris. We predicted that asrpaR overexpression should decrease RpaR from its already low level and, thus, have a measurable affect on rpaI expression, which depends on RpaR and on the enzymatic product of RpaI, pC-HSL. Our prediction was borne out by experiments with R. palustris containing a glyceraldehyde-3-phosphate dehydrogenase promoter-controlled asrpaR construct (Fig. 3 A and B). Furthermore, as discussed above, we also used a vector expressing asrpaR with a stop codon substituted for the glutamate-codon at position 11 in the potential polypeptide coding sequence of asrpaR, and cells with this vector had a phenotype identical to that of cells with the wild-type asrpaR vector (Fig. 3A). This finding supports our view that asrpaR function is at the level of RNA interactions and asrpaR does not code for a functional protein. The data suggest asrpaR base pairs with rpaR mRNA, and basepairing reduces rpaR translation.

Fig. 3.

Overexpression of asrpaR decreases RpaR levels and RpaR-dependent expression of rpaI. (A) Overexpression of asrpaR reduces rpaI-lacZ expression. β-Galactosidase activity in R. palustris CGA814 with the asrpaR expression vector pHH104 (asrpaR), the asrpaR STOP mutation vector pHH105 (asrpaR-STOP), or empty vector pBBPgdh (vector) grown for 16 h with 0.5 nM pC-HSL. (B) pC-HSL production by R. palustris CGA009 with pHH104 (filled bars; asrpaR) or pBBPgdh vector control (open bars; vector). Cells were grown in the presence of 0.5 mM p-coumarate. (C) Overexpression of asrpaR reduces hexaHisRpaR in recombinant P. aeruginosa. (C Upper) SDS/PAGE of soluble polypeptides from P. aeruginosa containing a hexaHis-RpaR expression plasmid (pHH101) plus either the asrpaR-expression vector pHH100 (AS) or the empty vector pQF5016b (V). Purified hexaHis-RpaR (predicted molecular mass 27,800) is included for comparison. Locations of molecular mass standards (in kilodaltons) shown on the left. (C Lower) HexaHis-RpaR was visualized by probing with anti–poly-His antibody. For A and B, data are the means of two biological replicates, and the error bars indicate ranges.

To gain more direct evidence that asrpaR interfered with RpaR expression, we investigated whether overproduction of asrpaR affected the level of a His-tagged RpaR expressed from a low-copy number plasmid in recombinant Pseudomonas aeruginosa, which has a high genomic GC content similar to that of R. palustris. For overexpression of asrpaR, we used a plasmid with an iso-propylthio-β-d-galactopyranoside-inducible asrpaR (pHH100). We assessed the level of hexaHis-RpaR by Western blotting with anti–poly-His serum. The hexaHis-RpaR was easily detected in cells that were not overexpressing asrpaR, whereas overexpression of asrpaR reduced the level of hexaHis-RpaR to below detection (Fig. 3C).

Reducing asrpaR Expression Increases RpaR Polypeptide Levels and rpaI Promoter Activity in R. palustris.

Construction of a specific asrpaR mutant is not straightforward: Deletion of the antisense-coding DNA also disrupts the corresponding rpaR gene (Fig. 1A), and deletion of the RpaR-binding site required for asrpaR expression (Fig. 2C) reduces rpaI expression (2). Therefore, to create the R. palustris asrpaR mutant CGA851, we inserted an Ω stem-loop terminator fragment (16) between the asrpaR transcript start site and the RpaR-box (Fig. 2B and Materials and Methods). Disruption of the R-protein binding site spacing in other AHL quorum sensing systems results in loss of transcriptional activation (17). As expected, we did not detect asrpaR RNA in the Ω insertion mutant (Fig. 4A). However, the asrpaR-Ω mutant had normal levels of rpaR mRNA (Fig. 4A), suggesting the asrpaR does not affect rpaR transcription or mRNA stability.

Fig. 4.

