Bacterial strains and culture conditions

LB medium (1% [w/v] Tryptone; 1% [w/v] NaCl and 0.5% [w/v] yeast extract) was used for culture of bacteria unless stated otherwise. The antibiotics ampicillin (100 μg mL−1), tetracycline (15 μg mL−1), kanamycin (50 μg mL−1), and chloramphenicol (25 μg mL−1) were included in growth media as needed. Bacterial cultures were stored at -80°C in medium containing ~15% glycerol. To revive cultures, LB medium (2 mL) was inoculated from the frozen stock cultures and incubated with shaking (250 rpm) at 37°C overnight. The overnight cultures served as inoculum (1:1000) for LB medium or Kornberg medium (1.1% [wt/vol] K₂HPO₄, 0.85% [wt/vol] KH₂PO₄, 0.6% [wt/vol] yeast extract containing 0.5% [wt/vol] glucose). Growth was determined by monitoring OD₆₀₀ and/or by assaying total cellular protein.

Construction of chromosomal deletions and FLAG^® fusion proteins

Gene deletions (of E. coli) and carboxy-terminally 3XFLAG^®-tagged constructs (of E. coli and Salmonella) were introduced by the Red recombinase method, as described [42, 43]. For gene deletions, primers carrying sequences (40 nt) for the beginning (forward primer) or end (reverse primer) of the open reading frame were designed (S1 Table) and plasmids pKD13 (or pKD3) were used as template DNAs for PCR amplification [43]. For constructing 3XFLAG tag constructs, primers carrying sequences (40 nt) matching the terminus of the targeted gene (forward primer) and the region downstream from it (reverse primer) were designed (S1 Table) and plasmid pSUB11 was used as template DNA for PCR amplification [42, 43]. PCR products were gel purified and used directly for electro-transformation. E. coli and Salmonella strains carrying pKD46 helper plasmid were grown at 30°C in SOB (0.5% [w/v] yeast extract; 2% [w/v] tryptone; 10 mM NaCl, 2.5 mM KCl; 10 mM MgCl₂; 10 mM MgSO₄) supplemented with 100 μg/mL ampicillin and 10 mM arabinose to mid-log (OD₆₀₀ of 0.5), collected by centrifugation, washed three times with ice-cold 10% glycerol and resuspended with ice-cold 10% glycerol. The transformation was then performed by electroporation. After 1h recovery at 37°C in SOB medium, bacteria were spread onto LB agar plates supplemented with antibiotics for the selection of Cm^R or Kn^R recombinants. Correct insertion of the marker and 3XFLAG sequence in the genome was confirmed by PCR amplification and by sequencing of the insertion site using primers listed in (S1 Table). When necessary, the FRT-flanked antibiotic resistance cassette was removed using pCP20 [43].

Construction and purification of carboxy-terminally His-tagged UvrY protein

His-tagged UvrY (UvrY-His₆) protein was constructed by PCR amplification of the coding sequence of uvrY gene using genomic DNA of E. coli MG1655 as template DNA and oligonucleotides UvrY-6xhis-F and UvrY-6xhis-R as primers (S1 Table). The amplicon was gel-purified, digested using Nde I and Xho I restriction enzymes (NEB), and cloned into a similarly digested and dephosphorylated pET24-a (+) vector DNA (NEB) using electrocompetent DH5α for the transformation. The pET-UvrY plasmid was confirmed by gel electrophoresis and sequencing of the inserted DNA using T7-promoter and T7 terminator primers. The pET-UvrY plasmid was moved into BL21 (DE3) E. coli strain for expression. The E. coli BL21_DE3/pET-UvrY cells carrying UvrY-His₆ were grown in 500 mL of LB containing 50 μg/mL kanamycin at 37°C. At OD₆₀₀ of 0.6, expression of the UvrY-His₆ was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to 1 mM, followed by 2h of shaking incubation at 37°C, 250 rpm.

The purification procedure for UvrY-His₆ was similar to the method previously described for isolation of DeaD-His₆ with some modification [4]. Cells were collected by centrifugation (600 x G, 4°C, 5min), suspended in buffer (20 mM Tris-HCl [pH 7.9], 500 mM NaCl, 20 mM imidazole) containing a protease inhibitor cocktail (cOmplete Mini, EDTA-free protease inhibitor cocktail, Roche Diagnostics), and lysed using a French Press. The lysates were centrifuged (20,000 x G, 15 min, 4°C) to remove unbroken cells and cell debris and the supernatant solutions were purified by affinity chromatography on a HisTrap HP column as instructed by the manufacturer (GE Healthcare Life Sciences). Fractions containing UvrY were pooled and dialyzed against UvrY storage buffer (20 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1 mM dithiothreitol and 10% glycerol). UvrY was then aliquoted, frozen using liquid nitrogen, and stored at −80°C. The bicinchoninic acid method was used for assaying protein concentration, as recommended (Pierce Biotechnology).

UvrY phosphorylation in vitro

For in vitro phosphorylation, the purified UvrY-His₆ protein was incubated with 100 mM acetyl-phosphate (Sigma) in a phosphorylation buffer containing 50 mM HEPES pH7.5, 100 mM NaCl and 10mM MgCl₂. The reaction mixtures were incubated for 60 min at room temperature. To determine relative amount of UvrY-P formed, the reactions were fractionated on Phos-tag^TM SDS-PAGE gels [1.0mm Protean 3 (Bio-Rad) minigels] of the following composition: 7.5% separating gel [7.5% (29:1) acrylamide: bis-acrylamide, 357mM Bis-Tris, pH 6.8, 100 μM Zn (NO₃)₂, and 50 μM Phos-tag reagent (Waco Pure Chemical Industries)] with a 4% stacking gel [4% (29:1) acrylamide:bis-acrylamide, 357 mM Bis-Tris, pH 6.8]. UvrY-P was resolved by electrophoresis at constant 150 V at 4°C for 70 min using modified MOPS running buffer (100 mM Tris, 100 mM MOPS, 0.5% SDS, and 5 mM NaHSO₃). Gels were subsequently stained with Coomassie blue and the signals were imaged using a ChemiDoc XRS+ system (Bio-Rad) and quantified using Quantity One image analysis software (Bio-Rad).

ChIP-exo and deep sequencing

E. coli and Salmonella strains carrying biologically functional UvrY-FLAG [4] or SirA-FLAG proteins (S1 Fig) were grown in Kornberg medium and LB (supplemented with 10 mM glucose), respectively at 37°C, 250 rpm. At mid-exponential phase of growth (OD₆₀₀ of 0.6), formaldehyde (1% final concentration) was added and the cultures were incubated for 20 min at 30°C, 150 rpm. The crosslinking reaction was then quenched by the addition of 5 mL of 1.0 M glycine, pH 8.0 to 10 mL of culture. The samples were kept at room temperature for 5 min with gentle swirling. The cells were harvested by centrifugation (6000 x G, 5 min, 4°C), washed twice with ice-cold 1x PBS, suspended in 500 μL lysis buffer (10 mM Tris-HCl pH 8.8, 50 mM NaCl, 20% [w/v] sucrose, 10 mM EDTA) containing protease inhibitor cocktail (cOmplete Mini, EDTA-free protease inhibitor cocktail, Roche Diagnostics) and 2 mg/mL lysozyme. After 30 min on ice, 500 μl of 2x IP buffer (100 mM Tris-HCl, pH 7.0, 300 mM NaCl, 2% Triton X-100, 2 mM EDTA) was added and the samples were incubated at 37°C for 10 min, followed by 2 min on ice. The samples were then sonicated on ice, (60 pulses total, 5 sec on 5 sec off per pulse, in a total of 6 sets, separated by 2 min on ice, using a sonicator (Fisher Scientific, Sonic Dismembrator Model 500, set at 20% amplitude). Unbroken cells and debris were removed by centrifugation. Sonicated samples (1 mL) were treated with 20 μL ANTI-FLAG® M2 beads (Sigma) by spinning on a tube rotating mixer (Thermo Scientific) at 4°C for 4 hr to immunoprecipitate UvrY-FLAG. The beads were washed three times with 1mL 1X IP buffer and two times with 1mL 1X TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The beads were resuspended in 100 μL TE buffer and the formaldehyde crosslinking was reversed by heating at 95°C for 20 min. Afterward, the samples were incubated with 8 μL of 10 mg/mL RNase A for 2 h at 37°C and with 4 μL of 20 mg/mL proteinase K at 55°C for 2h. The resulting DNA was purified using a Qiagen MinElute PCR Purification Kit according to manufacturer’s instructions. As a control, wild type strains expressing no FLAG epitope tagged protein were treated using the same procedures. The specificity of the ChIP assay was confirmed by amplifying promoter DNA of the csrB, lacY and 16s rRNA (rrsH) genes with primers (S1 Table) using the DNA recovered from the ChIP reactions as a template (S2 Fig). csrB was used as a positive control for UvrY/SirA binding while lacY (E. coli) and 16S rDNA (Salmonella) were used as negative controls. After confirming the specificity of the ChIP reactions, the DNA samples were frozen and shipped to Peconic LLC for the remainder of the ChIP-exo procedure and deep sequencing with Illumina Hi-seq 2000 as described previously [34].

