A novel CsrA titration mechanism regulates fimbrial gene expression in Salmonella typhimurium
2013; Springer Nature; Volume: 32; Issue: 21 Linguagem: Inglês
10.1038/emboj.2013.206
ISSN1460-2075
AutoresTorsten Sterzenbach, Kim Thuy Nguyen, Sean‐Paul Nuccio, Maria G. Winter, Christopher A. Vakulskas, Steven Clegg, Tony Romeo, Andreas J. Bäumler,
Tópico(s)Vibrio bacteria research studies
ResumoArticle20 September 2013free access Source Data A novel CsrA titration mechanism regulates fimbrial gene expression in Salmonella typhimurium Torsten Sterzenbach Torsten Sterzenbach Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Kim T Nguyen Kim T Nguyen Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Sean-Paul Nuccio Sean-Paul Nuccio Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Maria G Winter Maria G Winter Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Christopher A Vakulskas Christopher A Vakulskas Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA Search for more papers by this author Steven Clegg Steven Clegg Department of Microbiology, The University of Iowa, Iowa City, IA, USA Search for more papers by this author Tony Romeo Tony Romeo Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA Search for more papers by this author Andreas J Bäumler Corresponding Author Andreas J Bäumler Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Torsten Sterzenbach Torsten Sterzenbach Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Kim T Nguyen Kim T Nguyen Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Sean-Paul Nuccio Sean-Paul Nuccio Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Maria G Winter Maria G Winter Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Christopher A Vakulskas Christopher A Vakulskas Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA Search for more papers by this author Steven Clegg Steven Clegg Department of Microbiology, The University of Iowa, Iowa City, IA, USA Search for more papers by this author Tony Romeo Tony Romeo Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA Search for more papers by this author Andreas J Bäumler Corresponding Author Andreas J Bäumler Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Author Information Torsten Sterzenbach1, Kim T Nguyen1, Sean-Paul Nuccio1, Maria G Winter1, Christopher A Vakulskas2, Steven Clegg3, Tony Romeo2 and Andreas J Bäumler 1 1Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA, USA 2Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA 3Department of Microbiology, The University of Iowa, Iowa City, IA, USA *Corresponding author. Department of Medical Microbiology and Immunology, University of California Davis, One Shields Avenue, 3146 Tupper Hall, Davis, CA 95616, USA. Tel.:+1 530 754 7225; Fax:+1 530 754 7240; E-mail: [email protected] The EMBO Journal (2013)32:2872-2883https://doi.org/10.1038/emboj.2013.206 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A hierarchical control of fimbrial gene expression limits laboratory grown cultures of Salmonella enterica serovar typhimurium (S. typhimurium) to the production of type I fimbriae encoded by the fimAICDHF operon. Here we show that an unlikely culprit, namely the 5′-untranslated region (5′-UTR) of a messenger (m)RNA, coordinated the regulation. Binding of CsrA to the 5′-UTR of the pefACDEF transcript was required for expression of plasmid-encoded fimbriae. The 5′-UTR of the fimAICDHF transcript cooperated with two small untranslated RNAs, termed CsrB and CsrC, in antagonizing the activity of the RNA binding protein CsrA. Through this post-transcriptional mechanism, the 5′-UTR of the fimAICDHF transcript prevented production of PefA, the major structural subunit of plasmid-encoded fimbriae. This regulatory mechanism limits the costly expression of plasmid-encoded fimbriae to host environments in a mouse model. Collectively, our data suggest that the 5′-UTR of an mRNA coordinates a hierarchical control of fimbrial gene expression in S. typhimurium. Introduction The genomes of Escherichia coli and