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1.
Cuadrado Y Fernández M Recio E Aparicio JF Martín JF 《Applied microbiology and biotechnology》2004,64(2):228-236
The first two genes of the threonine pathway, ask and asd, were cloned and sequenced from the aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331. The two genes are organized in a bicistronic operon. ask, encoding the apartokinase (ASK), is located upstream from asd. The presence of a ribosome-binding site within the ask sequence suggests that this open reading frame encodes two overlapping proteins. The formation of both subunits of the aspartokinase from a single gene was studied using antibodies raised against the C-terminal end of the aspartokinase subunits. Disruption of asd results in a significant decrease of aminoethoxyvinylglycine production, thus supporting the involvement of the ask–asd operon in the biosynthesis of this metabolite. This is the first report in which a gene cluster for the first two steps of aminoethoxyvinylglycine biosynthesis is characterized.Abbreviations
ASD
Aspartate semialdehyde dehydrogenase
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ASK
Aspartokinase
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AVG
Aminoethoxyvinylglycine 相似文献
2.
Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria. 总被引:3,自引:1,他引:2
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J D Cirillo T R Weisbrod A Banerjee B R Bloom W R Jacobs Jr 《Journal of bacteriology》1994,176(14):4424-4429
The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase. 相似文献
3.
4.
Alternate sigma factor SigF controls the expression of virulence-associated genes and is believed to contribute to the pathology
of tuberculosis. It was reported to be absent in fast-growing nontuberculous mycobacteria until its orthologs were reported
recently in a database. In this study, we demonstrate the presence of sigF gene in few commonly studied nonpathogenic mycobacterial species. Further, we studied the sigF expression in Mycobacterium smegmatis and observed that unlike its late-stage expression in M. tuberculosis and M. bovis, found in earlier studies, sigF is expressed throughout the growth in M. smegmatis, by and large, at the same level, but its expression varies upon exposure to different stress conditions. The presence of
sigF orthologs in nontuberculous mycobacteria and its continued expression throughout the growth suggests that apart from regulating
the expression of virulence factor genes in pathogenic mycobacteria, SigF is likely to have more roles in the mycobacterial
physiology. 相似文献
5.
6.
Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor 总被引:8,自引:3,他引:5
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Ehrt S Guo XV Hickey CM Ryou M Monteleone M Riley LW Schnappinger D 《Nucleic acids research》2005,33(2):e21
Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome. 相似文献
7.
Background
The unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2′ epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium.Methodology/Principal Findings
In this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this “rescue” plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality.Conclusions/Significance
This study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist. Overall, this study further validates DprE1 as a promising target for new anti-mycobacterial drugs. 相似文献8.
Asma Javid Charlotte Cooper Albel Singh Steffen Schindler Milena Hänisch Robert L. Marshall Rainer Kalscheuer Vassiliy N. Bavro Apoorva Bhatt 《Molecular microbiology》2020,113(2):521-533
The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies. 相似文献
9.
Background
The usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis. 相似文献10.
Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways 总被引:10,自引:4,他引:6
Mycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl-glycineamide formyltransferases (GAR enzymes) and methionyl-tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M. 相似文献
11.
O.M. Barrientos E. Juárez Y. Gonzalez D.A. Castro-Villeda M. Torres S. Guzmán-Beltrán 《Letters in applied microbiology》2021,72(3):351-356
Tuberculosis (TB) is caused by Mycobacterium tuberculosis. TB is highly prevalent, characterized by the constant occurrence of drug-resistant cases, and confounded by the incidence of respiratory disease caused by non-tuberculous mycobacteria (NTB). Expanding the spectrum of drugs for the treatment of TB is indispensable. Loperamide, an antidiarrhoeal drug, enhances immune-driven antimycobacterial activity, and we aimed to evaluate its bactericidal activity against M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium terrae and Mycobacterium smegmatis. Loperamide exhibited an inhibitory effect against all mycobacterial species tested, with MICs of 100 and 150 μg ml−1. Thus, loperamide is a mycobactericidal drug with potential as adjunctive therapy for TB and NTB infections. 相似文献
12.
13.
