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Functional Disassociation Between the Protein Domains of MSMEG_4305 of Mycolicibacterium smegmatis (Mycobacterium smegmatis) in vivo.

ABSTRACT: MSMEG_4305 is a two-domain protein of Mycolicibacterium smegmatis (Mycobacterium smegmatis) (Mycolicibacterium smegmatis). The N-terminal domain of MSMEG_4305 encodes an RNase H type I. The C-terminal domain is a presumed CobC, predicted to be involved in the aerobic synthesis of vitamin B12. Both domains reach their maximum at distinct pH, approximately 8.5 and 4.5, respectively. The presence of the CobC domain influenced RNase activity in vitro in homolog Rv2228c. Here, we analyzed the role of MSMEG_4305 in vitamin B12 synthesis and the functional association between both domains in vivo in M. smegmatis. We used knock-out mutant of M. smegmatis, deficient in MSMEG_4305. Whole-cell lysates of the mutants strain contained a lower concentration of vitamin B12, as it determined with immunoenzimatic assay. We observed growth deficits, related to vitamin B12 production, on media containing sulfamethazine and propionate. Removal of the CobC domain of MSMEG_4305 in ?rnhA background hardly affected the growth rate of M. smegmatis in vivo. The strain carrying truncation showed no fitness deficit in the competitive assay and it did not show increased level of RNA/DNA hybrids in its genome. We show that homologs of MSMEG_4305 are present only in the Actinomycetales phylogenetic branch (according to the old classification system). The domains of MSMEG_4305 homologs accumulate mutations at a different rate, while the linker region is highly variable. We conclude that MSMEG_4305 is a multidomain protein that most probably was fixed in the phylogenetic tree of life due to genetic drift.

SUBMITTER: Czubat B

PROVIDER: S-EPMC7466739 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Functional Disassociation Between the Protein Domains of MSMEG_4305 of Mycolicibacterium smegmatis (Mycobacterium smegmatis) in vivo.

Czubat Bożena B Minias Alina A Brzostek Anna A Żaczek Anna A Struś Katarzyna K Zakrzewska-Czerwińska Jolanta J Dziadek Jarosław J

Frontiers in microbiology 20200819

MSMEG_4305 is a two-domain protein of Mycolicibacterium smegmatis (Mycobacterium smegmatis) (Mycolicibacterium smegmatis). The N-terminal domain of MSMEG_4305 encodes an RNase H type I. The C-terminal domain is a presumed CobC, predicted to be involved in the aerobic synthesis of vitamin B12. Both domains reach their maximum at distinct pH, approximately 8.5 and 4.5, respectively. The presence of the CobC domain influenced RNase activity in vitro in homolog Rv2228c. H ...[more]

PMID: 32973726

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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.

Dataset Information

Functional Disassociation Between the Protein Domains of MSMEG_4305 of Mycolicibacterium smegmatis (Mycobacterium smegmatis) in vivo.

Publications

Functional Disassociation Between the Protein Domains of MSMEG_4305 of <i>Mycolicibacterium smegmatis</i> (<i>Mycobacterium smegmatis</i>) <i>in vivo</i>.

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