Salmonella enterica contain a large number of fimbrial gene clusters belonging to the chaperone/usher assembly class, a group defined by sequence homology of the encoded periplasmic chaperone and outer membrane usher assembly proteins (Nuccio and Bäumler, 2007; Yue et al, 2012). Many of these operons are required for intestinal colonization and/or the pathogenesis of urinary tract infection (Bäumler et al, 1997; Bergsten et al, 2005; Nielubowicz and Mobley, 2010; Wagner and Hensel, 2011). Each operon encodes structural subunits that are assembled into a fimbrial filament on the cell surface by a periplasmic chaperone and an outer membrane usher protein (Hung and Hultgren, 1998; Proft and Baker, 2009; Waksman and Hultgren, 2009). Fimbriated bacteria can express >200 fimbrial filaments on their surface, each composed of up to 1000 copies of the major structural subunit (Klemm, 1994; Proft and Baker, 2009). Thus, upon expression of a fimbrial gene cluster, the respective major structural subunit becomes one of the most abundant proteins in the bacterial cell. The costly expression of these surface structures is tightly controlled by regulatory mechanisms that prevent their elaboration in vitro. For example, laboratory-grown cultures of S. enterica serovar typhimurium (S. typhimurium) commonly elaborate only type 1 fimbriae encoded by the fimAICDHF operon (Duguid et al, 1966; Clegg et al, 1987), while expression of the remaining 11 chaperone/usher (C/U)-type fimbrial operons that are present in its genome cannot be detected by western blotting (Humphries et al, 2005) or flow cytometry (Humphries et al, 2003). Similarly, 9 of the 13 C/U-type fimbrial operons present in the genome of enterohaemorrhagic E. coli are not expressed under in vitro growth conditions (Low et al, 2006). One of the reasons why only a selected few C/U-type fimbrial operons are expressed under standard laboratory conditions is the hierarchical control of fimbrial gene expression in E. coli and S. enterica (Xia et al, 2000; Snyder et al, 2005; Holden et al, 2006; Nuccio et al, 2007). For example, the elaboration of type I fimbriae by uropathogenic E. coli strain CFT073 suppresses the expression of pyelonephritis-associated fimbriae, which in turn suppresses expression of F1C fimbriae (Snyder et al, 2005). Similarly, expression of PefA, the major structural subunit of plasmid-encoded fimbriae, can be detected by western blotting in S. typhimurium after the biosynthesis genes for type 1 fimbriae (fimAICDHF) have been deleted (Nuccio et al, 2007). The S. typhimurium fimAICDHF operon encodes a periplasmic chaperone (FimC), an outer membrane usher (FimD), a major structural subunit (FimA), three minor structural subunits (FimI, FimF and FimH), but not regulatory proteins. It is thus not obvious by which mechanism deletion of the fimAICDHF operon induces expression of plasmid-encoded fimbriae. Here, we investigated the mechanism by which the presence of the fimAICDHF operon prevents expression of plasmid-encoded fimbriae. Our results identify a novel mechanism of bacterial gene regulation that ensures hierarchical expression of fimbrial biosynthesis genes. Results SirA and the fimAICDHF operon synergize in repressing plasmid-encoded fimbriae The goal of this study was to determine the mechanism by which the presence of type I fimbrial biosynthesis genes interferes with expression of plasmid-encoded fimbriae in S. typhimurium. Consistent with a previous report (Nuccio et al, 2007), deletion of the fimAICDHF genes induced expression of PefA as detected by western blotting (Figure 1A). Deletion of type I fimbrial biosynthesis genes was accompanied by an increase in pefA transcript levels, as determined by quantitative real-time PCR (Figure 1B). Two possible mechanisms could account for the observation that the fimAICDHF genes reduce pefA transcript levels. The first possibility was that the fimAICDHF messenger RNA (mRNA) interfered with pefA transcription or with pefA transcript stability. This scenario