ChIP-exo data analysis

Sequencing reads were mapped to their respective E. coli MG1655 (NC_000913.3) and Salmonella enterica subsp. enterica serovar Typhimurium 14028S (NC_016856.1) genomes with BWA (Burrows-Wheeler Aligner, available from http://bio-bwa.sourceforge.net). Alignments were visualized using the Integrated Genome Viewer (IGV, Broad Institute) [44, 45]. Peaks were identified in the mapped reads with GeneTrack [46]. Low confidence singleton peaks and those without matching peaks on the opposing strand were discarded. Enriched genomic loci were classified into three groups based on enrichment of read counts relative to read counts within 500 bp upstream of the transcription start sites of lacY and/or 16s rRNA (rrsH), which were determined not to be direct targets of UvrY/SirA (S2 Fig). Maps of enriched regions are shown as bedgraphs, which display in a continuous fashion the abundance of reads identified at particular position in the genome from the sequencing analysis. For motif discovery, DNA sequences from the peak-pair midpoint of each of the enriched genomic loci, with +/- 40nt upstream and downstream extension distance were extracted. Centered in the 81nt-long extracted DNA sequence is the UvrY/SirA crosslinking site. The extracted DNA sequences were uploaded into MEME Suite [47] for motif discovery analysis. The datasets from ChIP-exo analyses were submitted to the NCBI GEO repository under the accession number GSE74810.

RNA extraction and Northern blotting

Procedures for RNA extraction and Northern blotting were conducted as described previously, with minor modification [4]. Briefly, bacteria were harvested at mid-exponential phase (OD₆₀₀ of 0.6) or as otherwise stated and total cellular RNA was prepared (RNeasy mini kit, Qiagen). RNA was separated by electrophoresis on 5% polyacrylamide gels containing 7 M urea, transferred to a positively charged nylon membrane (Roche Diagnostics) by electro-blotting, and crosslinked to the membrane with UV light. Crosslinked RNA was hybridized to DIG-labeled antisense probe (68°C, overnight) and signal was developed using the DIG Northern Starter kit (Roche Diagnostics). Antisense RNAs were prepared from PCR products using the DIG Northern Starter kit (Roche Diagnostics). Blots were imaged using a ChemiDoc XRS+ system (Bio-Rad) and RNA signals quantified using Quantity One image analysis software (Bio-Rad). The 5S or 16S and 23S rRNAs served as loading controls, and were detected by hybridization or methylene blue staining, respectively.

Western blotting

Western blotting was conducted as previously described [4]. Briefly, proteins (10 μg) were separated using SDS-PAGE and electroblotted onto 0.2 mm polyvinylidene difluoride membranes.Anti-FLAG® M2 monoclonal antibody (Sigma) and anti-RpoB monoclonal antibody (Neoclone) were used for detection of the FLAG epitope and RpoB. Signal detection used treatment with horseradish peroxidase-linked secondary antibodies followed by SuperSignal® West Femto Chemiluminescent Substrate (Thermo Scientific). Blots were imaged using the ChemiDoc XRS+ system (Bio-Rad) and the signals were quantified using Quantity One image analysis software (Bio-Rad).

Analysis of UvrY-FLAG protein stability

Bacterial cells were grown to mid-exponential phase of growth, at which point protein synthesis was stopped by the addition of tetracycline (200 μg/mL) and chloramphenicol (100 μg/mL). Cells were collected at several times following the addition of the antibiotics, harvested by centrifugation and were immediately mixed with Laemmli sample buffer and lysed by sonication and boiling. Samples (10 μg protein) were subjected to SDS- PAGE and were analyzed by Western blotting as described above.

β-Galactosidase assays

The assays for specificβ-Galactosidase activity were conducted as described previously [2]. Values represent the averages from two independent experiments. Error bars represent standard errors of the means.

Analysis of UvrY phosphorylation in vivo

Studies to determine the extent of phosphorylation of the UvrY-FLAG protein in vivo were conducted as previously described [4]. Briefly, proteins from cell lysates were fractionated on Phos-tag^TM SDS-PAGE gels, which resolve UvrY from UvrY-P, and the FLAG epitope of the UvrY-FLAG protein was detected by Western blotting.

Quantitative RT-PCR

Quantitative Real Time-PCR (q-RT-PCR) was conducted in an iCycler™ thermocycler (Bio-Rad) using the iScript™ One-Step RT-PCR Kit with SYBR® Green (Bio- Rad) as described previously, with minor modifications [4]. Reactions (15 μl) contained immunoprecipitated DNA (50 ng/μl), primers (0.5 μM each; S1 Table) and SYBR® Green RT-PCR Reaction Mix. PCR cycle parameters were as follows: 40 cycles of PCR at 95°C denaturation for 10 s, 60°C of annealing, extension, and detection for 30 s. The specificity of the PCR product was determined by melting curve analysis with reference to the calculated Tm. For melting curve analysis, the temperature was increased from 60°C to 95°C at a rate of 0.5°C/10 s. PCR product concentration was determined using a standard curve prepared with iCycler iQ optical system software version 3.1 (Bio-Rad), according to the manufacturer’s instructions.

Analysis of UvrY binding to csrB DNA in vivo by ChIP-PCR

UvrY binding to csrB genomic DNA in vivo was analyzed by ChIP-quantitative PCR (ChIP-PCR). The first steps of the ChIP-PCR assay were conducted as described for the ChIP-exo assay. After releasing the formaldehyde crosslinks, the DNA was extracted and purified using Qiagen MinElute Purification Kit. The isolated csrB DNA was assayed using quantitative real time-PCR (q-RT-PCR) with primers listed in (S1 Table). The lacY gene served as a negative control for this reaction using primers also listed in (S1 Table).

Electrophoretic gel mobility shift assay (EMSA) for DNA binding

For DNA electrophoretic gel mobility shift assays, the regions from -246 to +49 and -247 to +56, with respect to the transcriptional start sites of csrB and csrC, respectively, were amplified by PCR and end-labeled with [γ-³²P] ATP using T4 polynucleotide kinase. Binding reactions (10 μl) contained 0.5 nM of end labeled DNA, 20 mM Tris HCl (pH 7.5), 10% (v/v) Glycerol, 50 mM KCl, 3 mM MgCl₂, 1 mM dithiothreitol, 100 μg/mL BSA and phosphorylated or non-phosphorylated UvrY-His₆ protein. Reactions were incubated for 30 min at 37°C, then 1μl xylene cyanol was added and samples were separated by electrophoresis on 7% non-denaturing polyacrylamide gels with 0.5X TBE as the running buffer. After electrophoresis, the gels were vacuum dried, the radioactive signals captured by phosphorimage analysis (PMI^TM, Bio-Rad) and analyzed using Quantity One software.

