Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA

Abstract

One of the prokaryotic post-transcriptional regulatory mechanisms involves the CsrA/RsmA family of proteins that act by modulating translation initiation at target mRNAs. In this study, we identified the regulon of RsmA of the Pseudomonas aeruginosa PAK strain by using cultures in the stationary phase of growth. The RsmA regulon includes over 500 genes, of which approximately one-third were affected by an rsmA mutation negatively, while the rest were affected positively. By isolating RsmA/mRNA complexes, analysing transcriptional and translational fusions, and performing gel-shift analyses, we identified 40 genes in six operons that are regulated by RsmA directly at the level of translation. All of these genes were affected by RsmA negatively and include genes encoding the type VI secretion system HSI-I, which has been implicated in the P. aeruginosa chronic infections. On the other hand, we were unable to demonstrate a direct interaction of RsmA with transcripts that are positively affected by this protein, including mRNAs encoding the type III secretion system and the type IV pili genes. Our work supports a model in which RsmA acts as a negative translational regulator, and where its positive effects are achieved indirectly by RsmA-mediated interference with translation of specific regulatory factors.

Figure 1

Growth phase dependent expression of RsmZ, RsmY and RsmA. A. Growth curves of the various P. aeruginosa strains; B. Time course of rsmY and rsmZ expression as determined by using transcriptional lacZ fusions and measuring levels of β-galactosidase activity; C. Time course of RsmA accumulation as determined by a Western immunoblot analysis using anti-RsmA antibodies.

Figure 2

Isolation and identification of transcripts bound to RsmA-H₆. RsmA-H₆ was purified from a culture of P. aeruginosa PAK rsmA (pMMB67-RsmA-H₆) using nickel-affinity chromatography. 5% of total RNA that was isolated from RsmA-H₆-containing fractions was visualized on a denaturing agarose gel (Upper panel, lane A). 5% of total RNA that was isolated from equivalent fractions obtained using the control strain P. aeruginosa PAK rsmA (pMMB67) was loaded in lane B. Lane M – Molecular size marker. Sections 1-4 of lane A of the denaturing agarose gel indicate equivalent sections of a denaturing polyacrylamide gel (not shown) that was used to separate the rest of the total RNA isolated from the RsmA-H6-containing fractions. RNA was extracted separately from each section of the polyacrylamide gel, converted to cDNA, followed by second strand synthesis and cloning. Shown are the identities of the isolated RNAs as determined by sequencing of 12 clones per gel section (Lower panel).

Figure 3

Organization of operons and features of leader sequences of the PA0081 and PA0082 operons (A); and the PA4492 operon (B). Asterisks represent the transcription initiation sites (+1 sites) identified by a bacterial sigma70 promoter recognition program BPROM, and confirmed by the 5′ RACE procedure. -10 and -35 represent the putative promoters corresponding to the indicated +1 sites. Open stars represent additional transcription initiation sites identified in the 5′RACE procedure. RBS: ribosome binding site. Dashed arrows: Sequences identified in RNA-RsmA-H₆ co-purification assay. GGA motifs are encircled, start codons are underlined. In A., the putative promoter and RBS are shown on the top strand for PA0081 and on the bottom strand for PA0082.

Figure 4

Secondary structures of leader sequences of genes identified as direct targets of RsmA regulation as predicted by M-fold (Zuker, 2003). Highlighted are the locations of the GGA sequences (thick lines) and of the ribosome binding sites (RBS).

Figure 5

RNA mobility shift assays with purified RsmA-H₆. A. Binding of RsmA-H₆ to RsmY, RsmZ, and the leader sequences of transcripts encoding PA0081, PA0082, PA4492, exsD, exsC, exoS, pilM, and the negative control lolB. Transcripts were radiolabeled and incubated in the absence or presence of various concentrations of RsmA-H₆ for 30 minutes at room temperature, before they were analyzed on an 8% native polyacrylamide gel. For each panel, reactions in lanes 1-3 contained 10 nM of radiolabeled RNA and 0, 1, and 3 μM RsmA-H₆ monomer, respectively. B. Competition experiments were conducted using 10 nM labeled PA0081 transcript and 0 (lane 1) or 2.5 μM (lanes 2-8) RsmA-H₆ monomer. The reaction shown in lane 2 was incubated in the absence of any competitor RNA. Reactions shown in lanes 3-8 were incubated in the presence of the unlabeled transcript of RsmY (lanes 3, 4), RsmZ (lanes 5, 6) and lolB (lanes 7, 8) at 2-fold (lanes 3, 5, 7) and 200-fold (lanes 4, 6, 8) excess relative to the labeled transcript.