seemed unlikely, since there was no precedent for such a regulatory mechanism. The second possible scenario was that expression of the fimbrial proteins encoded by the fimAICDHF operon reduced pefA transcript levels by an unknown mechanism. We reasoned that this mechanism might require signal transduction across the cytoplasmic membrane, because the periplasmic chaperone (FimC), the outer membrane usher (FimD), and the structural subunits (FimA, FimI, FimF and FimH) encoded by fimAICDHF operon are located in the cell envelope. Figure 1.SirA and the fimAICDHF operon synergize in repressing PefA expression. (A) Expression of PefA was detected in cell lysates of the indicated strains using western blot. (B) Relative expression of pefA-transcripts was assessed in RNA isolated from the indicated bacterial strains by real-time PCR. Bars represent the average of four independent experiments ±standard error. *P<0.05 (Student's t-test). (C) Expression of FimA was detected in cell lysates of the indicated strains using western blot. (D) Relative expression of fimA-transcripts was assessed in RNA isolated from the indicated bacterial strains by real-time PCR. Bars represent the average of four independent experiments ±standard error. n.d., not detected. (E) Surface expression of PefA was detected by flow cytometry in the indicated bacterial strains. Bars represent the average of four independent experiments ±standard error. (F) Representative images of PefA expression detected by flow cytometry. wt, S. typhimurium wild type (SR-11); sirA, S. typhimurium sirA mutant (TS23); fimAICDHF, S. typhimurium ΔfimAICDHF mutant (SPN342); fimAICDHF sirA, S. typhimurium sirA ΔfimAICDHF mutant (TS24).Source data for this figure is available on the online supplementary information page. Source Data for Figure 1 [embj2013206-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint When we investigated the possible contribution of two component regulatory systems to this process, we noted that inactivation of sirA had little effect on pefA transcript levels or PefA expression on its own, but a marked increase in PefA expression was observed in a sirA ΔfimAICDHF mutant (TS24) compared to a ΔfimAICDHF mutant (SPN342) (Figure 1A and B). Inactivation of sirA did not substantively alter expression of FimA (Figure 1C and D), suggesting that chromosomally encoded SirA did not exert its effect on pefA expression by altering expression of type I fimbriae. A marked increase in surface assembly of plasmid-encoded fimbriae was detected by flow cytometry with anti-PefA antiserum in the S. typhimurium sirA ΔfimAICDHF mutant (TS24) compared to the sirA mutant (TS23), the ΔfimAICDHF mutant (SPN342) or wild type (SR-11) (Figure 1E and F). These data suggested that suppression of plasmid-encoded fimbriae involved some synergistic interaction between SirA and the presence of the fimAICDHF genes, which provided our first lead for investigating the mechanism by which the presence of type I fimbrial biosynthesis genes interferes with pef expression. The fim operon cooperates with CsrB and CsrC to repress PefA expression SirA is the response regulator of the BarA/SirA two-component regulatory system in S. typhimurium (Ahmer et al, 1999), which is also known as BarA/UvrY in E. coli. In E. coli and S. typhimurium, SirA is a positive regulator of csrB and csrC, two genes encoding small untranslated RNAs termed CsrB and CsrC (Suzuki et al, 2002; Teplitski et al, 2003; Weilbacher et al, 2003). Consistent with these reports, we found that levels of CsrB and CsrC RNA were significantly reduced in a S. typhimurium sirA mutant (TS23) (Figure 2A and B). We next investigated whether increased PefA expression observed in a sirA ΔfimAICDHF mutant (TS24) was due to reduced expression of CsrB and CsrC RNA. Similar to the enhanced PefA expression observed in a sirA ΔfimAICDHF mutant (TS24) (Figure 1B), we found a profound increase in PefA expression in a csrB csrC ΔfimAICDHF mutant (TS131), while only marginal expression of PefA was