DNase I footprinting

Double stranded DNA for footprinting of csrB (466 bp long, extending from 420 bp upstream to 46 bp downstream of the TSS) or csrC (419 bp long, extending from 319 bp upstream to 100 bp downstream of the TSS) was prepared by PCR using strand-specific [³²P] 5’-end-primers (S1 Table). The 5’-end-labeled PCR products were gel-purified and the DNA (0.5 nM) was used for binding reactions (10 μL) containing 20 mM Tris HCl (pH 7.5), 10% (v/v) glycerol, 50 mM KCl, 3 mM MgCl₂, 1mM dithiothreitol and 100 μg/mL bovine serum albumen along with phosphorylated or non-phosphorylated UvrY-His₆ protein. The binding reactions were incubated at 37°C degrees for 30 min followed by treatment with 0.025 U DNase I (Roche) per reaction, for 1 min at 37°C. The DNase I was inactivated by heating at 75°C degrees for 10 min. Reactions were then mixed with formamide loading buffer (10 mL formamide, 10 mg xylene cyanol FF and 10 mg bromophenol blue), denatured at 95°C for 5 min, and separated by electrophoresis on a 6% denaturing polyacrylamide gel. The gel was vacuum dried, radioactive signals captured by phosphor imaging (PMI^TM, Bio-Rad) and analyzed with Quantity One software.

Construction of plasmid-borne csrB-lacZ and csrC-lacZ fusions for S-30 transcription-translation

The csrB-lacZ and csrC-lacZ carrying plasmids, pLFXcsrB-lacZ and pLFXcsrC-lacZ, were constructed by first amplifying the 502 nt (-500 to +2 with respect to csrB transcriptional start site) and 304 nt (-301 to +3 with respect to csrC transcriptional start site) genomic regions using the primer pairs csrB lacZ Fwd / csrB lacZ Rev and csrC lacZ Fwd / csrC lacZ Rev (S1 Table). The PCR products were gel purified, digested with PstI and KpnI, ligated to PstI- and KpnI-digested and dephosphorylated plasmid pLFX, and electroporated into DH5αλpir cells. DNA sequencing was used to confirm that the cloned regions did not contain any mutations.

In vitro coupled transcription-translation

In vitro transcription-translational assays used pLFXcsrB-lacZ and pLFXcsrC-lacZ supercoiled plasmids and were performed with S-30 extracts prepared from the UvrY deficient strain, CF7789 uvrY::cam, as described previously [16], except that reactions (32 μl) contained 0.5 U E. coli RNA polymerase holoenzyme and 3 μl of ³⁵S-methionine (1175 Ci/mmol). The UvrY-P (2.3 μM), ppGpp (250 μM) and DksA (2 μM) were included, as indicated, in the reactions. IHF (1μM) was added to all of the reactions that contained pLFXcsrB-lacZ plasmid. DksA protein was a generous gift of Prof. Richard Gourse, University of Wisconsin, Madison. IHF protein was a generous gift from Prof. Anca Segall, San Diego State University. Incorporation of ³⁵S-methionine into protein products was determined after SDS PAGE separation by using phosphorimaging. Signal intensity was determined using Quantity One software.

Phylogenetic analysis of BarA, UvrY, CsrA, and FliW distribution

We identified CsrA, BarA, UvrY and FliW orthologs from all fully-sequenced bacterial genomes in the NCBI genomes database (http://www.ncbi.nlm.nih.gov/genome/), using the NCBI Prokaryotic Genome Annotation Pipeline v2.0 to assign orthology [48]. This pipeline combines a sequence similarity-based approach with the comparison of the predicted gene products to the nonredundant protein database, Entrez Protein Clusters, the Conserved Domain Database (CDD) [48].

In order to filter out false positives and false negatives from the NCBI orthologs data set, we aligned a set of representative sequences against the genomes with predicted orthologs using tblastn [49]. The alignment results were filtered by sequence similarity and alignment coverage: BarA was filtered by 50% similarity and 50% coverage, UvrY was filtered by 75% similarity and 80% coverage, CsrA was filtered by 60% similarity and 50% coverage, and FliW was filtered by 50% similarity and 50% coverage. All the similarity and coverage thresholds were established by the minimum similarity and coverage observed between annotated sequences from NCBI nucleotide database and the following set of representatives used as query sequences: BarA from Escherichia coli (NCBI protein identification number PI: 190908466), UvrY from E. coli (PI: 190906390), CsrA from E. coli and Bacillus subtilis (PI: 257755450 and 459391362), and FliW from Bacillus amyloliquefaciens (PI: 307608134). After filtering the results by alignment similarity and coverage, we performed a manual refinement of the results by verifying the pairwise alignments, sequence annotations, and bibliographic information.

A reference phylogeny of fully-sequenced genomes encoding CsrA and at least one of BarA, UvrY or FliW was extracted from the NCBI taxonomy database [50] using NCBI-taxcollector [51] and PhyloT v2015.1 (http://phylot.biobyte.de), and gene presence/absence data were visualized using iTOL v3.0 [52]. Correlations among presence/absence of CsrA, BarA, UvrY and FliW were calculated using Pearson’s product moment correlation, and significance was assessed using Fisher’s Z transform (implemented in the R stats package, v3.2.0). For calculating the correlations, we clustered the results at species and genus level in order to minimize the number of false negatives and false positives. For clustering the results at species level, we established the presence/absence as the value that appears most often at strain level (statistical mode). For clustering the results at genus level, we established the presence/absence as the mean calculated at species level.

In order to determine if any strong biases in the methodology used to identify orthologous genes could affect our results we used two additional and widely-employed methods to identify alternative ortholog sets: KEGG orthology database (KO) [53], and UniProt reference clusters of orthologs (UniRef) [54]. KEGG database defines the orthologs by comparing experimental data with KEGG pathway maps, BRITE functional hierarchies and KEGG modules. UniProt defines the orthologs by clustering protein sequences by 50% identity. We compared the correlations calculated for these alternative approaches to the results obtained from the NCBI Prokaryotic Genome Annotation Pipeline, at genus and species level.

Determination of genome-wide UvrY/SirA-binding loci using ChIP-exo

To probe for UvrY/SirA DNA binding sites in E. coli and Salmonella, we used ChIP-exo, a comprehensive genomic DNA-protein interaction assay with single nucleotide resolution and high specificity [34]. This assay uses nuclease trimming reactions to remove nonspecific DNA from the immunoprecipitated complexes and to identify the crosslinking sites much more precisely than ChIP-seq [34]. The experiments were performed with mid-exponential phase cultures (OD₆₀₀ of 0.6) of strains carrying FLAG-tagged UvrY or SirA proteins, respectively, expressed from the native genomic loci. Altogether, 44 million sequencing reads for UvrY (E. coli) and 31 million for SirA (Salmonella) were mapped to their respective genomes. From these analyses, two highly enriched genomic loci were identified at the csrB and csrC genes of both E. coli (Fig 1A and 1B) and Salmonella (Fig 2A and 2B). These results indicated that the csrB and csrC genes represent the strongest targets of UvrY/SirA binding in these species.

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Fig 1. Genomic binding sites for UvrY identified by ChIP-exo.