Construction and use of integrative vectors to express foreign genes in mycobacteria 总被引:5,自引:2,他引:3
Odir A. Dellagostin Sue Wall Elizabeth Norman Tina O'Shaughnessy Jeremy W. Dale JohnJoe McFadden 《Molecular microbiology》1993,10(5):983-993
We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained. 相似文献
14.
15.
Cloning and Characterization of Arylamine N-Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance 总被引:2,自引:0,他引:2
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Mark Payton Roy Auty Rupika Delgoda Martin Everett Edith Sim 《Journal of bacteriology》1999,181(4):1343-1347
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 from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as 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. 相似文献
16.
Varun Shenoy Rashmika Gunda Cora Noble Annalisa Haraguchi Steven Stevenson Jaiyanth Daniel 《Cell biochemistry and function》2024,42(2):e3963
Mycobacterium tuberculosis and nontuberculous mycobacteria such as Mycobacterium abscessus cause diseases that are becoming increasingly difficult to treat due to emerging antibiotic resistance. The development of new antimicrobial molecules is vital for combating these pathogens. Carbon nanomaterials (CNMs) are a class of carbon-containing nanoparticles with promising antimicrobial effects. Fullertubes (C90) are novel carbon allotropes with a structure unique among CNMs. The effects of fullertubes on any living cell have not been studied. In this study, we demonstrate that pristine fullertube dispersions show antimicrobial effects on Mycobacterium smegmatis and M. abscessus. Using scanning electron microscopy, light microscopy, and molecular probes, we investigated the effects of these CNMs on mycobacterial cell viability, cellular integrity, and biofilm formation. C90 fullertubes at 1 µM inhibited mycobacterial viability by 97%. Scanning electron microscopy revealed that the cell wall structure of M. smegmatis and M. abscessus was severely damaged within 24 h of exposure to fullertubes. Additionally, exposure to fullertubes nearly abrogated the acid-fast staining property of M. smegmatis. Using SYTO-9 and propidium iodide, we show that exposure to the novel fullertubes compromises the integrity of the mycobacterial cell. We also show that the permeability of the mycobacterial cell wall was increased after exposure to fullertubes from our assays utilizing the molecular probe dichlorofluorescein and ethidium bromide transport. C90 fullertubes at 0.37 µM and C60 fullerenes at 0.56 µM inhibited pellicle biofilm formation by 70% and 90%, respectively. This is the first report on the antimycobacterial activities of fullertubes and fullerenes. 相似文献
17.
Paul K. Crellin Julian P. Vivian Judith Scoble Frances M. Chow Nicholas P. West Rajini Brammananth Nicholas I. Proellocks Adam Shahine Jerome Le Nours Matthew C. J. Wilce Warwick J. Britton Ross L. Coppel Jamie Rossjohn Travis Beddoe 《The Journal of biological chemistry》2010,285(39):30050-30060
The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria. 相似文献
18.
Quanxin Long Xiaohong Xiang Qingqin Yin Shuangjiang Li Wenmin Yang Hang Sun Qi Liu Jianping Xie Wanyan Deng 《Journal of cellular physiology》2019,234(11):19774-19784
Mycobacterium tuberculosis, the leading causative agent of tuberculosis, remains one of the most deadly infectious pathogens. PE_PGRS proteins become a new focus as their species specificity in mycobacteria, especially in pathogenic mycobacteria. Despite intensive research, PE_PGRS proteins are still a mysterious aspect of mycobacterial pathogenesis with unknown mechanism. Herein, we focused on a PE_PGRS member from M. tuberculosis, PE_PGRS62, characterized by a surface-exposed protein function in disrupting phagolysosome maturation. Expression of PE_PGRS62 in Mycobacterium smegmatis, a nonpathogenic species naturally deficient in PE_PGRS genes, resulted in enhanced resistance to various in vitro stresses and cellular survival in macrophage. As a consequence, the cytokine profiles of macrophage were disturbed and cell apoptosis were inhibited via decreasing endoplasmic reticulum stress response. 相似文献
19.
Sophia A. Pacheco Fong-Fu Hsu Katelyn M. Powers Georgiana E. Purdy 《The Journal of biological chemistry》2013,288(33):24213-24222
A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis. 相似文献