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. 2021 Feb 26;49(4):2357-2374.
doi: 10.1093/nar/gkab060.

Structure-based analyses of Salmonella RcsB variants unravel new features of the Rcs regulon

Juanjo Huesa  1   2 , Joaquín Giner-Lamia  3   4   5 , M Graciela Pucciarelli  3   6 , Francisco Paredes-Martínez  1   2 , Francisco García-del Portillo  3 , Alberto Marina  7   8 , Patricia Casino  1   2   8
Affiliations

Structure-based analyses of Salmonella RcsB variants unravel new features of the Rcs regulon

Juanjo Huesa et al. Nucleic Acids Res. .

Abstract

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.

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Figures

Figure 1.
Figure 1.
Structure of RcsB bound to PrprA and P1flhDC promoters. (A) RcsB dimer (subunits in pink and magenta) bound to promoter PrprA (rprA-RcsB-P) and RcsB dimer (subunits in orange and purple) bound to promoter P1flhDC (flhDC-RcsB-P). The REC domain from each subunit is labelled and the loop connecting the REC and DBD domains is colored in cyan. (B) Superposition of the dimerization REC domains for RcsB-P dimer (in blue hues; PDB: 5O8Z) and rprA-RcsB-P complex (pink and magenta subunits). DBD domains rotate to dock at the rprA promoter shown as surface.
Figure 2.
Figure 2.
Mechanism of RcsB activation. (A) Superposition of the dimer formed by the REC domains of RcsB-P (in blue hues; PDB: 5O8Z) with the isolated REC domain (RcsBREC-P in green hues) bound to phosphomimetic BeF3− and as well as with (B) the rprA-RcsB-P complex (pink and magenta subunits) and flhDC-RcsB-P complex (subunits in orange and purple). (C, D) Zoom of the active site in A and B, highlighting the phosphorylatable residue D56, residues involved in the β5-T mechanism (T87 and L108 as well as N90 and Q110), residue H12 involved in REC dimerization and loop β3α3 (Lβ3α3), loop β4α4 (Lβ4α4) and loop β5α5 (Lβ5α5). (E) Zoom of the active site for the superposition of the isolated REC domain of RcsB in apo (RcsBREC in red) and phosphorylated form (RcsBREC-P in green) with REC domain of RcsB-P (in blue; PDB: 5O8Z). Residues are labelled as in C and D. (F) Structures superposed in E, showing the dimer for the phosphorylated forms. The residues that are involved in stabilizing the dimer interface (H12, M88 and Q110) are shown in the apo form avoiding proper disposition of the dimer interface.
Figure 3.
Figure 3.
Role of L108 in the activation mechanism of RcsB. (A) Phosphorylation of RcsB-WT and mutant variants RcsB-L108A and RcsB-L108F using radioactive phosphodonor AcP and incubating at room temperature for 5, 10, 20, 40 and 60 min. (B) EMSA experiments performed with RcsB-WT and mutant variants L108A and L108F in order to test binding to P1flhDC in the absence and presence of phosphodonor AcP. (C) Effect of L108A and L108F mutations in capsule formation and motility. Overexpression of RcsB mutants in the S. Typhimurium strain MD4821 (igaA1 ΔrcsB) to monitor effect on colanic capsule production and motility in vivo. Expression of the wild-type and empty vector (as control).
Figure 4.
Figure 4.
Dissecting binding specificity of RcsB to DNA. (A) rprA-RcsB-P complex showing how α9 is oriented at each subunit of the dimer with respect to dimerization of the REC domain. (B) Interaction of each DBD domain of the RcsB dimer (in pink and magenta) with PrprA promoter showing the residues involved in binding located at α7, α8 and α9. At each DBD domain, residue K180 inserts specifically in the major groove of DNA. (C) Detail interaction of each DBD domain of the RcsB dimer (in pink and magenta) with the PrprA promoter indicating the residues involved in the binding with the nucleotides at each strand of the promoter (in green and yellow). Labelled residues are colour coded as the DBD chains and show location at the corresponding helix. Nitrogenous bases are represented as boxes, deoxyribose sugar as small pentagons and phosphate groups as red circles. Lines colour coded as DBD chains represent binding interactions between residue and DNA. Nucleotides at motif (G/A)GA in the direct strand are colored in red and at motif AGA in the complementary strand are colored in blue. (D) Superposition of all DBD domains bound to PrprA and P1flhDC to show the flexibility of the side chain for K180.
Figure 5.
Figure 5.
Transcriptional response in S. Typhimurium producing RcsB variants having distinct phosphorylation states. (A) Hierarchical complete-linkage clustering was applied to 578 genes that were differentially expressed (adjusted P-value < 0.05) in S. Typhimurium actively growing in LB medium (OD600 ∼ 0.25) as denoted by the RNA-seq data (Supplementary Table S6). Positive or negative expression values in RPKM (reads per kilobase million) with respect to the reference sample (ΔrcsB null mutant strain harbouring empty vector) are indicated by shades of yellow or blue, respectively. Differential expression values on the colour bar are shown in log2 form. (B) Venn diagram showing number of genes differentially expressed in bacteria producing RcsB-WT or the indicated variants. Black numbers indicate the number of genes that S. Typhimurium expressing each RcsB variant (M88A, L108A and D56A) shares with those producing RcsB-WT. (C) GO enrichment analysis of differentially expressed genes. Only GO of biological process with adjusted P-value < 0.05 are shown. (D) Read coverage of both colanic acid capsule-related (wca) and flagella synthesis (flg) genes. Values in the X-axis indicate genome coordinates. Values in the Y-axis indicate RNA-seq read density profiles. Blue arrows represent gene orientation.
Figure 6.
Figure 6.
Distribution of predicted RcsB motifs found in the genome of S. Typhimurium strain SL1344. Two main categories: intra-CDS (green) and intergenic (blue). Transcriptional start sites (TSS) are indicated in pink, and initial ATG codon in yellow.
Figure 7.
Figure 7.
Genome-wide analysis of predicted RcsB binding motifs. (A) Read coverage of potential RcsB target genes. Examples of genes with RcsB binding sites are displayed: internal TSS and downstream gene regulation (rpoS and flgM); located in the iron cluster-related genes fes, entF and fepE; located in the promoter regions osmB and ytfk genes. Values in the X-axis indicate genome coordinates. Values in the Y-axis indicate RNA-seq read density profiles. Blue arrows represent gene positions. Black arrows indicate TSS positions that were retrieved from SalCom (45). (B) Intersection of RcsB motif sites of potential target genes with RNAseq transcriptional expression. Scatter-plot of log2 fold-change (RcsB-WT/null ΔrcsB) versus RcsB binding sites located from -300 bp upstream to +100 bp downstream of either TSS or initial ATG codon. Expressed and repressed genes are coloured in red and blue respectively. Top five most induced/repressed genes are indicated. (C) EMSA experiments were performed with RcsB wild-type and the pseudopalindromic box of RcsB found in promoter regions of fepE, ytfK and osmB as well as in the Intra-CDS regions of nlpD and flgA in order to test binding in the absence and presence of phosphodonor AcP. The sequence of the pseudopalindromic box of RcsB used in the experiments is shown for comparison highlighting in red the nucleotides that compose the box.
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