Bedgraphs depict genomic loci enriched by co-immunoprecipitation with cross-linked UvrY-FLAG, proximal to the csrB (A), csrC (B), spf (B) and cspA (C) genes. Two UvrY crosslinking sites were discovered in the promoter regions of csrB (panel A, underlined in panel D) and csrC (panel B, underlined in panel E). One site is within the region extending from -222 to -142 in csrB or from -223 to -143 in csrC. The other crosslinking site lies close to the promoter, extending from -56 to +25 in csrB and from -49 to +32 in csrC promoter regions. A 9 bp inverted repeat DNA sequence within the upstream crosslinking regions is bolded and capitalized. A putative IHF binding site (broken underline) is located between the two UvrY crosslinking sites in the promoter region of csrB (D), but was not apparent in csrC. The DNA used for DNase I footprinting included 466 bp (-420 to +46) for csrB and 419 bp (-319 to +100) for csrC. DNA fragments used for electrophoretic mobility shift assay included (-246 to +49) for csrB and (-247 to +56) for csrC. The images were constructed using the Integrated Genome Viewer (IGV, Broad Institute) (44, 45).

https://doi.org/10.1371/journal.pone.0145035.g001

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Fig 2. Genomic binding sites for SirA discovered by ChIP-exo.

Bedgraphs show enriched genomic loci, proximal to the regulatory regions of csrB (A), csrC (B) and spf (B). Two putative SirA binding sites are shown for the promoter regions of csrB (rna62) (panel A, underlined in panel C) and csrC (panel B, underlined in panel D). One site is located in the region from -215 to -135 in csrB and from -181 to -101 csrC promoter regions, respectively. The other putative binding site is close to the promoter, within the -41 to +40 (csrB) and -81 to -1 in csrC promoter regions, respectively. A 9 bp-long inverted repeat DNA sequence in the upstream sites is shown in bold and capitalized. A putative IHF binding site is marked between the two putative UvrY binding sites in the promoter region of csrB (C, broken underline), but was not apparent in csrC. The images were constructed using the Integrated Genome Viewer (IGV, Broad Institute) (44, 45).

https://doi.org/10.1371/journal.pone.0145035.g002

In addition to the csrB and csrC genes, weakly enriched genomic loci were identified proximal to the promoter regions of 286 genes in E. coli and 301 genes in Salmonella (S2 Table). These enriched genomic loci were arbitrarily classified into three groups based on enrichment of their occupancy peaks over background (S2 Table). Accordingly, genomic loci enriched 5-fold or greater than the lacY and/or 16s rRNA (rrsH) genes were included in group one. Besides csrB and csrC, the promoter regions of fhuF and spf from E. coli and spf from Salmonella fell into group one. Genomic loci that were enriched 2- to 5-fold comprised group two, which included the promoter regions of 9 genes in E. coli and 8 genes in Salmonella. The rest of the genomic loci, which include regions proximal to the promoter regions of 275 genes in E. coli and 292 genes in Salmonella, have only1.5- to 2.0 occupancy read fold over that of lacY or 16s rDNA (rrsH) and were grouped into group three.

UvrY/SirA consensus DNA binding motif

Further inspection of the ChIP-exo results showed enriched crosslinking in the upstream locations of csrB and csrC, with sequences extending from -222 to -142 from the start of transcription (csrB) or -223 to -143 (csrC) in E. coli (Fig 1D and 1E) and from -215 to -135 (csrB) and -181 to -101 (csrC) in Salmonella (Fig 2C and 2D). Distinct sites of crosslinking in these genes were also observed at downstream sequences that extended from -56 to +25 (csrB) and from -49 to +32 (csrC) in E. coli (Fig 1D and 1E) and from -41 to +40 (csrB) and from -81 to -1 (csrC) in Salmonella (Fig 2C and 2D). In the center of each of the upstream putative binding sites is an 18 nt nearly perfect palindrome or inverted repeat sequence (IR). In csrB, the sequence, TGTGAGAGATCTCTTACA, is centered at -183/-182 in E. coli (Fig 1D) and -182/-181 in Salmonella (Fig 2C). Moreover, a partially conserved additional sequence (TGTAGGAGA) located 5 bp downstream of the IR, is seen in the csrB promoter of E. coli (Fig 1D) and Salmonella (Fig 2C). The IR of csrC shows weaker symmetry in both E. coli (TGTGAGACATTGCCGATA) (Fig 1E) and Salmonella (TGTGAGACATTGACCATT) (Fig 2D). Moreover, a partially conserved sequence (TGTAAG) representing half of the IR, located 16 bp upstream of the IR, is also seen in the csrC promoter of E. coli (Fig 1E) and Salmonella (Fig 2D). In contrast, the IR is not preserved in the downstream crosslinking sites of the csrB or csrC genes.

To search for the conserved IR at the weaker targets of UvrY/SirA (S2 Table), DNA sequences from these enriched promoter regions were analyzed using MEME Suite software [47]. These analyses showed that the IR sequence is not conserved in any of the weaker putative binding sites.

UvrY requires phosphorylation for DNA binding

In the BarA-UvrY TCS, BarA is the histidine kinase, which upon signal detection, autophosphorylates and transfers the phosphoryl group to a conserved aspartate residue of UvrY [20]. The phosphorylated UvrY is then thought to bind specific DNA sequences in the promoters of its target genes and regulate their transcription [18, 20, 22]. MBP-SirA was previously found to bind similarly to csrB DNA in vitro regardless of whether it had been phosphorylated or not, starting at a concentration of 1.5 μM [25], suggesting that MBP-SirA phosphorylation might not be required for DNA binding in vitro. This observation was consistent with another report, showing that phosphorylation only increased the DNA binding affinity of SirA-His₆ by approximately two-fold in vitro [19].

In contrast to results from in vitro DNA binding experiments, deletion of the gene for BarA sensor kinase caused more than a 10-fold decrease in the level of CsrB RNA [16, 22, 35]. Similarly, in Salmonella, substitution of alanine for the predicted phosphorylated histine residue of SirA, Asp54, caused the loss of CsrB expression [25]. Thus, it appears that phosphorylation is critical for UvrY activity in vivo. Whether UvrY requires phosphorylation for efficient DNA binding in vivo or for later steps in transcription initiation was not clear.

To address this issue, we used both in vitro and in vivo assays to investigate the role of UvrY phosphorylation in DNA binding. We performed electrophoretic mobility shift assay (EMSA) using phosphorylated (UvrY-P) and non-phosphorylated forms of the recombinant protein, UvrY-His₆, which was determined to be functional in vivo (S1 Fig). DNA fragments that encompass the ChIP-exo-derived putative UvrY binding sites in the promoter regions of csrB (Fig 1D) and csrC (Fig 1E) were used as probes. Unlabeled csrB and rrlE DNA fragments were used as competitive and non-competitive DNA probes, respectively. Similar to the previous observations [19, 25], our results showed that in vitro phosphorylation led to a modest (~2-fold) increase in DNA binding affinity of UvrY to csrB (Fig 3A and 3B) and csrC DNA (Fig 3C), as compared to the non-phosphorylated UvrY. However, we also observed that the UvrY protein preparation contained around 7% of UvrY-P prior to the in vitro phosphorylation reaction (Fig 3D). It is possible that this contaminating UvrY-P was entirely responsible for binding to csrB and csrC DNA in vitro (Fig 3B).

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Fig 3. Electrophoretic gel mobility shift assay showing UvrY binding to csrB and csrC DNA.