Reduced asrpaR correlates with increased RpaR levels but not rpaR mRNA. (A) asrpaR (Upper) and rpaR mRNA (Lower) from wild type (WT), asrpaR-Ω (ASΩ) mutant, wild type containing pBBPgdh vector control (WT vect), and wild type containing the asrpaR expression vector pHH104 (WT asrpaR) grown in the presence and absence of pC-HSL. (B) An R. palustris asrpaR mutant has increased VSVG-RpaR levels. (B Upper) SDS/PAGE of soluble polypeptides from the wild type or asrpaR-Ω mutant grown in the presence of 250 nM pC-HSL. Purified VSVG-RpaR (predicted molecular mass 28,200) is included (rightmost lane) for comparison. Locations of molecular mass standards (in kilodaltons) are indicated on the left. (B Lower) VSVG-tagged RpaR was visualized by Western blotting with VSVG antibodies.

We hypothesized the asrpaR-Ω mutation would affect RpaR polypeptide levels and activity. Because we were unable to detect RpaR in R. palustris by Western blotting with RpaR antiserum, we replaced the native rpaR gene with a gene coding for RpaR with a N-terminal vesicular stomatis virus glyocoprotein (VSVG) epitope (Materials and Methods) in the wild type and the asrpaR-Ω mutant. We then detected VSVG-RpaR in R. palustris cell lysates by Western blotting with anti-VSVG serum (Fig. 4B). As predicted, there was more VSVG-RpaR in the asrpaR-Ω mutant than in the wild type (Fig. 4B). By using the rpaI-lacZ reporter we also show that the rpaI promoter is more active in the asrpaR-Ω strain than its parent (Fig. 5A). These data are consistent with the idea that RpaR polypeptide levels are increased when there is no asrpaR present to interfere with translation. We also found that rpaI-lacZ levels were indistinguishable between wild type and CGA854 (Fig. 5A), which harbors an asrpaR gene with a stop codon in the potential coding sequence. As discussed previously, this finding supports our view that asrpaR is not translated.

Fig. 5.

Reduced asrpaR correlates with increased rpaI expression and pC-HSL production. (A) β-Galactosidase levels of R. palustris containing an rpaI-lacZ plasmid (pPrpaI) and grown with pC-HSL. Shown are the wild type CGA009 (WT), the asrpaR-Ω mutant CGA851 (ASΩ), the asrpaR-STOP mutant CGA853 (AS-STOP), WT with the vector control for the anti-asrpaR, pBBPgdh (V), or WT with the anti-asrpaR expression vector pHH106 (antiAS). Data for the WT, ASΩ, and AS-STOP are reported as a percent of WT and for the WT carrying the antiAS plasmid data are reported as a percent of the vector control. (B) pC-HSL production by R. palustris CGA009 strains carrying the anti-asrpaR expression construct pHH106 (filled bars; antiAS) or the vector alone (open bars; vector). Cells were grown with 0.5 mM p-coumarate. Data are the means of two biological replicates, and the error bars indicate ranges.

We generated an anti-asrpaR expression vector (pHH106) plasmid coding for 204 bases near the 3′ end of the rpaR sense mRNA (the full-length rpaR ORF is 732 bases). As predicted, R. palustris containing the anti-asrpaR expression vector showed a modest increase in rpaI-lacZ activity (Fig. 5A) and pC-HSL levels (Fig. 5B).

Discussion

Bacterial regulatory RNAs are important in gene control. Most studies of these regulatory RNAs have focused on trans-encoded small RNAs (sRNAs) known to control a range of functions. The list of sRNA-controlled functions includes examples of AHL signal receptors (18, 19). With the advent of RNAseq methods, there have been reports showing cis-encoded asRNAs can be quite common in bacteria (10, 11). With a few exceptions, the functional importance of these asRNAs is not understood (10, 11), and one interpretation of the RNAseq results is that in many cases, the asRNAs might have little or no biological significance. In a previously published study of quorum sensing in R. palustris, we discovered antisense rpaR RNA and an RNAseq analysis indicated its synthesis was activated by RpaR and pC-HSL, the quorum-sensing signal to which RpaR responds (2). The fact that the synthesis of this asRNA was regulated suggested that it might be of biological significance. We suspected that an asrpaR transcript might regulate some feature of quorum sensing or it might code for a regulator of quorum sensing. Thus, we chose to study it in more detail.