detected by western blotting in a csrB csrC mutant (TS130) (Figure 2C). A similar trend was observed when pefA transcripts were quantified by real-time PCR (Figure 2D). No further increase in PefA expression was apparent in a sirA csrB csrC ΔfimAICDHF mutant (TS133) (Figure 2C), thus the lack of SirA or inactivation of the SirA-regulated genes csrB and csrC produced similar effects on PefA expression. These data suggested that the fimAICDHF genes and the small regulatory RNAs CsrB and CsrC synergized in suppressing expression of PefA. Figure 2.SirA represses expression of PefA via downregulation of CsrB and CsrC. Expression of CsrB RNA (A) and CsrC RNA (B) was quantified by real-time PCR using RNA isolated from S. typhimurium wild type (wt) and the sirA mutant (sirA). Bars represent the average of four independent experiments ±standard error. (C) Expression of PefA was detected in cell lysates of the indicated strains using western blot. (D) Relative expression of pefA transcripts was assessed in RNA isolated from the indicated bacterial strains by real-time PCR. Bars represent the average of three independent experiments ±standard error. (E) Expression of csrA mRNA was quantified by real-time PCR using RNA isolated from the indicated strains. Bars represent the average of four independent experiments ±standard error. Statistical analysis was performed by a Student's t-test.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013206-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint CsrA is a positive regulator of plasmid-encoded fimbriae CsrB and CsrC contain multiple high-affinity binding sites for CsrA (carbon storage regulator), a post-transcriptional regulator that binds the 5′-untranslated region (UTR) of mRNAs, thereby either reducing translation (by preventing ribosome binding or enhancing mRNA decay) or enhancing translation (by increasing translation and/or RNA stability) (Wei et al, 2001; Jonas et al, 2008; Bhatt et al, 2009). Binding of CsrB and CsrC sequesters CsrA, thereby repressing the activity of this RNA-binding protein (Liu et al, 1997; Weilbacher et al, 2003). In light of this connection, we next investigated whether CsrA was involved in regulating fimbrial expression in S. typhimurium. Expression of FimA was not markedly changed in a csrA mutant (TS112) or a csrB csrC mutant (TS130) compared to the S. typhimurium wild type (SR-11) (Figure 3A and B), although inactivation of csrA reduced fimA transcript levels modestly (Figure 3C). Interestingly, expression of PefA was abrogated in a csrA ΔfimAICDHF mutant (TS113) compared to the ΔfimAICDHF mutant (SPN342) (Figure 3D). Similarly, the profound PefA expression observed in a sirA ΔfimAICDHF mutant (TS24) was abrogated in a csrA sirA ΔfimAICDHF mutant (TS115). These data suggested that inactivation of sirA and deletion of the fimAICDHF genes increased PefA expression through a mechanism that was fully dependent on the positive regulator CsrA. Figure 3.CsrA regulates expression of PefA. (A) Expression of FimA was detected in cell lysates of the indicated S. typhimurium strains using western blot. (B) Quantification of FimA levels in western blots (N=3) by densitometry. (C) Relative expression of fimA mRNA was quantified in RNA isolated from the indicated bacterial strains by real-time PCR. Bars represent the average of three independent experiments ±standard error. (D) Expression of PefA was detected in cell lysates of the indicated S. typhimurium strains using western blot. wt, S. typhimurium wild type (SR-11). (E–G) Electrophoretic mobility shift assays (EMSAs). 3′-end biotinylated pefA 5′-UTR RNA (E), pefACCA 5′-UTR RNA (F) or pefA 5′-UTR RNA mutated at the indicated positions (G) was incubated with the indicated concentrations of CsrA-6xHis dimers. RNA protein complexes were separated on a native 5% TBE gel to perform EMSA. (H) Expression of PefA was detected in cell lysates of the indicated S. typhimurium strains using western blot.