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Salmonella enterica serovar Typhimurium harbors five pathogenicity islands (SPI) required for infection in vertebrate hosts. Although the role of SPI1 in promoting epithelial invasion and proinflammatory cell death has been amply documented, SPI4 has only more recently been implicated in Salmonella virulence. SPI4 is a 24-kb pathogenicity island containing six open reading frames, siiA to siiF. Secretion of the 595-kDa SiiE protein requires a type I secretory system encoded by siiC, siiD, and siiF. An operon polarity suppressor (ops) sequence within the 5 untranslated region upstream of siiA is required for optimal SPI4 expression and predicted to bind the antiterminator RfaH. SiiE concentrations are decreased in a SPI1 mutant strain, suggesting that SPI1 and SPI4 may have common regulatory inputs. SPI1 gene expression is positively regulated by the transcriptional activators HilA, HilC, and HilD, encoded within SPI1, and negatively regulated by the regulators HilE and PhoP. Here, we show that mutations in hilA, hilC, or hilD similarly reduce expression of siiE, and mutations in hilE or phoP enhance siiE expression. Individual overexpression of HilA, HilC, or HilD in the absence of SPI1 cannot activate siiE expression, suggesting that these transcriptional regulators act in concert or in combination with additional SPI1-encoded regulatory loci to activate SPI4. HilA is no longer required for siiE expression in an hns mutant strain, suggesting that HilA promotes SPI4 expression by antagonizing the global transcriptional silencer H-NS. Coordinate regulation suggests that SPI1 and SPI4 play complementary roles in the interaction of S. enterica serovar Typhimurium with the host intestinal mucosa.

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution ofEnterobacteriaceaeby horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of bothcis-regulatory sequences and intransinteractions with modulatory ligands.IMPORTANCEBacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, theircis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.

31Gene duplication and subsequent evolutionary divergence have allowed conserved proteins 32 to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive 33 duplication and diversification in bacteria, where they act as environmentally-responsive 34 repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many 35 antimicrobial agents. We have performed structural, functional, and genetic analyses of 36 representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral 37 functions, including repression of their own expression and that of divergently-transcribed multi-38 drug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, 39 SlyA and RovA have acquired the ability to counter-silence horizontally-acquired genes, which 40 has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. 41 SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to 42 provide the enhanced levels of expression required for their new role. Moreover, in contrast to 43 MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to 44 acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and 45 in trans interactions with modulatory ligands.46 Evolution of SlyA Transcription Factors Will, et al. 109been conserved not due to their primordial role in regulating antimicrobial resistance but rather as 110 a consequence of their counter-silencing function, which is essential to maintain the regulated 111 expression of horizontally-acquired genes in Enterobacteriaceae. 112 113 Results 114 Evolution of SlyA Transcription Factors Will, et al. 6 Salicylate-mediated inhibition of SlyA activity. As environmentally-responsive repressors 115 whose conformation and regulatory actions are modulated by small aromatic carboxylates (12, 116 14), MarR family TFs are inhibited by salicylate in vitro (12). In their structural analyses of SlyA-117 DNA interactions, Dolan et al, (33) inferred from our structural data (see below) that salicylate 118 might regulate SlyA. Using electrophoretic mobility shift assays, they demonstrated that salicylate 119 inhibits DNA-binding by SlyA. To confirm that this influences the function of SlyA as a 120 transcriptional regulator, we performed in vitro transcription assays (IVTs) of slyA and the 121 divergently transcribed ydhIJK efflux pump operon. Supercoiled plasmid DNA containing the 122 slyA-ydhIJK region was incubated with RNA polymerase (RNAP) and increasing SlyA 123 concentrations in the presence or absence of salicylate. SlyA repressed slyA transcription 124 approximately 5.3-fold, while ydhI transcription was inhibited ~19-fold (Figure 1A, B). The 125 addition of 2mM sodium salicylate reduced SlyA-mediated repression to 2.8-fold and 3.2-fold, 126 respectively, indicating that the sensitivity to aromatic carboxylates observed in classical MarR 127TFs...