scholarly journals Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae

2005 ◽  
Vol 387 (2) ◽  
pp. 541-551 ◽  
Author(s):  
Avinash R. SHENOY ◽  
Nandini P. SREENATH ◽  
Mohana MAHALINGAM ◽  
Sandhya S. VISWESWARIAH
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Adenylate Cyclase ◽  
Biochemical Properties ◽  
The Other ◽  
Gene Products ◽  
Class Iii ◽  
Expression And Purification ◽  
Mycobacterium Tuberculosis H37rv ◽  
High Concentrations

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.

scholarly journals Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv

FEBS Journal ◽  
2010 ◽  
Vol 278 (2) ◽  
pp. 341-353 ◽  
Author(s):  
Anjum Mahmood ◽  
Shubhra Srivastava ◽  
Sarita Tripathi ◽  
Mairaj Ahmed Ansari ◽  
Mohammad Owais ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Characterization ◽  
Tuberculosis H37rv ◽  
Secretory Proteins ◽  
Mycobacterium Tuberculosis H37rv

scholarly journals Characterization of the Two Mycobacterium tuberculosis recA Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 6005-6015 ◽  
Author(s):  
Krishna K. Gopaul ◽  
Patricia C. Brooks ◽  
Jean-François Prost ◽  
Elaine O. Davis
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
The Other ◽  
Expression Level ◽  
Promoter Elements ◽  
Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

Ochrobactrum tritici strain 5bvl1 — characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain

2004 ◽  
Vol 50 (9) ◽  
pp. 697-703 ◽  
Author(s):  
Rita Branco ◽  
M Carmen Alpoim ◽  
Paula V Morais
Keyword(s):  
Heavy Metal ◽  
Growth Rate ◽  
Type Strain ◽  
Treatment Plant ◽  
The Other ◽  
Reducing Ability ◽  
High Concentrations ◽  
Ph Range ◽  
Ochrobactrum Tritici

Bacterial strain 5bvl1, isolated from a chromium-contaminated wastewater treatment plant and identified as Ochrobactrum tritici, was resistant to a broad range of antibiotics, to Cr(VI), Ni(II), Co(II), Cd(II), and Zn(II), and was able to grow in the presence of 5% NaCl and within the pH range 4–10. Characterization showed that strain 5bvl1 could be considered a halotolerant and alkalitolerant microorganism resistant to high concentrations of Cr(VI). This strain was able to grow aerobically in up to 10 mmol·L–1 Cr(VI). Cr(VI) resistance was independent of sulphate concentration. Under aerobic conditions strain 5bvl1 was also able to reduce high Cr(VI) concentrations (up to 1.7 mmol·L–1). Increasing concentrations of Cr(VI) in the medium lowered the growth rate of strain 5bv11 but the reduction in growth rate could not be directly correlated with the amount of Cr(VI) reduced. Unlike the type strain, which was only able to reduce Cr(VI), strain 5bvl1 was resistant to Cr(VI) and able to reduce it. Moreover, in strain 5bvl1, the rate and extent of Cr(VI)-reduction were higher than in the other strains of the genus Ochrobactrum. Ochrobactrum strain 5bvl1 resists high Cr(VI) concentrations and has a high Cr(VI)-reducing ability, making it a valuable tool in bioremediation.Key words: Ochrobactrum, Cr(VI) resistance, Cr(VI)-reduction, heavy metal, bioremediation.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

Characterization of linezolid-resistance-associated mutations in Mycobacterium tuberculosis through WGS

2019 ◽  
Vol 74 (7) ◽  
pp. 1795-1798 ◽  
Author(s):  
Rui Pi ◽  
Qingyun Liu ◽  
Qi Jiang ◽  
Qian Gao
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome ◽  
Single Mutation ◽  
Spontaneous Mutant ◽  
Mutant Selection ◽  
Mycobacterium Tuberculosis H37rv ◽  
Linezolid Resistance ◽  
Genome Scale

Abstract Objectives Linezolid is becoming an important antibiotic for treating MDR/XDR TB, but the mutations conferring resistance to linezolid remain inadequately characterized. Herein, we investigated the linezolid-resistance-associated mutations on a whole-genome scale through parallel selections of resistant isolates in vitro. Methods Ten parallel Mycobacterium tuberculosis H37Rv cultures were subjected to spontaneous mutant selection on 7H11 agar plates containing 2.5 mg/L linezolid. The linezolid resistance of resulting colonies was confirmed by growth on a second linezolid plate. WGS was then performed to identify mutations associated with linezolid resistance. Results Of 181 colonies appearing on the initial linezolid plates, 154 were confirmed to be linezolid resistant. WGS showed that 88.3% (136/154) of these isolates had a T460C mutation in rplC, resulting in a C154R substitution. The other 18 isolates harboured a single mutation in the rrl gene, with G2814T and G2270T mutations accounting for 7.8% (12/154) and 3.9% (6/154), respectively. Conclusions No mutations in novel genes were associated with linezolid resistance in a whole-genome investigation of 154 linezolid-resistant isolates selected in vitro. These results emphasize that rrl and rplC genes should be the major targets for molecular detection of linezolid resistance.

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