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013206-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint CsrA induces expression of plasmid-encoded fimbriae by binding the pefA 5′-UTR Since the substantial PefA expression observed in a sirA ΔfimAICDHF mutant was CsrA dependent (Figure 3D), we wanted to study the mechanism by which CsrA activates expression of plasmid-encoded fimbriae. We reasoned that this regulatory effect could be either indirect or a direct consequence of CsrA binding to the 5′-UTR of the pefA transcript. We performed 5′-rapid amplification of cDNA ends (5′-RACE) to determine the transcriptional start site of the pefACDEF transcript, which was located 76 bp upstream of the pefA start codon (RefSeq accession NC_003277.1 coordinate 14163). We next synthesized a biotinylated transcript containing nucleotides +1 to +148 relative to the transcriptional start site using in vitro transcription and investigated binding of CsrA to this transcript employing an RNA electrophoretic mobility shift assay (EMSA). When the biotinylated pefA 5′-UTR transcript was incubated with increasing concentrations of CsrA-6xHis, we observed a mobility shift at a CsrA-6xHis dimer concentration of 512 nM (Figure 3E). CsrA binds to a conserved sequence present in CsrB RNA, CsrC RNA and in the 5′-UTR of its target mRNAs, which contains a central GGA motif that is 100% conserved (Dubey et al, 2005; Mercante et al, 2009). Inspection of the pefA 5′-UTR nucleotide sequence revealed a single site (nucleotides 9–16, GCTGGAAA) with conserved GGA motif but otherwise marginal similarity to the proposed CsrA consensus sequence (ACAGGATG). To determine whether this potential CsrA-binding site was responsible for the observed mobility shift, we generated a transcript (pefACCA 5′-UTR) in which the conserved GGA motif was mutagenized to CCA. No mobility shift was observed when the biotinylated pefACCA 5′-UTR transcript was incubated with increasing concentrations of CsrA-6xHis (Figure 3F). These data suggested that CsrA bound specifically to a single GGA binding site in the pefA 5′-UTR. To further map the CsrA-binding site in the pefA 5′-UTR, we mutated nucleotides directly adjacent to the GGA motif (T11A, A15T) or in a distance of one nucleotide (C10G, A16T) or two nucleotides (G9C, A17T) from the GGA motif. An altered mobility suggested that the respective mutations altered the secondary structure of the resulting biotinylated constructs. Importantly, no electrophoretic shift was observed when these biotinylated constructs were incubated with increasing concentrations of CsrA-6xHis (Figure 3G). In contrast, mutation of nucleotides located in a distance of three bases from the GGA motif (T8A, T18A) did no longer prevent an electrophoretic shift with CsrA-6xHis. To further investigate whether binding of CsrA to the GGA motif in the pefA 5′-UTR was required for expression of plasmid-encoded fimbriae, we introduced point mutations into the S. typhimurium genome to change the central GGA motif in the pefA 5′-UTR to CCA. The resulting pefACCA mutation was introduced into a ΔfimAICDHF mutant and a ΔfimAICDHF csrB csrC mutant and PefA expression was determined by western blotting. Introduction of a pefACCA mutation into a ΔfimAICDHF mutant (TS139) or a ΔfimAICDHF csrB csrC mutant (TS 140) markedly lowered PefA protein levels (Figure 3H). Collectively, these data suggested that CsrA activates expression of plasmid-encoded fimbriae by binding a GGA motif in the 5′-UTR of the pefACDEF transcript. The 5′-UTR of the fimAICDHF transcript is sufficient for suppressing PefA expression We next investigated the mechanism by which SirA and the fimAICDHF genes suppressed the CsrA-mediated activation of PefA expression. Inactivation of sirA did not alter the transcript levels of csrA (Figure 2E), but lowered the levels of CsrB RNA (Figure 2A) and CsrC RNA (Figure 2B), two known inhibitors of CsrA activity (Suzuki et al, 2002; Weilbacher et al, 2003). These data were consistent with the idea that the mechanism by which SirA suppressed CsrA activity was by