Binding of phosphorylated (UvrY-P) and non-phosphorylated (UvrY) UvrY-His₆ to csrB DNA (A and B) and csrC DNA (C) was tested as shown. The csrB and csrC DNA probes (0.5 nM) used for this experiment (depicted in Fig 1D and 1E, respectively) were incubated with increasing concentrations of in vitro phosphorylated or non-phosphorylated UvrY-His6 protein for 30 min at room temperature. The DNA-protein complexes were resolved by electrophoresis on a non-denaturing 7% polyacrylamide gel. The phosphorylation state of the UvrY-His₆ protein used in these experiments was determined by Phos-tag SDS PAGE gel analysis (D).

https://doi.org/10.1371/journal.pone.0145035.g003

To determine whether UvrY requires phosphorylation for in vivo DNA binding, we tested UvrY-FLAG binding to csrB in vivo in a strain that lacked BarA (∆barA) and its isogenic barA wild-type strain. For this analysis, we first determined the phosphorylation state of UvrY-FLAG protein in the presence and absence of BarA. The results indicated that in the presence of BarA, approximately 7% of the UvrY protein was phosphorylated (S3 Fig), similar to a previous determination [4]. In the absence of BarA, no detectable phosphorylation of UvrY-FLAG protein was observed in LB medium at mid-exponential phase of growth (S3 Fig). We next used ChIP-PCR to test for in vivo binding of UvrY to csrB DNA under this growth condition. In this experiment, an approximate 35-fold reduction in DNA binding was observed in the strain that lacked BarA (∆barA) relative to the isogenic barA wild-type strain (Fig 4). These results show for the first time that UvrY requires phosphorylation for effective DNA binding in vivo.

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Fig 4. Effects of BarA and IHF on in vivo binding of UvrY to csrB.

The effects of BarA, IhfA and IhfB on in vivo binding of UvrY to csrB promoter were determined by ChIP-quantitative PCR (ChIP-PCR) in a WT strain (MG1655 expressing UvrY-FLAG) and isogenic ∆barA, ∆ihfA, ∆ihfB and ∆ihfA∆ihfB strains, as described in the Experimental Procedures. The lacY gene served as a negative control for this reaction. Data depict the results of three independent experiments. Error bars represent the standard errors of the means.

https://doi.org/10.1371/journal.pone.0145035.g004

UvrY specifically binds to an 18 nt-long IR DNA sequence

In order to more precisely define the UvrY DNA binding sites, we performed in vitro DNase I footprinting experiments on csrB and csrC DNA using phosphorylated and non-phosphorylated UvrY-His₆ protein. The DNA probes for this experiment encompassed both the upstream and downstream putative binding sites of these genes (Fig 1D and 1E). The results of these experiments showed protection of only the upstream binding sites containing the IR sequences of both csrB (Fig 5) and csrC (Fig 6 and S4 Fig). The putative downstream binding sites that were observed in vivo (Fig 1A and 1B & 1D and 1E) were not protected in vitro. In addition, only the phosphorylated UvrY-His6 was observed to protect the IR, which further indicates that UvrY requires phosphorylation for tight, specific binding. Together, these results suggest that UvrY-P binds specifically and directly to the IR sequence of these genes. However, in vivo binding of UvrY-P to the downstream sequences of these genes either requires conditions or factor(s) that were absent from the footprinting reactions or perhaps more likely, UvrY-P becomes cross-linked to DNA indirectly through interactions with DNA binding proteins such as RNA polymerase, which must bind to the downstream regions of these genes in order to initiate transcription.

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Fig 5. DNase I footprinting of E. coli csrB DNA using phosphorylated and non-phosphorylated UvrY.

A ³²P-end labeled DNA probe that included both the upstream and downstream putative UvrY binding sites was used for these experiments (shown in Fig 1D). Reactions in all lanes except 1 contained DNase I (0.025U/12.5ul reaction). Reactions in lanes 3–6 and lanes 7–10 contained 0.25, 0.35, 0.5, 0.7 μM of phosphorylated or non-phosphorylated UvrY-His₆, respectively. Lane 2 reaction contained no UvrY protein. A vertical black bar indicates a protected region, and the sequence corresponding to the protected region is shown in a vertical rectangular box. An alignment of sequences corresponding to the protected regions from DNase I and the ChIP-exo results is shown in the horizontal rectangular box. The 18nt-long palindromic sequence and the partially conserved palindromic sequences are marked with broken black lines.

https://doi.org/10.1371/journal.pone.0145035.g005

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Fig 6. DNase I footprinting of E. coli csrC DNA by phosphorylated and non-phosphorylated UvrY.

A ³²P-end labeled DNA probe that included both the upstream and downstream putative UvrY binding sites was used for these experiments (Fig 1E). Reactions in all lanes except lanes 1 contained DNase I (0.025U/12.5ul reaction). Reactions in lanes 3–6 and lanes 7–10 contained 0.25, 0.35, 0.5, 0.7 μM of phosphorylated or non-phosphorylated UvrY-His₆, respectively. Lane 2 reaction contained no UvrY. A vertical black bar indicates a protected region, and the sequence corresponding to the protected region is shown in a vertical rectangular box. An alignment of sequences corresponding to the protected regions from DNase I and ChIP-exo results is shown in the horizontal rectangular box. The 18nt-long palindromic sequence and the partially conserved palindromic sequences are marked with broken black lines.

https://doi.org/10.1371/journal.pone.0145035.g006

UvrY requires IHF for optimal binding to and expression of csrB but not csrC

The nucleoid-associated protein Integration Host Factor (IHF) is a heterodimeric protein composed of two homologous subunits, IHFα (IhfA) and IHFβ (IhfB), which facilitates the transcription of many genes by bending the DNA [55–57]. In the Csr/Rsm system, IHF was shown to directly bind to the promoter of the csrB gene of Salmonella) [18] and the rsmZ promoter of Pseudomonas fluorescens [58]. In Salmonella, deletion of ihfA was also shown to decrease csrB expression [18]. However, whether the observed effect of IHF in sRNA expression in those bacterial species is UvrY-mediated and if so, whether IHF is required for UvrY to bind to DNA or for later transcription initiation steps is not clear.

In this study, we identified a putative IHF binding site in the promoter region of csrB of E. coli (Fig 1D) and Salmonella (Fig 2C), in agreement with an earlier report [18], located between the upstream and downstream in vivo crosslinking sites for UvrY-P, but we did not identify a similar site within the csrC genes. To determine whether IHF affects expression of csrB in E. coli, we measured the levels of CsrB in the presence and absence of IHF (∆ihfA and/or ∆ihfB). Our results revealed a ~10-fold reduction in CsrB RNA levels in the absence of IhfA, IhfB, or both, compared to the isogenic wild type strain (Fig 7A). We also performed epistasis experiments in which we measured the effect of IHF (∆ihfA and/or ∆ihfB) on the expression of csrB in a strain lacking uvrY (∆uvrY). Our results showed that neither IhfA nor IhfB affected the levels of CsrB in the uvrY mutant strain (Fig 7A), suggesting that UvrY mediates the effect of IHF on the expression of csrB. Analysis of csrB DNA binding by UvrY-FLAG using in vivo ChIP-PCR analyses showed that binding was reduced by approximately 2.5-fold in the absence of either IhfA or IhfB (Fig 4), suggesting that IHF is required for optimal binding of UvrY-P to csrB DNA in vivo. However, this modest effect of IHF on UvrY DNA binding does not appear to account for its 10 to 12-fold effect on csrB expression. Therefore, IHF also appears to affect later steps in csrB transcription (Fig 7A). Western blotting experiments demonstrated that IHF did not affect the phosphorylation (S3 Fig) or expression of UvrY-FLAG (S5 Fig). Hence, our results reveal that IHF is required for optimum binding of UvrY-P to csrB DNA and activation of csrB transcription.

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Fig 7. Effects of UvrY, BarA and IHF on CsrB/C sRNAs levels.

