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H N Jayaram R Ramakrishnan C S Vaidyanathan 《Archives of biochemistry and biophysics》1968,126(1):165-174
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Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra 总被引:3,自引:0,他引:3
To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations. 相似文献
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Hiwa M?len Gustavo A De Souza Sharad Pathak Tina S?fteland Harald G Wiker 《BMC microbiology》2011,11(1):18
Background
The potential causes for variation in virulence between distinct M. tuberculosis strains are still not fully known. However, differences in protein expression are probably an important factor. In this study we used a label-free quantitative proteomic approach to estimate differences in protein abundance between two closely related M. tuberculosis strains; the virulent H37Rv strain and its attenuated counterpart H37Ra. 相似文献4.
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H37Rv是结核分枝杆菌标准有毒株,H37Ra是从H37Rv获得的稳定减毒株,但目前H37Ra毒力减弱原因尚不完全清楚。本研究利用表型芯片系统,高通量分析H37Ra生长表型,并与H37Rv表型比较,筛选两菌株表型差异,分析与H37Ra毒力减弱可能的相关表型及分子机制。结果发现,与H37Rv相比,H37Ra耐酸及耐渗透压能力显著下降,且不能利用丁二酸单甲酯和吐温40作为碳源。结核分枝杆菌耐酸能力直接影响其在吞噬体中的生存和代谢,耐高渗能力影响其必需营养物质的跨膜运输,代谢途径的改变影响其在宿主内的能量摄取,三者改变均可能与H37Ra毒力减弱相关。 相似文献
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Polynucleotide phosphorylase of Mycobacterium tuberculosis H37Rv 总被引:2,自引:0,他引:2
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为了解结核病的致病分子机理和筛选结核病致病菌的毒力基因,利用抑制消减杂交(SSH)技术分析了结核分支杆菌强毒株H37Rv和弱毒株H37Ra间的基因组DNA间差异。通过Southern杂交验证及序列分析得到仅在强毒株H37Rv基因组中有的DNA片段8个,其中一个编码已知的毒力因子mce蛋白,1个编码PE家族蛋白,1个编码purC合成酶,和4个潜在蛋白,另一个为非编码区片段。其中有2个基因经PCR方法已证实在强毒株H37Rv和临床分离的强毒株中存在,而在H37Ra和临床弱毒株中无;仅在弱毒株H37Ra基因组中的DNA片段3个,其中2个为新基因片段,已被GenBank收录。 相似文献
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【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。 相似文献
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By comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence. 相似文献
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Kovács L Csanádi A Kiss E Miczak A 《Acta microbiologica et immunologica Hungarica》2005,52(3-4):363-371
Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule. 相似文献
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The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA
deoxyribonucleic acid
- td
delay in initiation
- OD
optical density
- CAM
chloramphenicol
- RIF
rifampicin 相似文献
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K S Sundaram T A Venkitasubramanian 《Biochemical and biophysical research communications》1977,78(2):839-845
The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by H37Rv grown and was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)7 of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis. 相似文献
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1. The biosynthesis of nucleic acid purine in Mycobacterium tuberculosis H(37)R(v) has been studied by using (14)C-labelled precursors. 2. The results indicate that C-2 and C-8 of the purine ring are derived most efficiently from serine and glycine and not from formate. 3. [(14)C]Methionine is not incorporated into the ureide carbon atoms of the purine ring. 相似文献
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Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement. 相似文献