increasing the levels of the small untranslated RNAs CsrB and CsrC, which contain multiple high-affinity binding sites for CsrA and sequester this RNA binding protein (Suzuki et al, 2002; Weilbacher et al, 2003). However, the mechanism by which the fimAICDHF genes suppressed CsrA activity remained obscure. As mentioned above, one possibility was that the fimAICDHF mRNA was responsible for the observed suppression of PefA expression (Figure 1A). Interestingly, we noticed that the 5′-UTR of the fimAICDHF transcript contained two potential CsrA-binding sites that matched the proposed consensus sequence (ACAGGAUG, Figure 4A). To investigate whether the 5′-UTR of the fimAICDHF transcript was sufficient for preventing PefA expression, we cloned the corresponding DNA region in the vector pBAD30 under the control of the E. coli arabinose operon promoter (Figure 4B). The resulting plasmid (pTS30) or the vector control (pBAD30) was introduced into wild-type S. typhimurium (SR-11) or the ΔfimAICDHF mutant (SPN342) and expression of PefA detected by western blotting after growing bacteria in the presence of the inducer arabinose. Expression of the 5′-UTR of the fimAICDHF transcript fully suppressed PefA expression in the S. typhimurium ΔfimAICDHF mutant (SPN342) (Figure 4C). In contrast, introduction of the vector control (pBAD30) did not prevent expression of PefA in the ΔfimAICDHF mutant (SPN342). These data provided compelling evidence that proteins encoded by the fimAICDHF operon were not required for suppression of PefA expression. Instead, our data raised the surprising possibility that the 5′-UTR of a chromosomally encoded mRNA (i.e., the fimAICDHF transcript) could regulate the expression of a gene product (i.e., PefA) encoded on the virulence plasmid. Figure 4.The 5′-UTR of the fimA transcript suppresses PefA expression and binds to CsrA. (A) Sequence of the 5′-UTR of the fimA transcript according to Kroger et al (2012). Predicted CsrA-binding sites are underlined. Capital letters indicate the start of the pefA open reading frame. (B) Schematic depiction of the fimAICDHF operon and the cloning strategy for overexpressing the 5′-UTR of the fimA transcript. (C) Expression of PefA was detected in cell lysates of the indicated strains using western blot. Bacteria were grown for 48 h statically in the presence of 0.2% arabinose and carbenicillin. (D–G) Electrophoretic mobility shift assays (EMSAs). RNA protein complexes were separated on a native 5% TBE gel to perform EMSAs. 3′-end biotinylated fimA 5′-UTR RNA was incubated with the indicated concentrations of CsrA-6xHis dimers in the absence (D) or presence (E) of unlabelled competitor RNA. The concentrations of specific (fim) or non-specific (phoB) competitor RNA are indicated on the bottom of each lane. (F, G) 3′-end biotinylated fimA 5′-UTR RNA mutated at the indicated positions was incubated with the indicated concentrations of CsrA-6xHis dimers.Source data for this figure is available on the online supplementary information page. Source Data for Figure 4 [embj2013206-sup-0004-SourceData-S4.pdf] Download figure Download PowerPoint CsrA binds the 5′-UTR of the fimAICDHF transcript To further investigate the mechanism by which the fimAICDHF genes suppress PefA expression, we determined whether CsrA can bind to the 5′-UTR of the fimAICDHF transcript by an EMSA. A biotinylated transcript containing nucleotides +1 to +264 relative to the published start site of the fimAICDHF transcript (Kroger et al, 2012) was synthesized by in vitro transcription. When this biotinylated 5′-UTR transcript was incubated with increasing concentrations of purified His-tagged CsrA protein (CsrA-6xHis) (Mercante et al, 2006), we observed a partial mobility shift starting at a CsrA-6xHis dimer concentration of 16 nM, whereas a complete mobility shift was observed at a CsrA-6xHis dimer concentration of 256 nM (Figure 4D). To verify the specificity of the binding reaction, 256 nM CsrA-6xHis dimer was incubated with biotinylated in vitro transcribed 