The rpaR gene is upstream of rpaI, and both genes are transcribed in the same direction. There are 86 bp between the rpaR translational stop codon and the rpaI transcription start site. There is an RpaR-binding site situated in the rpaR-rpaI intergenic region that is required for activation of rpaI by pC-HSL-bound RpaR (Fig. 1A). Our evidence indicates that asrpaR is approximately 300–600 bases in length with a transcript start site located 41 bp into the rpaR ORF and confirms that pC-HSL and RpaR activate expression of asrpaR, and this activation depends on the RpaR-binding site required for rpaI induction by pC-HSL and RpaR (Fig. 2). We present several lines of evidence indicating that asrpaR is not translated and that asrpaR functions as a regulatory RNA (Figs. 2–5).

Our studies of asrpaR overexpression strains and asrpaR mutants indicate that asrpaR represses RpaR levels, but not rpaR transcription, and it affects rpaI expression and timing of pC-HSL production under our experimental conditions. Thus, asrpaR has a measurable biological activity. We propose it serves as a mechanism to fine-tune cellular levels of RpaR in populations that have achieved a quorum. This proposal is supported by our finding that rpaR mRNA levels are not affected by induction of asrpaR or by inactivating mutations of asrpaR (Figs. 1B and 4A). We suppose the regulatory mechanism involves asrpaR base-pairing with rpaR message. Other asRNAs exert activity either by modifying levels of target transcripts or by interfering with target translation (10, 11). It does not appear that asrpaR affects rpaR mRNA levels, either via transcription interference, attenuation, or altering RNase susceptibility, because rpaR sense transcript levels are indistiguishable in wild-type and asrpaR-mutant strains (Fig. 4A). In fact, asrpaR overexpression does not affect rpaR mRNA levels (Fig. 4A) although RpaR activity is reduced (Fig. 3).

Many regulatory RNAs, including the handful of characterized asRNAs, affect translation by base pairing with the target mRNA ribosome-binding site (rbs) and interfering with ribosome binding (10, 11). This situation is unlikely to be the case for asrpaR, which is predicted to base pair with the 3′ region of rpaR mRNA. We do not know how asrpaR interferes with translation. There are examples of regulatory RNAs that bind their targets up- or downstream of the rbs, yet still affect translation (20–22), and it has been suggested that extended stretches of sense-asRNA duplexes might be sufficiently stable to interfere with translation (10).

In AHL quorum-sensing systems, the cellular concentration of the LuxR homolog affects the sensitivity of the system to the signal (23–25). A variety of regulatory mechanisms maintain these transcription factors at specific levels. Active Agrobacterium tumefaciens TraR levels are influenced posttranslationally by TrlR, a TraR homolog that does not possess a DNA binding domain. TrlR forms inactive heterodimers with full-length TraR (3). The A. tumefaciens quorum-sensing circuit also includes TraM an antiactivator protein that interacts with TraR to prevent its DNA binding (5, 8). A. tumefaciens TraM mutants respond to much lower levels of the quorum-sensing signal than does the parent (26). Antiactivator proteins have been identified in P. aeruginosa. The QteE (7) and QslA (6) proteins function to reduce levels of active LasR and decrease sensitivity to the quorum-sensing signal. Here, we show an additional form of quorum-sensing transcription factor control in R. palustris. The R. palustris quorum-sensing circuit can use asrpaR to fine-tune RpaR levels. Compared with control of a quorum-sensing signal receptor by polypeptides, control of RpaR pools by an asRNA might increase target specificity and, obviously, there could be energy efficiency because there is no translation. It has also been suggested that asRNA regulation might have an advantage over transcription regulator proteins when fast responses are useful (27, 28).

