Transcriptional Regulation of the assT - dsbL - dsbI Gene Cluster in Salmonella enterica Serovar Typhi IMSS-1 Depends on LeuO, H-NS, and Specific Growth Conditions

Abstract

The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ⌬leuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions. T he assT gene encodes an arylsulfate sulfotransferase that is present in several organisms, including mammals and a wide spectrum of microbial genomes (2, 25, 55). Mammalian sulfotransferases are located in the cytosol and in the Golgi apparatus membrane; their activities have been detected in liver, brain, kidney, and intestinal epithelial cells (53,55). These sulfotransferases use 3=-phosphoadenosine-5=-phosphosulfate (PAPS) as sulfate donor and are involved in a wide variety of biological processes such as cell communication, growth, development, defense, and detoxification (6,19,43,63,65). Unlike mammals, bacterial sulfotransferases are periplasmic enzymes that require phenolic sulfate esters as donor substrates, and it has been suggested that the intestinal microflora uses this enzyme to detoxify phenolic compounds (31,32,34,35).In Salmonella enterica serovar Typhi, the assT gene is located upstream of dsbL and dsbI, a couple of genes encoding a reduction-oxidation (red-ox) pair of proteins involved in the disulfide bond formation required for AssT activation in the periplasm (25,40,44). The nomenclature was adopted from the orthologous genes previously described in uropathogenic Escherichia coli (UPEC) and Salmonella enterica serovar Typhimurium (25,40). In contrast to the highly conserved assT, the genomic organization of the assT-dsbL-dsbI gene cluster is only conserved in Enterobacter, Yersinia, Citrobacter, and Escherichia strains, as well as within the Salmonella genus (5,12,25,40,41,62).The crystal structures for AssT and DsbL proteins have been solved. AssT forms a homodimer, whose Cys-418 and Cys-424 are involved in disulfide bond formation and its His-436 undergoes the transient sulfurylation necessary for the ping-pong reaction mechanism (44). DsbL is a periplasmic monomer, forming a disulfide bond between Cys-29 and Cys-32, which is oxidized by the transmembrane protein DsbI (25). DsbI contains four predicted transmembrane helixes with two cysteine pairs .In vivo, assT, dsbL, and dsbI expression has been detected in macrophages infected with Salmonella; additionally, a transcriptome assay showed assT and ds...

LeuO is a global regulator of gene expression in Salmonella enterica serovar Typhimurium

“…If LeuO binds as a dimer, there may be as many as 1000–2000 LeuO binding sites (see Genome‐wide prediction and validation of LeuO binding sites ). It is not inconceivable that under appropriate growth conditions Salmonella synthesizes large amounts of LeuO to occupy even more binding sites than documented here as we are only beginning to understand the growth conditions under which LeuO plays a regulatory role (Gallego‐Hernandez et al ., 2012).…”

“…In order to validate our genome‐wide prediction of LeuO binding sites we searched for sites in other known LeuO‐regulated genes that were not identified in our ChIP‐chip study. We correctly predicted sites in the 5′ regulatory region of leuO itself (Chen and Wu, 2005), in the yjjQ‐bglJ operon (Stratmann et al ., 2008), in ompS1 , assT ( stm3192 ), and in the CRISPR‐associated casA and cas3 genes (Westra et al ., 2010; Medina‐Aparicio et al ., 2011; Gallego‐Hernandez et al ., 2012). Furthermore LeuO binding sites have been precisely mapped in the regulatory regions of Salmonella Typhi casA (Medina‐Aparicio et al ., 2011) and ompS1 (De la Cruz et al ., 2007) and our predicted binding sites map to these locations.…”

The Salmonella enterica serovar Typhi ltrR‐ompR‐ompC‐ompF genes are involved in resistance to the bile salt sodium deoxycholate and in bacterial transformation

“…Reverse transcription‐PCR (RT‐PCR) was performed according Gallego‐Hernández et al . (). Total RNA was isolated with acid phenol from 20 ml of bacterial cells grown in 50 ml of N‐MM to an OD 595 of 0.6.…”

“…In Salmonella , LTTRs are involved in multiple biological processes including metabolism (Borum and Monty, ; Hansen et al ., ; Hernández‐Lucas et al ., ; Kim et al ., ; Lewis et al ., ; Baños et al ., ; Turnbull et al ., ), porin synthesis (Fernández‐Mora et al ., ; De la Cruz et al ., ; Hernández‐Lucas et al ., ), detoxification (Hernández‐Lucas et al ., ; Gallego‐Hernández et al ., ), regulation of CRISPR/Cas system (Hernández‐Lucas et al ., ; Medina‐Aparicio et al ., ), virulence (Coynault et al ., ; Tenor et al ., ; Lawley et al ., ; Rodríguez‐Morales et al ., ; Jakomin et al ., ; Lahiri et al ., ; Dillon et al ., ) and resistance to stress conditions (Christman et al ., ; Fang et al ., ; Lahiri et al ., ; Jennings et al ., ). Although these transcriptional factors have a global role in pathogenic bacteria, in S. Typhi only the function of the LeuO regulator has been explored (Fernández‐Mora et al ., ; Hernández‐Lucas et al ., ; Medina‐Aparicio et al ., ; Gallego‐Hernández et al ., ) and the performance of a large number of these proteins remain to be established. Hence, the putative LysR‐type protein STY0036 was selected for study since it is only found in the Salmonella genus and genome‐wide screenings suggest a role of its orthologous (STM0030) in the pathogenicity of Salmonella Typhimurium (Eriksson et al ., ; Lawley et al ., ).…”

Stationary‐phase induction of vvpS expression by three transcription factors: repression by LeuO and activation by SmcR and CRP

“…Recent studies have increased the number of loci ascribed to the LeuO regulon (Shimada et al, 2011;Dillon et al, 2012;Hernandez-Lucas and Calva, 2012). One of the interesting aspects of LeuO regulation is that LeuO is often viewed as an antagonist of another transcription factor, histone-like nucleoid structuring protein (H-NS), as the repression of expression of some genes by H-NS is found to be relieved by LeuO (Chen and Wu, 2005;Hernandez-Lucas et al, 2008;Shimada et al, 2009;Gallego-Hernandez et al, 2012). In this regard, the finding that the activation of vvpS expression is achieved by SmcR and CRP (Figs 6A and 8B) via competition with LeuO in binding to LBS-I and -II (Figs 7C and 9C) provides another example of the antagonistic nature of the regulatory mechanism exerted by LeuO.…”