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

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.

The short chain fatty acid propionic acid activates the Rcs stress response system partially through inhibition of D-alanine racemase

“…A plasmid-borne luminescence reporter construct was used to screen for molecules that activate the Rcs system (Table 1, Figure S1, Supplemental Data). The previously described plasmid includes the promoter for the SMDB11_1637 open reading frame that is predicted to code for the conserved osmB gene (29, 34, 35). This promoter is induced by the Rcs system in S. marcescens and other bacterial genera (29, 34, 35).…”

“…The previously described plasmid includes the promoter for the SMDB11_1637 open reading frame that is predicted to code for the conserved osmB gene (29, 34, 35). This promoter is induced by the Rcs system in S. marcescens and other bacterial genera (29, 34, 35). A clinical isolate of S. marcescens bearing a plasmid with this reporter, pMQ747, was grown in LB medium and added to Biolog Phenotypic Microarray plates PM11-20.…”

Antibiotics Used in Empiric Treatment of Ocular Infections Trigger the Bacterial Rcs Stress Response System Independent of Antibiotic Susceptibility

“…All of these previously uncharacterized open reading frames bear high similarity to Rcs-regulated genes in other bacteria. SMDB11_1637 is similar to osmotically inducible lipoprotein B ( osmB ), which is positively regulated by the Rcs system in Erwinia amylovora [ 24 ], E. coli [ 25 , 26 ], P. mirabilis [ 27 ], S. enterica [ 28 ], and Yersinia pseudotuberculosis [ 29 ]. SMDB11_1194 is highly similar to umoD , which is Rcs-regulated in P. mirabilis [ 27 ], as is its ortholog YPO1624 in Y. pseudotuberculosis .…”

“…This is not unexpected, as several envelope stress response systems, beyond Rcs, are conserved among the Enterobacterales. For example, in Salmonella , the promoter of the osmB gene (similar to SMDB11_1687) is activated by both the Rcs and the RpoS stress response systems [ 28 ], suggesting that individual stress response genes are controlled by multiple regulatory systems. The use of the ∆ rcsB strain in addition to the WT enabled clear identification of Rcs-dependent activation of the reporters by ocular antibiotics.…”

2-Aminoimidazole Analogs Target PhoP Altering DNA Binding Activity and Affect Outer Membrane Stability in Gram-Negative Bacteria

“…Generally, activated (phosphorylated) response regulators dimerize and, to this point, have been seen to bind DNA in two different modeseither with a “tucked/extended” conformation or a “tucked/tucked” conformation. In each case, the protein domains must be able to move between these states correctly in order to adopt the optimum DNA-binding state. ,,, Anything that interferes with the population distribution and equilibrium between these conformational states will affect DNA binding. Under direct DNA-binding analysis, we observed no statistical difference in the binding affinity of PhoP and DNA in the presence of 2-AI; this is consistent with our mechanism from the standpoint of conformational dynamics.…”