Northern blots showing effect of several gene deletions on the levels of E. coli CsrB (A) and CsrC (B). Cultures were grown in LB to mid-exponential growth phase (OD₆₀₀ of 0.6). The 16S/23S rRNA loading controls are also shown.

https://doi.org/10.1371/journal.pone.0145035.g007

IHF facilitates the transcription of many genes by bringing relatively distant DNA sites (~200 bp) closer together in space by bending the DNA (~140°) [59, 60]. Therefore, we propose that UvrY-P bound at the upstream IR site (centered at -183/-182 in csrB of E. coli and at -182/-181 in the csrB of Salmonella) is brought into the proximity of the csrB promoter-RNA polymerase complex by IHF-mediated bending of the DNA. Such DNA bending may lead to UvrY-P binding to the downstream DNA crosslinking site directly or indirectly through interactions with RNA polymerase or perhaps with other unknown DNA binding factor(s). This type of transcriptional activation, known as repositioning, is observed in promoters where the primary activator is unable to make a productive contact with RNA polymerase without being repositioned by a secondary activator [60, 61]. Such a mechanism appears to be required for the activation of the E. coli narG promoter by NarL, another FixJ family response regulator, which binds at –190, while IHF binds at -125 and Fnr binds at – 41 of narG [59, 60].

In contrast to its effect on csrB, IHF was not needed for expression of csrC (Fig 7B). In fact, in the single and double IHF deletion strains, CsrC RNA levels were 2-fold higher than in the isogenic wild type strain (Fig 7B). This may be because in the ∆ihfA ∆ihfB strains, the lower levels of CsrB RNA (Fig 7A) lead to an increase in the concentration of free CsrA in the cell, which in turn leads to increased levels of CsrC via a negative feedback loop in the Csr system [13].

UvrY effects on the expression of other putative in vivo targets

To examine the regulatory effects of UvrY on the expression of the potential new target genes discovered by ChIP-exo (S2 Table), we tested several gene products and transcripts from groups one, two and three by Western and Northern blotting. Our results showed little or no regulatory effects of UvrY on the expression of the genes that were tested (S6A and S6C Fig), with the exception of cspA. UvrY showed a relatively modest, but reproducible negative effect on the expression of the cold-shock protein CspA (Fig 8).

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Fig 8. Effects of genes encoding UvrY and Csr factors on CspA protein levels.

Western blot showing effects of uvrY deletion and csrA::kan disruption on the levels of CspA-FLAG protein in an MG1655 derivative expressing CspA-FLAG (WT) from the cspA genomic locus. Effect of csrA::kan ΔuvrY complemented with UvrY (pUY14), CsrA (pCRA16) or control (pBR322) expression plasmids is also shown. Cultures were grown at 37°C in LB to mid-exponential growth phase (OD₆₀₀ of 0.6). The RpoB protein served as a loading control. This experiment was repeated at least three times with reproducible results.

https://doi.org/10.1371/journal.pone.0145035.g008

A 2-fold increase in the levels of CspA-FLAG was observed in a ΔuvrY strain compared to the isogenic wild type strain (Fig 8). Furthermore, disruption of csrA caused a modest decrease in CspA-FLAG levels, suggesting that CsrA may activate cspA expression. To determine whether UvrY regulates cspA indirectly through its effects on CsrB/C, and therefore CsrA activity, we conducted an epistasis analysis (Fig 8). A strain that was disrupted in both uvrY and csrA was transformed with plasmids expressing uvrY or csrA from the plasmid cloning vector pBR322, and CspA-FLAG protein levels were determined in the resulting strains. We observed that CspA-FLAG levels were essentially identical in strains containing pBR322 and the csrA-expression plasmid pCRA16. In contrast, the uvrY-expression plasmid (pUY14) led to modest decrease in CspA-FLAG levels in this strain. These results are consistent with a model in which UvrY inhibits cspA expression by binding to the cspA promoter, while the effect of CsrA may be mediated indirectly. Also consistent with this model, in silico analyses did not reveal typical CsrA binding sequences (GGA) in the 160 NT 5’-UTR of the cspA transcript (data not shown) and an RNA-seq analysis conducted previously did not reveal cspA mRNA among the 721 different transcripts that copurified with CsrA [2].

Based on all of these observations, we conclude that, under the growth conditions tested, the BarA-UvrY TCS directly exerts its global effect on gene expression primarily through the Csr system by activating the transcription of the CsrB and CsrC sRNAs, with cspA representing a likely exception. This raises the question of why UvrY-FLAG crosslinked in vivo (though weakly) to the regulatory regions of 286 genes in E. coli and 301 genes in Salmonella (S2 Table), but exerted little or no regulatory effect in the examples that were tested. To explain these observations, we propose three possible hypotheses: (i) Perhaps other unknown activator(s) are required along with UvrY/SirA for the expression of these genes, but were not available or functioning under the chosen growth conditions. Thus, UvrY/SirA might activate some of these genes under other growth conditions. (ii) Perhaps one or more repressors overrides the influence of UvrY/SirA on the expression of these genes under the conditions tested [62]. To address these two hypotheses, we searched for factors known to regulate the expression of the putative UvrY target genes. This analysis revealed several DNA binding proteins including CRP, FNR and IHF appear to regulate the greatest number of potential UvrY targets (S7 Fig). Hence, it is conceivable that such factors may mask the effects of UvrY on the expression of these genes under our growth conditions. (iii) Because the putative UvrY targets from the ChIP lack the 18nt-long IR sequence found in the regulatory regions of csrB/C, it is also possible that some of these genes have degenerate UvrY/SirA binding sites with no functional relevance or perhaps serving as nonspecific holding sites for UvrY/SirA [62, 63].

UvrY expression is activated by CsrA via a putative posttranscriptional mechanism that does not involve the RNA helicase DeaD, a known regulator of uvrY translation

In addition to BarA-UvrY, the transcription of CsrB/C is also strongly activated by CsrA [13, 16, 35] by mechanisms that are yet to be determined. Recent evidence suggested that CsrA positively affects uvrY expression both at the transcriptional and translational levels [35]. Moreover, CsrA is required for switching BarA from a protein possessing phosphatase activity to kinase activity on UvrY [35]. Thus, we tested the effect of CsrA on the expression, stability and phosphorylation of UvrY-FLAG. The results showed that while CsrA does not affect UvrY-FLAG protein stability (S8A Fig), it has a strong positive effect on UvrY-FLAG expression (Fig 9B). We observed a 5-fold reduction in UvrY-FLAG levels in a csrA::kan mutant strain compared to the isogenic wild type strain. These results confirm that CsrA regulates the expression of CsrB and CsrC by activating UvrY expression. Moreover, in silico analyses did not reveal any typical CsrA binding sites in the 5’-UTR of the uvrY transcript (data not shown) and uvrY mRNA was not among the 721 different transcripts that co-purified with CsrA [2], suggesting that the CsrA effect on uvrY expression is likely to be indirect.

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Fig 9. Effect of CsrA on uvrY expression.

Gene fusions used in this study: (i) native uvrY regulatory region and coding sequence containing a FLAG^® tag; (ii) araB promoter and mRNA nocoding leader fused to the uvrY-FLAG coding sequence and (iii) araB promoter fused at the transcription start site to the uvrY noncoding leader and uvrY-FLAG coding sequence (A) as previously depicted (4). Effects of csrA disruption on expression of the native uvrY-FLAG construct (panel A, i) in LB at different growth phases (B). Effects of csrA disruption on expression of the uvrY-FLAG constructs shown in panel A (C). Strains were grown in the presence of arabinose, and UvrY-FLAG was detected at mid-log phase of growth by Western blotting. RpoB served as a loading control for these experiments.

https://doi.org/10.1371/journal.pone.0145035.g009

To assess the genetic effects of CsrA on UvrY expression, we analyzed fusion derivatives that replaced the uvrY promoter or the promoter and noncoding leader with those from araB (Fig 9A) [4]. Results from these experiments showed that regulation by CsrA requires the uvrY noncoding leader but not the promoter region (Fig 9C). To test whether CsrA requires sequence from the uvrY coding region for regulation, we measured the expression of a uvrY’-’lacZ translational reporter fusion containing the leader and 12 or 22 codons of uvrY in wild type and csrA::kan strains (Fig 10A). The two different leaders were chosen because previous studies showed that the DeaD RNA helicase is needed to override inhibitory long-distance mRNA base-pairing between the uvrY noncoding leader and the proximal uvrY coding segment, and that DeaD regulates uvrY expression from gene fusions with 22 or more uvrY codons, but not from fusions with less than this, e.g. 12 codons, which do not permit the inhibitory base-pairing [4]. Our results showed that expression of both fusions (22 and 12 codon) was decreased in the csrA::kan strain by 2.8 and 2.6-fold, respectively (Fig 10B), suggesting that the effects of CsrA are not mediated via DeaD. We also tested the effect of CsrA on the expression of a DeaD-FLAG fusion, expressed from the native chromosomal locus, and confirmed that CsrA does not regulate deaD expression (Fig 10C).

