Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

Rns regulates nonpilus adhesin EtpA in human EnterotoxigenicE.coli(ETEC)

Rns, an araC family of transcriptional activator (AFTR) is known to regulate many of the known pili in human ETEC. Apart from pili, Rns is also known to regulate some nonpilus genes believed to have role in virulence. EtpA is a nonpilus adhesin, encoded with inetpBACoperon in ETEC genome. Using a combination of qRT-PCR and gel shift assay, we show that Rns binds to upstream of etpBAC operon and upregulates the expression of EtpA. This is the first report of Rns regulating a known virulence factor in ETEC.

1.65 Å resolution structure of the AraC-family transcriptional activator ToxT fromVibrio cholerae

ToxT is an AraC-family transcriptional activator protein that controls the expression of key virulence factors inVibrio cholerae, the causative agent of cholera. ToxT directly activates the expression of the genes that encode the toxin-coregulated pilus and cholera toxin, and also positively auto-regulates its own expression from thetcppromoter. The crystal structure of ToxT has previously been solved at 1.9 Å resolution (PDB entry 3gbg). In this study, a crystal structure of ToxT at 1.65 Å resolution with a similar overall structure to the previously determined structure is reported. However, there are distinct differences between the two structures, particularly in the region that extends from Asp101 to Glu110. This region, which can influence ToxT activity but was disordered in the previous structure, can be traced entirely in the current structure.

Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators

ABSTRACTBurkholderia thailandensisis a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant,Pseudomonas aeruginosa. Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar toP. aeruginosa. Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator inB. thailandensis. In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of theB. thailandensischoline catabolic pathway (gbdR1,gbdR2, andsouR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons ofgbdR1(BTH_II1869) andgbdR2(BTH_II0968). We also identified a functional ortholog forP. aeruginosasouR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar toP. aeruginosaGbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, theB. thailandensisSouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas inP. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed byB. thailandensisrepresents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection.IMPORTANCEMany proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential regulation of these components can help us understand both the evolution of these systems and the potential roles these pathways have in the biology of each bacterium. Here, we describe the transcriptome response ofBurkholderia thailandensisto the eukaryote-enriched molecule choline, identify the regulatory pathway governing choline catabolism, and compare the pathway to that previously described forPseudomonas aeruginosa. These data support a multitiered regulatory network inB. thailandensis, with conserved orthologs in the select agentsBurkholderia pseudomalleiandBurkholderia mallei, as well as the opportunistic lung pathogens in theBurkholderia cepaciaclade.

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

ABSTRACTCYP153, one of the most common medium-chainn-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed inn-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading variousn-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrumn-alkane-degrading Gram-positive bacterium,Dietziasp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the −10 and −35 regions inActinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZfusion cell indicated that the CYP153 gene promoter was induced byn-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed acypRmutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence ofn-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements inActinobacteriaandProteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by manyActinobacteria.