It is possible that an asRNA fine-tunes other quorum-sensing systems in a manner similar to asrpaR; antisense RNAs have been identified for Pseudomonas syringae psyR (29). They are believed to be read-through products from the adjacent psyI gene, which is convergently transcribed from and overlaps 69 bp of the 3′-end of psyR. One wonders whether this reflects an asRNA regulatory mechanism.

Materials and Methods

Bacteria Strains, Strain Construction, and Growth Conditions.

Escherichia coli S17-1 (30) and P. aeruginosa PA0-T7 (31) were grown in Luria–Bertani broth with aeration at 37 °C. R. palustris CGA009 (32) and its derivatives were grown photoheterotrophically at 30 °C in photosynthetic medium (PM) with 10 mM succinate (33) and p-coumarate or pC-HSL as indicated. Antibiotics were used at the following concentrations: for E. coli, 100 μg/mL ampicillin, 25 μg/mL kanamycin, and 20 μg/mL gentamicin; for P. aeruginosa, 300 μg/mL carbenicillin and 50 μg/mL gentamicin; and for R. palustris, 100 μg/mL gentamicin and 100 μg/mL kanamycin.

We used a variety of R. palustris CGA009 mutants including CGA814, which has a rpaI::lacZ mutation, and CGA850, a rpaR deletion mutant (1, 2). Other mutants were constructed as described below. The asrpaR mutant CGA851 was created by inserting an Ω stem-loop terminator fragment (16) between the asrpaR transcript start site and the RpaR box (Fig. 2B) without disruption of the rpaR gene. We used a knockout plasmid to make this mutant. The DNA fragment used to construct the knockout plasmid was created by PCR-sequence overlap extension (SOEing) (34) and corresponded to R. palustris genome coordinates 349794–348951. We inserted the 25-bp Ω stem-loop terminator fragment plus a repeat of the last three codons of rpaR (TGCTTGTGA) at coordinate 349350 to minimize the effect of the Ω terminator in RpaR activation of rpaI transcription. This fragment was cloned into the suicide plasmid pJQ200KS (35) and mated into R. palustris CGA009 by conjugation with E. coli S17-1 (30) to create strain CGA851.

To engineer R. palustris strains that encode a VSVG-tagged RpaR polypeptide, we replaced the native rpaR gene with a VSVG-tagged rpaR gene. By using SOEing, a 1,616-bp DNA fragment (coordinates 350481–348866) including the VSVG epitope (YTDIEMNRLGK) inserted between the first and second codons of rpaR was cloned into pJQ200KS and transferred into either wild-type R. palustris to create strain CGA852 or the asrpaR-Ω mutant CGA851 to create strain CGA853.

We created the R. palustris asrpaR-STOP mutant strain CGA854 by replacing the glutamate codon at position 11 in the potential ORF with a stop codon by PCR amplification of a 1,531-bp DNA fragment containing asrpaR and flanking sequence (coordinates 350481–348951), cloning this fragment into pJQ200KS, using QuikChange Site-Directed Mutagenesis (Stratagene) to make the mutation, and mating into R. palustris CGA009.

Plasmids.

The asrpaR-expression vector for use in P. aeruginosa, pHH100, was constructed by PCR amplification of a 721-bp DNA fragment from coordinates 349349–350069, and cloning in pQF5016b (36). The N-terminal hexaHis-tagged RpaR expression plasmid, pHH101, was constructed by SOEing to yield a DNA fragment containing the entire rpaR ORF (coordinates 350081–349350) with six histidine codons inserted between the first and second codons, and cloning into pBBR1MCS5 (37). The asrpaR-lacZ construct with an intact RpaR box (pHH102) was created by cloning a 110-bp PCR product containing the asrpaR promoter (coordinates 349311–349421) into pBBR1MCS5-lacZ (1). The asrpaR-lacZ reporter construct lacking the RpaR box (pHH103) was created in a similar fashion except that the PCR product was shorter (73 bp) because it lacked the RpaR-binding site (coordinates 349348–349421). An asrpaR-expression vector for use in R. palustris, pHH104, was constructed by using PCR amplification to generate a 722-bp DNA fragment from coordinates 349350–350071, which was cloned into pBBPgdh (38). pHH105 is identical to pHH104, except we mutated the nucleotide at coordinate 349467 from a G to a T by using a QuikChange Site-Directed Mutagenesis kit (Agilent Technologies). This base change modified the 11th codon of the potential ORF identified in the asrpaR from a glutamate to a stop codon but did not alter the sequence of RpaR. To generate the anti-asrpaR expression vector, pHH106, we PCR amplified a 204-bp DNA fragment that encodes an interior portion of rpaR sense mRNA (coordinates 349592–349389) and cloned it into pBBPgdh.