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Fig 10. CsrA activates uvrY expression without affecting DeaD or SrmB RNA helicase levels.

The uvrY gene fusions used in this study were previously depicted in (4): lacUV5 promoter fused at the transcription start site to the uvrY mRNA leader and N (12 or 22) uvrY codons fused in frame to lacZ (A). The effect of csrA disruption on expression of PlacUV5-uvrY’–’lacZ reporter fusions is shown (B). Cells were grown in LB and harvested at various times throughout growth and assayed for β-galactosidase specific activity (A₄₂₀/mg protein). The values represent the average of two independent experiments. Error bars depict standard error of the means. Western blots showing effects of csrA on the level of DeaD-FLAG (C) or SrmB-FLAG (D) in an MG1655 derivative that expresses the corresponding FLAG-tagged gene from its native genomic locus are shown. Effect of csrA::kan complemented by a csrA expression plasmid (pCRA16) or with a control plasmid (pBR322) is also shown for DEAD-FLAG. RpoB served as a loading control for these blots.

https://doi.org/10.1371/journal.pone.0145035.g010

Furthermore, another RNA helicase, SrmB, was shown to regulate the transcription of CsrB and CsrC RNAs by an undefined mechanism, which is different than that of DeaD [4]. We, therefore, examined the effect of CsrA on the levels of SrmB-FLAG and observed no effect on the expression of this RNA helicase (Fig 10D). Thus, CsrA does not appear to regulate expression of either of the DeaD-box RNA helicases that activate CsrB and CsrC transcription.

Finally, in a screen to find novel direct CsrA targets by RNAseq analysis, ihfA mRNA was found to co-purify with CsrA [2], suggesting that CsrA might regulate production of the IHF protein. Therefore, we hypothesized that in addition to activating uvrY expression, CsrA might also activate the expression of csrB by activating the expression of IHF. However, we observed no effect of CsrA on IhfA-FLAG or IhfB-FLAG levels when these fusions were expressed from their native chromosomal loci (S8B Fig). Altogether, our results indicate that CsrA activates csrB/C transcription by activating uvrY expression, most likely through a posttranscriptional mechanism that is yet to be defined.

Effects of ppGpp and DksA, mediators of the stringent response, on csrB expression

Previously, ppGpp and DksA were shown to strongly activate expression of CsrB and CsrC in E. coli [2]. However, the mechanism for these effects is yet to be elucidated. In an attempt to define this mechanism, we hypothesized that ppGpp and DksA might activate the expression of factor(s) that are known to activate CsrB/C transcription i.e., UvrY, IHF, CsrA, DeaD and/or SrmB. Previous studies showed that ppGpp and DksA positively affect CsrA levels, but this effect appears to be too modest to account for their strong effects on CsrB/C levels [2]. Therefore, we first determined whether DksA and ppGpp affect the in vivo levels of UvrY-FLAG protein (Western blot) and the in vivo csrB DNA binding (ChIP-PCR) by UvrY in ΔdksA, ΔrelA (encoding the major ppGpp synthase) and isogenic wild type strains. It was found that ppGpp and DksA had weak or negligible effects on UvrY levels (Fig 11), in vivo binding of UvrY to csrB (Fig 12), and the in vivo phosphorylation status of the UvrY protein (S3 Fig). Next, we tested the effects of DksA and ppGpp on IHF expression. The results showed no effect of DksA or ppGpp on IhfA-FLAG (Fig 13A) or IhfB-FLAG levels under our growth conditions (Fig 13B).

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Fig 11. Effect of DksA and RelA on UvrY-FLAG levels.

Western blotting of UvrY-FLAG levels in strains MG1655 (no FLAG fusion), WT (MG1655 expressing UvrY-FLAG from the uvrY genomic locus), and isogenic ∆dksA and ∆relA strains. Proteins were collected from cultures grown in LB medium to mid-exponential growth phase (~OD₆₀₀ of 0.6). RpoB served as a loading control.

https://doi.org/10.1371/journal.pone.0145035.g011

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Fig 12. Effect of DksA and RelA on in vivo binding of UvrY to csrB promoter.

The effect of DksA and RelA on in vivo binding of UvrY to csrB promoter was determined by ChIP-PCR assay in a WT (MG1655 expressing UvrY-FLAG) and isogenic ∆dksA, ∆relA and ∆barA strains. Agarose gel showing PCR amplification of csrB promoter region recovered from each strain. The lacY gene served as a negative control in this experiment.

https://doi.org/10.1371/journal.pone.0145035.g012

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Fig 13. Effect of DksA, RelA and UvrY on the expression of IHF subunits.

Western blotting of IhfA-FLAG (A) and IhfB-FLAG (B) proteins examined in WT (MG1655 expressing ihfB-FLAG or ihfA-FLAG fusions from the native genomic loci) and an MG1655 control lacking the FLAG fusions. Proteins from isogenic strains with ∆dksA, ∆relA and ∆uvrY disruption are as shown. Proteins were collected from cultures grown in LB medium to mid-exponential growth phase (~OD₆₀₀ of 0.6). RpoB loading controls for these analyses are also shown.

https://doi.org/10.1371/journal.pone.0145035.g013

Because ppGpp and DksA did not substantially affect the in vivo expression of IHF or alter the in vivo binding of UvrY-P to csrB DNA, we decided to test their direct effects on the in vitro transcription of csrB and csrC genes. However, neither gene was expressed in defined transcription reactions that contained UvrY-P and IHF in the case of csrB and basal reaction components (5nM RNAP; 40 mM Tris·HCl, pH 7.9; NaCl (165 mM); 5% glycerol; 10 mM MgCl₂; 1 mM DTT; 0.1 μg/μl BSA; 500 μMATP; 200 μM CTP and UTP; 10 μMGTP and [α-32P] GTP (2.5 μCi). This suggests that unknown factors may be required for csrB/C transcription (data not shown). We next used coupled transcription-translation in S-30 extracts to determine whether the addition of ppGpp and DksA would directly regulate expression of csrB/C in the presence of other cellular factors present in the S-30 extracts but not the defined transcription reactions. As previously reported [13, 16], UvrY-P strongly stimulated the expression of the full-length β-galactosidase from csrB-lacZ (Fig 14) and csrC-lacZ (S9 Fig) transcriptional fusions in this assay. A series of truncated β-galactosidase products was also observed in these reactions, which quantitatively responded to activators similarly to the full-length protein. While expression in the absence of UvrY-P was weak, the addition of ppGpp or ppGpp and DksA to the UvrY-deficient csrB-lacZ reaction caused a modest increase in expression (Fig 14). In the presence of UvrY-P, addition of ppGpp alone modestly activated csrB-lacZ expression (1.5-fold). DksA alone also had weak effects in the presence of UvrY-P (1.4-fold), while the addition of both ppGpp and DksA led to a 1.7-fold increase in csrB-lacZ expression. Addition of ppGpp alone, in the absence of UvrY-P, resulted in a slight increase in csrC-lacZ expression (S9 Fig). However, ppGpp and/or DksA failed to activate csrC-lacZ expression in reactions containing UvrY-P. We conclude that ppGpp and DksA directly activate csrB expression, at least partially accounting for the stimulatory effect of these regulators on the in vivo expression of this gene [2]. However, ppGpp and/or DksA may be indirectly involved in or require a factor that was deficient in our assays for csrC expression.