5′-UTR of the fimAICDHF transcript in the presence of increasing concentrations of unlabelled in vitro transcribed 5′-UTR of the fimAICDHF transcript. The unlabelled in vitro transcribed 5′-UTR of the fimAICDHF transcript was able to compete for CsrA binding as indicated by the disappearance of the mobility shift at higher concentrations of the competitor (Figure 4E). In contrast, competition for CsrA binding with a non-specific competitor (an in vitro transcribed fragment of the phoB gene) (Martinez et al, 2011) did not result in the disappearance of the mobility shift. These results suggested that CsrA specifically binds the 5′-UTR of the fimAICDHF transcript. To determine whether the potential CsrA-binding sites were responsible for the observed mobility shift, we generated a transcript in which both GGA motifs were mutagenized to CCA (G102C, G103C, G157C, G158C). No mobility shift was observed when the mutated (G102C, G103C, G157C, G158C) biotinylated 5′-UTR transcript of the fimAICDHF transcript was incubated with increasing concentrations of CsrA-6xHis (Figure 4F). These data suggested that CsrA bound specifically to GGA binding sites in the 5′-UTR of the fimAICDHF transcript. To further map the CsrA-binding sites in the 5′-UTR of the fimAICDHF transcript, we mutated nucleotides directly adjacent to each GGA motif (A99T, C100G, A101T, T105A, G106C, C107G, A154T, C155G, A156T, T160A, G161C, C162G). An electrophoretic shift was observed when the resulting biotinylated construct was incubated with increasing concentrations of CsrA-6xHis (Figure 4G). Finally, we mutated nucleotides in a distance of three nucleotides from each GGA motif (T96A, T97A, T98A, A108T, G109C, A110T, C151G, C152G, G153C, C163G, G164C, A165T). Mutation of nucleotides located in a distance of three bases from the GGA motif did not prevent an electrophoretic shift with CsrA-6xHis (Figure 4G). The 5′-UTR of the fimAICDHF transcript antagonizes the regulatory effects of CsrA The small untranslated RNAs CsrB and CsrC antagonize the regulatory effects of CsrA presumably by sequestering this RNA binding protein. Our finding that CsrA binds the 5′-UTR of the fimAICDHF transcript (Figure 4D and E) raised the possibility that the fimAICDHF genes antagonized the regulatory effects of CsrA by a similar mechanism. CsrB and CsrC are highly abundant in the cell, which explains in part why these small untranslated RNAs can antagonize CsrA activity. However, no antagonistic activity has been ascribed to mRNA targets of CsrA, presumably because these mRNAs are expressed at considerably lower levels than CsrB RNA and CsrC RNA. We thus compared expression levels of the 5′-UTR of the fimAICDHF transcript with those of CsrB RNA, CsrC RNA and the mRNAs of known CsrA targets (hilD, flhD and glgA) by quantitative real-time PCR (Yang et al, 1996; Altier et al, 2000; Jackson et al, 2002; Teplitski et al, 2003; Teplitski et al, 2006; Jonas et al, 2010; Yakhnin et al, 2013). As expected, the levels of CsrB RNA and CsrC RNA were ∼10- to 100-fold higher than transcript levels of hilD, flhD or glgA (Figure 5A). However, the levels of the fimAICDHF 5′-UTR were not only higher than those of other target mRNAs (i.e., hilD, flhD or glgA), but exceeded even the levels of the highly abundant CsrB RNA and CsrC RNA. Transcript levels of fimA, fimI and fimC were lower than that of the fimAICDHF 5′-UTR. Figure 5.The 5′-UTR of the fimA transcript regulates gene expression. RNA was isolated from the indicated bacterial strains grown in LB5.5. (A) Absolute copy numbers of the indicated transcript per microgram of total RNA were determined by quantitative real-time PCR for the indicated transcripts. (B–D) Relative changes in transcript levels were determined by quantitative real-time PCR for the indicated transcripts. Bars represent geometric means±standard error from three (A) or six (B–D) different experiments. Statistical analysis was performed by a Student's t-test and statistical significance of differences is indi
Referência(s)