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Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis.

ABSTRACT: Vitamin C is a critical dietary nutrient in human which has a wide range of regulatory effects on gene expression and physiology of Mycobacterium tuberculosis that leads to a dormant drug-tolerant phenotype. In the presence of iron, vitamin C shows a high bactericidal activity even in the drug resistant phenotype of M. tuberculosis. The regulatory mechanisms underlying vitamin C induced adaptations are largely unknown due to lack of functional genomics data in this field. In this study, we attempt to characterize the direct effect of vitamin C treatment on the physiology of actively growing Mycobacterium smegmatis. The study chose M. smegmatis as it is a fast-growing bacterium and a non-pathogenic model system which shares many physiological features with the pathogenic M. tuberculosis including dormancy and its regulation. The proteomic adaptation of M. smegmatis on vitamin C treatment demonstrates the important changes in cellular and metabolic process such as reversal of tricarboxylic acid cycle, decrease in ATP synthesis, decrease in iron acquisition and storage, and induction of dormancy regulators WhiB3, PhoP, and Lsr2.

SUBMITTER: Mishra A

PROVIDER: S-EPMC4435235 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis.

Mishra Abhishek A Sarkar Dhiman D

Frontiers in microbiology 20150518

Vitamin C is a critical dietary nutrient in human which has a wide range of regulatory effects on gene expression and physiology of Mycobacterium tuberculosis that leads to a dormant drug-tolerant phenotype. In the presence of iron, vitamin C shows a high bactericidal activity even in the drug resistant phenotype of M. tuberculosis. The regulatory mechanisms underlying vitamin C induced adaptations are largely unknown due to lack of functional genomics data in this field. In this study, we attem ...[more]

PMID: 26042100

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Project description:The DNA sequence of the promoter region of the Mycobacterium smegmatis rpsL gene, which encodes the S12 ribosomal protein, was determined. Primer extension analysis and S1 nuclease protection experiments identified the 5' end of the rpsL mRNA to be 199 bp upstream of the translation initiation codon. The rpsL promoter contained sequences upstream of this start point for transcription that were similar to the canonical hexamers found at the -10 and -35 regions of promoters recognized by Esigma70, the major form of RNA polymerase in Escherichia coli. To define the promoter of the rpsL gene, DNA fragments containing progressive deletions of the upstream region of the rpsL gene were inserted into a plasmid vector containing a promoterless xylE gene. These insertions revealed that the 200 bp of DNA sequence immediately upstream from the translation initiation codon was not essential for promoter function. In addition, 5' deletions removing all but 34 bp upstream of the transcription start point retained greater than 90% promoter activity, suggesting that the -35 hexamer was not essential for promoter activity. To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription. Single base substitutions in the -10 hexamer, but not in the -35 hexamer, severely reduced rpsL promoter activity in vivo. Within the -10 hexamer, nucleotide substitutions causing divergence from the E. Coli sigma70 consensus reduced promoter activity. The DNA sequence immediately upstream from the - 10 hexamer contained the TGn motif described as an extended -10 region in prokaryotic promoters. Mutations in this motif, in combination with a transition at either the -38 or -37 position within the -35 hexamer, severely reduced promoter activity, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer. Comparison of the nucleotide sequence of the rpsL promoter region of M. smegmatis with the homologous sequences from Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium tuberculosis showed the presence in these slowly growing mycobacterial species of conserved promoter elements a similar distance upstream of the translation initiation codon of the rpsL gene, but these other mycobacterial promoters did not contain the extended -10 motif.

Project description:A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

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Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis.

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Qualitative and quantitative proteomic analysis of Vitamin C induced changes in Mycobacterium smegmatis.

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