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Tuberculosis (TB), the leading cause of death from bacterial infections worldwide, results from infection with Mycobacterium tuberculosis (Mtb). The antitubercular agents delamanid (DLM) and pretomanid (PMD) are nitroimidazole prodrugs that require activation by an enzyme intrinsic to Mtb; however, the mechanism(s) of action and the associated metabolic pathways are largely unclear. Profiling of the chemical‐genetic interactions of PMD and DLM in Mtb using combined CRISPR screening reveals that the mutation of rv2073c increases susceptibility of Mtb to these nitroimidazole drugs both in vitro and in infected mice, whereas mutation of rv0078 increases drug resistance. Further assays show that Rv2073c might confer intrinsic resistance to DLM/PMD by interfering with inhibition of the drug target, decaprenylphophoryl‐2‐keto‐b‐D‐erythro‐pentose reductase (DprE2), by active nicotinamide adenine dinucleotide (NAD) adducts. Characterization of the metabolic pathways of DLM/PMD in Mtb using a combination of chemical genetics and comparative liquid chromatography‐mass spectrometry (LC‐MS) analysis of DLM/PMD metabolites reveals that Rv0077c, which is negatively regulated by Rv0078, mediates drug resistance by metabolizing activated DLM/PMD. These results might guide development of new nitroimidazole prodrugs and new regimens for TB treatment. [ABSTRACT FROM AUTHOR]

Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3′ untranslated region (3′UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3′UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3′UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3′UTR-mediated mRNA decay. In addition, the hmsT 3′UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3′UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment. [ABSTRACT FROM AUTHOR]

Y ersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate ( c- di- GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c- di- GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon ( y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c- di- GMP synthesis and biofilm formation in Y . pestis. [ABSTRACT FROM AUTHOR]

The expression of σ54-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed. [ABSTRACT FROM AUTHOR]

The genus Xanthomonas contains plant pathogens exhibiting innate resistance to a range of antimicrobial agents. In other genera, multidrug resistance is mediated by a synergy between a low-permeability outer membrane and expression of a number of multidrug efflux systems. This report describes the isolation of a novel gene cluster xmeRSA from Xanthomonas strain IG-8 that mediates copper chloride resistance. Subsequent analysis of these genes showed that they were responsible for the high level of multiple resistance in this strain and were homologues of the sme system of Stenotrophomonas maltophilia. Knock-out mutants of this gene cluster indicate that these genes are required for the copper resistance phenotype of strain IG-8. Expression analysis using lacZ fusions indicates that the genes are regulated by copper and other antimicrobials. Bioinformatic analysis suggests that these genes were acquired by horizontal gene transfer. [ABSTRACT FROM AUTHOR]

The expression of σ54-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed. [ABSTRACT FROM AUTHOR]

A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans, but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP, which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans, deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis, a phoP mutant made more extensive biofilms in vitro than did the wild type. Expression of HmsT, a diguanylate cyclase that positively regulates biofilms, is diminished in Y. pseudotuberculosis strains with functional PhoP.