Figure 6

Effect of RsmA on expression of PA0081, PA0082, and PA4492. Levels of β-galactosidase activity were determined in the P. aerugionosa PAK wild type and rsmA mutant strains carrying chromosomal lacZ fusions to PA0081, PA0082, and PA4492. A. Transcriptional fusions; B. Translational fusions; and C. Translational fusions driven by the constitutively active PlacUV5 promoter. D. Schematic representation of the transcriptional (i), translational (ii) and PlacUV5-driven translational (iii) lacZ fusions used in the study. See text for details.

Figure 7

Effects of various mutations in the RetS/GacSA/RsmYZ/RsmA regulatory pathway on the expression of PA0081 and exsD. Levels of β-galactosidase activity were determined in the P. aeruginosa PAK wild type and mutant strains carrying transcriptional (A and C) or translational (B and D) chromosomal lacZ fusions to PA0081 (A and B) or exsD (C and D).

Figure 8

Effect of RsmA on the expression of genes involved in iron homeostasis. Levels of β-galactosidase were measured in P. aeruginosa PAK wild type (white bars) and P. aeruginosa PAK rsmA mutant (black bars) carrying translational lacZ fusions to PA4228, PA1300, PA0081, and exsD (A), and transcriptional lacZ fusions to PA4228 and PA1300 (B). All assays were carried out after 4 hours of growth in LB with or without 300 μM iron chelator 2,2′-dipyridyl as indicated in the figure.

Figure 9

Immunoblot analysis of RsmA-regulated proteins. A. Cultures of P. aeruginosa PAK wild type and rsmA mutant were incubated for 7 hrs in LB, at 37°C and at 300 rpm shaking. Aliquots of cell and secreted fractions were analyzed by Western immunoblotting using antibodies against Hcp1, ExoS, ExoT, PilA and PilQ. B. Immunoblot analysis of secreted exotoxin A (ToxA). P. aeruginosa PAK wild type and rsmA mutant were incubated for 7 hrs in LB with 600 μM 2,2′-dipyridyl, at 37°C and at 300 rpm shaking. Secreted fractions were analyzed by Western immunobloting using antibodies against ToxA.

Figure 10

Alignment of sequences around the ribosome binding site (RBS) that represent the putative RsmA binding site in the leader sequences of the seven known direct targets of the P. aeruginosa RsmA. The sequence that was used to screen the intergenic sequences of the P. aeruginosa PAO1 genome to identify additional candidate targets of direct RsmA regulation is shown below the alignments. The putative RBS sequences are boxed.

Figure 11

General model for the RsmA-mediated global regulation in P. aeruginosa. P. aeruginosa RsmA is a part of the LadS/RetS/GacS/A/RsmY/Z/RsmA regulatory pathway in which LadS, RetS, and GacS/A modulate RsmA activity by regulating transcription of two small regulatory RNAs, RsmY and RsmZ. RsmY and RsmZ antagonize RsmA activity by competing with target mRNAs for RsmA binding. RsmA controls expression of more than 500 genes. Transcript levels of about two thirds these genes increase in the rsmA mutant or when RsmA is antagonized by RsmY and RsmZ. We propose that many of these genes are regulated by RsmA directly. On the other hand, we propose that the genes that are downregulated in the rsmA mutant or in the presence of high levels of RsmY and RsmZ are controlled by RsmA via indirect mechanisms. We propose that these mechanisms are specific to individual groups of genes and involve regulatory factors that are regulated by RsmA directly. Dashed lines – indirect regulation; Solid lines – direct regulation; white background – directly regulated genes; grey background – indirectly regulated genes.