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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]

Abstract: 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, and is the target of the herbicide glyphosate. Glyphosate tolerance activity of the enzyme could be obtained by natural occurrence or by site-directed mutagenesis. A functional Pseudomonas putida AroA was obtained by co-expression of two protein fragments AroA P. putida -N210 and AroA P. putida -C212 in Escherichia coli aroA mutant strain AB2829. From sequence analysis, the equivalent split site on E. coli AroA was chosen for further study. The result indicated that functional E. coli AroA could also be reconstituted from two protein fragments AroA E. coli -N218 and AroA E. coli -C219, under both in vivo and in vitro conditions. This result suggested that the fragment complementation property of this family of enzyme may be general. Additional experiments indicated that the glyphosate tolerance property of AroA could also be reconstituted in parallel with its enzyme activity. The implication of this finding is discussed. [Copyright &y& Elsevier]