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      Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility

      1 , 1 , 1 , 1 , 1

      Microbiology

      Microbiology Society

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          Most cited references 54

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          Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors

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            A common mechanism of cellular death induced by bactericidal antibiotics.

            Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.
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              Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.

              Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.
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                Author and article information

                Journal
                Microbiology
                Microbiology Society
                1350-0872
                1465-2080
                June 01 2019
                June 01 2019
                : 165
                : 6
                : 668-682
                Affiliations
                [1 ] Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India
                Article
                10.1099/mic.0.000797
                © 2019

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