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Fig 14. In vitro transcription-translation of a supercoiled plasmid-encoded csrB-lacZ transcriptional fusion.

Reactions contained pLFXcsrB-lacZ plasmid (2 μg), UvrY-P (2.3 μM), ppGpp (250 μM) and/or DksA (2 μM) as indicated. Incorporation of ³⁵S-labeled methionine into protein products was detected by SDS PAGE followed by phosphorimaging. Signal intensity of the full length protein was determined using Quantity One software. The fold-effects of regulatory factors were determined with respect to the control reaction lacking the factors, after normalization against the internal control, β-lactamase, which was encoded on the same plasmid. Absolute deviation for each reaction was determined from two independent experiments.

https://doi.org/10.1371/journal.pone.0145035.g014

As mentioned above, DeaD and SrmB activate csrB and csrC transcription by distinct mechanisms [4]. While DeaD activates uvrY translation, the mechanism by which SrmB activates csrB/C transcription is still not defined. Our ChIP-PCR data revealed that SrmB is required for normal binding of UvrY to csrB in vivo (S2 Fig). This effect of SrmB on UvrY binding occurs without it altering UvrY or UvrY-P levels [4]. Because neither ppGpp nor DksA had substantial effects on in vivo binding of UvrY to csrB DNA (Fig 12) we infer that the mechanism by which ppGpp and DksA activate csrB/C transcription does not involve SrmB and vice versa.

In silico analysis of other possible targets of UvrY (SirA) binding

In many cases, transcription factors compete for binding to their DNA binding sites with other transcription factors, which may play antagonistic roles in the regulation of the target gene(s) [64]. Hence, we reasoned that there could be additional targets of UvrY/SirA in the genomes of E. coli / Salmonella that were not captured by ChIP-exo. To test this hypothesis, we performed in silico analysis using the Ab Initio Motif Identification Environment (AIMIE) database [65]. We scanned the E. coli genome using the first six bases of the 18 bp (TGTAAGNNNNNNCTTACA) UvrY binding sequence, followed by manually checking the presence of the rest of the IR DNA sequence in the regulatory region of each discovered putative target. In this way, we identified 19 putative target genes containing the 18-bp UvrY-P IR sequence either perfectly or near perfectly conserved in their regulatory regions (S10 Fig). Next, we searched for factors known to directly regulate the expression of these putative targets and compared the respective DNA binding motif of each factor with the 18 bp IR UvrY binding motif. Out of the 19 putative targets, regulatory factors were previously established for 5 of them (S11 Fig). When we compared the respective DNA binding motifs of each factor with the IR binding motif of UvrY, we found an overlap in all of them (S11 Fig), supporting the possibility that undiscovered direct target sequences for UvrY binding might have gone undetected in our experiments. Whether these putative target sequences function in UvrY regulation under other growth conditions will require future investigation.

Phylogenetic distribution of BarA, UvrY, CsrA and FliW

The BarA-UvrY TCS exerts global effects on gene expression by activating CsrB and CsrC transcription, thus controlling CsrA activity [16, 35]. This signaling pathway is common to the commensal bacterium E. coli, the pathogen Salmonella, as well as a variety of other γ-proteobacterial pathogens [66]. In contrast, little is known about the workings of the Csr system in other species that possess csrA homologs, but have not been shown to express CsrA-inhibitory sRNAs. Recently, Mukherjee et al. have shown that the FliW protein of B. subtilis binds to CsrA and antagonizes its activity, thus preventing it from binding to the flagellin mRNA, hag [41, 67]. Because BarA-UvrY is devoted to transcription of CsrB/C sRNAs in E. coli and Salmonella and fliW is absent in species known to produce CsrA-inhibitory sRNAs [41], we hypothesized that BarA-UvrY and FliW might represent different modules for regulating CsrA activity in different bacterial species. To test this hypothesis, we examined the phylogenetic distributions of CsrA, BarA-UvrY and FliW across fully-sequenced bacteria (S3 Table). We found that, for species encoding at least one readily identifiable CsrA ortholog, the presence of BarA-UvrY and FliW were strongly anti-correlated at species level (Spearman correlation = -0.97, p = 1.10x10^-205) and genus level (Spearman correlation = -0.95, p = 7.31x10^-74) (Fig 15). Of the 346 genomes encoding CsrA, 340 also encoded either BarA-UvrY or FliW, but not both, and only 6 species might encode both BarA-UvrY and FliW systems: Desulfosporosinus acidiphilus, D. meridiei, D. orientis, D. baculatum, Magnetococcus marinus, and Candidatus Sulfuricurvum sp (Fig 15 and S3 Table). Interestingly, in all cases where both BarA/UvrY and FliW may function, only the BarA component appears to be present, which may indicate that this two-component system has lost or is losing its function in these species. Additional experimental data are needed in order to confirm this hypothesis.

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Fig 15. The distribution of CsrA, BarA, UvrY and FliW across phylogenies indicates a strong anti-correlation between BarA/UvrY and FliW.

Depicted is a reference phylogeny of fully-sequenced genomes encoding CsrA and at least one of BarA, UvrY or FliW, plotting their presence/absence data obtained from orthology databases. The results show that the presence of BarA-UvrY and FliW are significantly anti-correlated.

https://doi.org/10.1371/journal.pone.0145035.g015

Accurate and complete identification of orthologs across a broad taxonomic range is a challenging problem, with potential for both false-positives and false-negatives [68]. Although difficult to detect, any strong biases in the methodology we used to identify orthologous genes could affect our results. To address this concern, we used two additional and widely-used methods to identify alternative ortholog sets: KEGG orthology database (KO) [53], and UniProt reference clusters of orthologs (UniRef) [54]. We found that, regardless of the method used to identify orthologs, FliW and BarA-UvrY were strongly negatively correlated, particularly when conditioning on the presence of CsrA (S3 Table). In addition, we applied sequence similarity filtering and manual curation to reduce false positives and false negatives in the NCBI orthologs data set.

After the sequence similarity filtering and manual curation, we found several false positives for CsrA in the Streptococcus genus, such as misannotated heavy metal stress response proteins (NCBI identification numbers (GI): 15675047, 13622200, 21904470), peptide methionine sulfoxide reductases (GI: 222114035, 134272076, 209540564, 24638057, 342165139, 342165138), and putative CsrA homologs that lack the N-terminal and C-terminal conserved regions of CsrA (GI: 895760047, 882844105, 882819224). Most of the main false negatives that we identified are BarA and UvrY from genomes that match the representative homologs from E. coli within the similarity and coverage thresholds (see methods) such as Pseudomonas (NCBI genome accession numbers: NC_022594, NC_022591, NC_022361, NC_022360), Xanthomonas (NC_020815, NC_017271, NC_017267, NC_016010, NC_013722), Pseudoxanthomonas (NC_014924), Shewanella (NC_009052, NC_009665, NC_008321, NC_008322, NC_017566, NC_016901), and other members of the γ-Proteobacteria class (S3 Table).

These false positives and false negatives caused a small increase of 2% in the negative correlation, reinforcing the present conclusions (Spearman correlation = -0.95 vs. -0.97, comparing the unfiltered and filtered data sets, respectively) (S3 Table). These results argue against methodological bias as strongly affecting our results. Together, these results indicate that the negative correlation between FliW and BarA-UvrY regulatory systems is highly unlikely to be artifactual and thus represents a biologically relevant observation. The implications of this observation for the possible regulation of CsrA activity by inhibitory sRNAs in FliW-encoding species will require additional investigation to unravel.