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      Suppression of capsule expression in Δlon strains of Escherichia coli by two novel rpoB mutations in concert with HNS: possible role for DNA bending at rcsA promoter

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      MicrobiologyOpen
      John Wiley & Sons, Ltd
      Ces, HNS, rcsA, rpoB, Δlon

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          Abstract

          Analyses of mutations in genes coding for subunits of RNA polymerase always throw more light on the intricate events that regulate the expression of gene(s). Lon protease of Escherichia coli is implicated in the turnover of RcsA (positive regulator of genes involved in capsular polysaccharide synthesis) and SulA (cell division inhibitor induced upon DNA damage). Failure to degrade RcsA and SulA makes lon mutant cells to overproduce capsular polysaccharides and to become sensitive to DNA damaging agents. Earlier reports on suppressors for these characteristic lon phenotypes related the role of cochaperon DnaJ and tmRNA. Here, we report the isolation and characterization of two novel mutations in rpoB gene capable of modulating the expression of cps genes in Δlon strains of E. coli in concert with HNS. clpA, clpB, clpY, and clpQ mutations do not affect this capsule expression suppressor (Ces) phenotype. These mutant RNA polymerases affect rcsA transcription, but per se are not defective either at rcsA or at cps promoters. The results combined with bioinformatics analyses indicate that the weaker interaction between the enzyme and DNA::RNA hybrid during transcription might play a vital role in the lower level expression of rcsA. These results might have relevance to pathogenesis in related bacteria.

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          A simple and rapid method for the preparation of gram-negative bacterial genomic DNA.

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            Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli

            Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these—H-NS and Fis—bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.
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              Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance.

              Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.
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                Author and article information

                Journal
                Microbiologyopen
                Microbiologyopen
                mbo3
                MicrobiologyOpen
                John Wiley & Sons, Ltd (Chichester, UK )
                2045-8827
                2045-8827
                October 2015
                25 September 2015
                : 4
                : 5
                : 712-729
                Affiliations
                Department of Molecular Biology, School of Biological Sciences, Centre for Advanced Studies in Functional and Organismal Genomics, Madurai Kamaraj University [University with Potential for Excellence] Madurai, Tamil Nadu, 625 021, India
                Author notes
                Correspondence M. Hussain Munavar, Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Palkalai Nagar, Madurai, Tamil Nadu 625 021, India. Tel/Fax: ++91-452-2458210; E-mail: munavar61@ 123456gmail.com

                Funding Information This work was funded by common programs given to School of Biological Sciences namely, UGC-Centre for Excellence in Genomic Sciences, Centre for Advanced Studies in Functional and Organismal Genomics, Networking Resource Centre in Biological Sciences, DBT-IPLS of SBS, MKU and DST-PURSE program given to Madurai Kamaraj University.

                Article
                10.1002/mbo3.268
                4618605
                26403574
                089e6a4f-5e77-4401-875e-65909eed155f
                © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 09 March 2015
                : 23 April 2015
                : 04 May 2015
                Categories
                Original Research

                Microbiology & Virology
                ces,hns,rcsa,rpob,δlon
                Microbiology & Virology
                ces, hns, rcsa, rpob, δlon

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