Identification of genes differentially expressed inMycobacterium tuberculosisby differential display PCR

Rivera-Marrero, Carlos A.; Burroughs, Mark; Masse, Rupa A.; Vannberg, Fredrik; Leimbach, Darcy L.; Roman, Jesse; Murtagh, James J.

doi:10.1006/mpat.1998.0235

Cited by 36 publications

(26 citation statements)

References 19 publications

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“…For example, by transforming M. tuberculosis H37Ra with a cosmid library of M. tuberculosis H37Rv, Pascopella et al (1994) identified a locus that enhanced growth and survival of M. tuberculosis H37Rv in the spleens of infected mice. Furthermore, comparison of M. tuberculosis H37Rv with M. tuberculosis H37Ra revealed differences in gene expression (Kinger & Tyagi, 1993 ;Rindi et al, 1999 ;Rivera-Marrero et al, 1998), transposition of insertion elements ) and genomic polymorphisms and deletions (Brosch et al, 1999), but the reason(s) for the decreased virulence of M. tuberculosis H37Ra have not been determined.…”

Section: Abbreviationmentioning

confidence: 99%

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice

et al. 2002

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The potential pathogenic role of Mycobacterium tuberculosis H37Rv fadD33, a gene encoding an acyl-CoA synthase that is underexpressed in the attenuated strain H37Ra, was investigated. In a first approach, fadD33 was cloned and expressed in strain H37Ra to restore gene expression and fadD33-complemented bacteria were used to investigate whether fadD33 might confer any growth advantage to M. tuberculosis H37Ra in an infection model of BALB/c mice. No differences were found in the growth rates of M. tuberculosis H37Rv, H37Ra and fadD33-complemented H37Ra in the lungs and spleen. In contrast, in the liver, where the attenuated strain H37Ra showed impaired growth compared to the virulent strain H37Rv, complementation of the attenuated strain H37Ra with fadD33 restored bacterial replication. In a further approach, the fadD33 gene of strain H37Rv was disrupted by allelic exchange mutagenesis and the virulence of the mutant strain was tested by mouse infection. It was found that disruption of fadD33 decreased M. tuberculosis H37Rv growth in the liver, but not in the lungs or spleen, and complementation of the fadD33-disrupted mutant with fadD33 restored bacterial replication in the liver, but did not affect replication in the lungs and spleen. These findings suggest that fadD33 plays a role in M. tuberculosis virulence by supporting bacterial growth in the liver.

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Section: Abbreviationmentioning

confidence: 99%

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice

et al. 2002

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“…In this study, we used random oligonucleotide primers to create a unique cDNA fi ngerprint for the strain TPY grown on Fe 2+ or S 0 , thus providing an effi cient tool for assessing differential gene expression in the strain TPY under different energy resources (Rivera-Marrero et al, 1998;Welsh et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…Electrophoresis was performed as described (Rivera-Marrero et al, 1998) with some modifi cations: 3.5 μl of RAP-PCR products was mixed with 2 μl loading dye (95% formamide, 20 mM EDTA, 0.05% xylene cyanol, and 0.05% bromophenol blue) and were denatured at 95 C for 3 min. Then each sample (three parallel lanes) was loaded on a 6% polyacrylamide sequencing gel containing 7 M urea prepared in TBE buffer.…”

Section: Methodsmentioning

confidence: 99%

Identification of differentially expressed genes in Sulfobacillus sp. TPY grown on either elemental sulphur or Fe2+

Chen²,

Ao³

et al. 2010

J. Gen. Appl. Microbiol.

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IntroductionBacterial leaching of metal sulfi des has developed rapidly during the last decade. The recovery of heavy metals via the application of microorganisms is now an established biotechnological technique. The mobilization of metal cations from insoluble ores by biological oxidation and complexation processes is referred to as bioleaching (Brierley and Brierley, 2001;Rohwerder et al., 2003).Currently, mesophilic microorganisms have been applied successfully for the bioleaching of gold, copper, zinc, and uranium (Brierley and Brierley, 2001;Ewart and Hugues, 1991;Merroun et al., 2003;Rawlings et al., 2003). However, the rate of bioleaching is limiting, due, in part, to the fact that the bioleaching microorganisms cannot adapt to the complicated leaching circumstances such as high concentrations J. Gen. Appl. Microbiol., 56, 389 397 (2010) Sulfobacillus sp. TPY is a moderately thermophilic and acidophilic bacterium found in hydrothermal vents in the Pacifi c Ocean. This bacterium can oxidize ferrous sulfate (Fe 2+ ) and elemental sulfur (S 0 ) under separate conditions. We used random arbitrarily primed polymerase chain reaction (RAP-PCR) to screen and identify differentially expressed genes from bacteria grown on Fe 2+ or S 0 as the energy source. Fifty-fi ve differential cDNA fragments were isolated and subjected to single-pass sequencing. Thirty-fi ve fragments were identifi ed as orthologs of known genes in the GenBank databases, of which 19 were confi rmed to be differentially expressed at the transcriptional level by Northern blot analysis. Among these 19 genes, 14 genes, including isocitrate dehydrogenase, formyltetrahydrofolate deformylase, 3-hydroxybutyryl-CoA dehydrogenase, and GTP-binding protein, were upregulated in TPY grown on Fe 2+ or downregulated in TPY grown on S 0 , while fi ve genes such as the outer membrane adhesion-like protein, phosphomannomutase, and cysteine desulfurase sufS were upregulated in TPY strain grown on S 0 or downregulated in TPY grown on Fe 2+ . These altered genes are involved in metabolism, osmotic stress, cell membrane alterations, oxidative stress, and the regulatory adaptive response. These results will aid our understanding of the molecular basis of Fe 2+ or S 0 oxidation by the moderately thermophilic and acidophilic bacteria.

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“…The application of techniques such as differential display and subtractive hybridization, commonly used for the analysis of eukaryotic gene expression, has been limited in prokaryotes, owing to the generally accepted view that 3h-polyadenylylation of mRNA is a eukaryotic feature. Differential display in prokaryotes therefore requires the use of non-specific, random arbitrary primers (RAP) for cDNA synthesis, often involving extensive testing and optimization of primers (Kwaik & Pederson, 1996 ;Fislage et al, 1997 ;Rivera-Marrero et al, 1998). In addition, reamplification by PCR and subcloning of the isolated cDNA fragments is often difficult and results in failure.…”

Section: Discussionmentioning

confidence: 99%

Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis

2000

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The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria.

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Identification of genes differentially expressed inMycobacterium tuberculosisby differential display PCR

Cited by 36 publications

References 19 publications

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice

Identification of differentially expressed genes in Sulfobacillus sp. TPY grown on either elemental sulphur or Fe2+

Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis

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