Northern Blotting.

We isolated total RNA from R. palustris cultures at an optical density at 660 nm of 0.45 as described (15, 39). RNA (10 μg) was denatured, separated by agarose-gel electrophoresis [1.2% (wt/vol) agarose], and transferred to a positively charged nylon membrane by using a NorthernMax-Gly Gel Prep kit (Ambion). Membranes were probed with either a 207-bp, strand-specific, DIG-labeled RNA probe specific to rpaR sense or to asrpaR RNA (coordinates 349386–349592). The RNA probes were created by transcription-labeling and hybridization as described by the manufacturer (DIG Northern Starter kit; Roche Applied Science).

Primer Extension Analysis.

We annealed 20 μg of total RNA from log-phase R. palustris cultures with 10 nM of a 6-FAM–labeled primer complementary to the terminal 118 bp of the rpaR ORF. Reverse transcription was with SuperScript III reverse transcriptase (Life Technologies), and the cDNA was purified for GeneScan sequencing analysis as described (2).

Analysis with 3′-RACE.

We used 10 μg of total RNA isolated from log-phase R. palustris grown in the presence of 250 nM pC-HSL and attached a poly-A linker to the 3′-terminal of RNA using E. coli Poly(A) polymerase (New England Biolabs). After cleanup using the RNAeasy kit (Qiagen), 25% of the reaction volume was further processed by using the 3′-RACE System for Rapid Amplification of cDNA Ends as described by the manufacturer (Life Technologies). Two rounds of 3′-RACE were performed by using a distinct primer unique to asrpaR each time (round 1 unique primer corresponded to genome coordinate 349662–349680; round 2–349469-349489). Eleven 3′-RACE products were TA-cloned into pCR2.1 TOPO (Life Technologies) and sequenced (Table S1).

Promoter Activity Assays.

To monitor the rpaI promoter levels, we used either R. palustris CGA814, which harbors a chromosomal rpaI-lacZ fusion, or the plasmid pPrpaI (1), and to monitor asrpaR promoter activity, we used lacZ transcriptional fusion plasmids (pHH102 and pHH103). β-Galactosidase levels were measured with a Tropix Galacto-Light Plus kit (Applied Biosystems).

Measurement of pC-HSL.

To measure pC-HSL levels in R. palustris cultures, bacteria were grown with 0.5 mM p-coumarate (starting OD₆₆₀ 0.04) for the indicated times. pC-HSL was extracted from cultures with acidified ethyl acetate (0.1 mL of glacial acetic acid per liter) and measured by using a described pC-HSL bioassay (1).

Western Blotting.

To detect VSVG-RpaR from R. palustris and hexaHis-RpaR from P. aeruginosa, cells were grown to late-log phase, harvested by centrifugation, suspended in SDS/PAGE buffer, and lysed by boiling and sonication. Cell lysates (7.5 μg of protein per lane for P. aeruginosa; 18 μg per lane for R. palustris) were separated on duplicate 12% acrylamide Tris-glycine SDS/PAGE gels. One gel was stained with Coomassie brilliant blue stain, and the other was electroblotted onto a PVDF membrane (Millipore). VSVG-tagged RpaR was detected with VSVG antiserum (Sigma Chemical), which was detected with secondary anti-rabbit horseradish peroxidase-conjugated IgG and chemiluminescent substrate (Pierce Protein Research). HexaHis-RpaR was visualized by using a SuperSignal West HisProbe kit (Pierce Protein Research).