Faculty Opinions recommendation of Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis.

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.

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Electrophysiological Characterization of Mechanosensitive Channels in the Native Membrane of Corynebacterium Glutamicum

Biophysical Journal ◽

10.1016/j.bpj.2016.11.2888 ◽

2017 ◽

Vol 112 (3) ◽

pp. 534a

Author(s):

Yoshitaka Nakayama ◽

Kosuke Komazawa ◽

Navid Bavi ◽

Ken-ichi Hashimoto ◽

Hisashi Kawasaki ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Mechanosensitive Channels ◽

Electrophysiological Characterization

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Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance

Journal of Bacteriology ◽

10.1128/jb.181.4.1343-1347.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1343-1347 ◽

Cited By ~ 89

Author(s):

Mark Payton ◽

Roy Auty ◽

Rupika Delgoda ◽

Martin Everett ◽

Edith Sim

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Antitubercular Drug ◽

Isoniazid Resistance ◽

E Coli ◽

Monospecific Antiserum ◽

Many Sources ◽

Eukaryotic Organisms

ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

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Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

The Journal of Membrane Biology ◽

10.1007/bf00238414 ◽

1995 ◽

Vol 144 (1) ◽

Cited By ~ 55

Author(s):

C. Cui ◽

D.O. Smith ◽

J. Adler

Keyword(s):

Escherichia Coli ◽

Patch Clamp ◽

Cytoplasmic Membrane ◽

Mechanosensitive Channels ◽

Patch Clamp Recording ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp

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Characterization of pncA Mutations and Prediction of PZA Resistance in Mycobacterium tuberculosis Clinical Isolates From Chongqing, China

Frontiers in Microbiology ◽

10.3389/fmicb.2020.594171 ◽

2021 ◽

Vol 11 ◽

Author(s):

Kun Li ◽

Zhongping Yang ◽

Jing Gu ◽

Ming Luo ◽

Jiaoyu Deng ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Multidrug Resistance ◽

Gene Knockout ◽

Clinical Isolates ◽

Drug Resistant ◽

Full Spectrum ◽

Susceptibility Tests ◽

New Mutations

Pyrazinamide (PZA) is widely used to treat drug-sensitive or multidrug resistance tuberculosis. However, conventional PZA susceptibility tests of clinical isolates are rather difficult because of the requirement of acid pH. Since resistance to pyrazinamide is primary mediated by mutation of pncA, an alternative way of PZA susceptibility test is to analyze the pyrazinamidase activities of Mycobacterium tuberculosis clinical isolates. Therefore, a database containing the full spectrum of pncA mutations along with pyrazinamidase activities will be beneficial. To characterize mutations of pncA in M. tuberculosis from Chongqing, China, the pncA gene was sequenced and analyzed in 465 clinical isolates. A total of 124 types of mutations were identified in 424 drug-resistant isolates, while no mutation was identified in the 31 pan-susceptible isolates. Ninety-four of the 124 mutations had previously been reported, and 30 new mutations were identified. Based on reported literatures, 294 isolates could be predicted resistant to pyrazinamide. Furthermore, pyrazinamidase activities of the 30 new mutations were tested using the Escherichia coli pncA gene knockout strain. The results showed that 24 of these new mutations (28 isolates) led to loss of pyrazinamidase activity and six (8 isolates) of them did not. Taken together, 322 isolates with pncA mutations could be predicted to be PZA resistant among the 424 drug-resistant isolates tested. Analysis of pncA mutations and their effects on pyrazinamidase activity will not only enrich our knowledge of comprehensive pncA mutations related with PZA resistance but also facilitate rapid molecular diagnosis of pyrazinamide resistance in M. tuberculosis.

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Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104820118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2104820118

Author(s):

Charles D. Cox ◽

Yixiao Zhang ◽

Zijing Zhou ◽

Thomas Walz ◽

Boris Martinac

Keyword(s):

Escherichia Coli ◽

Conformational Changes ◽

Functional Characterization ◽

Mechanosensitive Channel ◽

Mechanical Forces ◽

Mechanosensitive Channels ◽

Membrane Tension ◽

Lipid Environment ◽

Small Conductance

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

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Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

Central Asian Journal of Global Health ◽

10.5195/cajgh.2013.107 ◽

2014 ◽

Vol 2 ◽

Author(s):

Sailau Abeldenov ◽

Murat Saparbayev ◽

Bekbolat Khassenov

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Mycobacterium Tuberculosis ◽

Recombinant Proteins ◽

Biochemical Characterization ◽

Ion Trap ◽

Affinity Column ◽

Dna Repair Enzymes ◽

Repair Enzymes

Introduction: Tuberculosis (TB) is a human disease caused by Mycobacterium tuberculosis (Mtb). Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER) pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway.Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670), xthA (Rv0427c), and nei (Rv3297) were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+) expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3) strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by protein mass spectrometry by ion trap tandem MS/MS on nLC-ESI-Ion-Trap platform. Biochemical characterization of DNA repair protein-catalyzed activity was carried out by measuring apurinic/apyrimidinic endonuclease, DNA glycosylase, exonuclease, and 3'-repair diesterase functions. In addition, effect of the opposite base and the influence of metal ion cofactors were measured. Conclusion: Results of the ongoing study will help us define the role of DNA repair enzymes in the emergence of mutations in the mycobacterial genome and, possibly, the origins of multi-drug resistance in mycobacteria.

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