Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria

Wards, Barry J.; Collins, D M

doi:10.1111/j.1574-6968.1996.tb08563.x

Cited by 109 publications

(57 citation statements)

References 10 publications

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“…Genomic regions (Ϸ800 bp each) flanking the gene of interest were amplified by PCR and cloned into pKO so as to flank the kan R determinant. For targeted disruptions, constructs were electroporated into mycobacteria as described (26) and selected on 7H10 plates with hygromycin. In the first screening step, each colony was tested by PCR with two primer pairs, one specific for integration upstream of the gene of interest and the other specific for integration downstream.…”

Section: Methodsmentioning

confidence: 99%

Regulation of theMycobacterium tuberculosishypoxic response gene encoding α-crystallin

Sherman

Voskuil

Schnappinger

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

679

720

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Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin ( acr ) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

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Section: Methodsmentioning

confidence: 99%

Regulation of theMycobacterium tuberculosishypoxic response gene encoding α-crystallin

Sherman

Voskuil

Schnappinger

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

679

720

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“…The sequence of the cloned fragments was confirmed by DNA sequencing. The plasmids were electroporated into M. tuberculosis as described by Wards & Collins (1996).…”

Section: Methodsmentioning

confidence: 99%

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

et al. 2009

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Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C 6 or C 2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the DilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the DilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

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“…H37Rv and M. bovis ATCC35723 were cultivated as described (10,16). Mycobacterial strains were transformed by using a previously described method (19). Escherichia coli DH10B, M15, and Tuner cells were grown in LB supplemented with carbenicillin (80 g͞ml), kanamycin (50 g͞ml), or hygromycin (180 g͞ml).…”

Section: Methodsmentioning

confidence: 99%

“…Logarithmically grown E. coli XL-Blue cells were transduced with the extract for 2 h at 30°C and plated out on LB containing hygromycin. Colonies were scraped from the plates, and plasmid DNA was isolated and electroporated (19) into Mycobacterium smegmatis mc 2 155. The resulting phage ( WhiB3) was used for the transduction of H37Rv, as described (20).…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

Steyn

Collins

Hondalus

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

223

210

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Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-5153 His) in the 4.2 domain of RpoV. We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M. tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-5153 His) allele. Infection of mice and guinea pigs with a M. tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model. In contrast, an M. bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs. However, we found that immunocompetent mice infected with the M. tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M. tuberculosis. Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection. Our results imply that M. tuberculosis replication per se is not a sufficient condition for virulence in vivo. They also indicate a different role for M. bovis and M. tuberculosis whiB3 genes in pathogenesis generated in different animal models. We propose that M. tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host.T he increased susceptibility of HIV-infected individuals and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) results in the death of 2-3 million people each year (1) and underscores the urgency of deciphering the molecular mechanisms of virulence of this pathogen. The highly variable protective efficacy of Mycobacterium bovis bacillus Calmette-Guérin in adults (0-80%; ref. 2) emphasizes the urgency for developing second-generation antituberculosis antimicrobial agents and vaccines. With these aims in mind, research stimulated by the advances in mycobacterial genetics (3, 4) has led to the identification of several genes that have been implicated in virulence (5-12).MTB requires sophisticated genetic mechanisms to recognize appropriate environmental signals and to convey this information to the transcriptional apparatus of the organism. The activation of bacterial sigma factors to regulate gene expression is an effective response mechanism that enables pathogens to respond instantly to a multitude of environmental signals. Bacterial 70 -type sigma factors are composed of four major regions, called regions 1, 2, 3 and 4 (13). Region 4 is subdivided further into sub regions 4.1 and 4.2; the latter is known to interact with the Ϫ35 region of promoters (13) and other transcription factors. Mutations in or close to the helix-turn-helix (HTH) motif in region 4.2 can result in either positive or negative effects on activation by transcri...

show abstract

Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria

Cited by 109 publications

References 10 publications

Regulation of theMycobacterium tuberculosishypoxic response gene encoding α-crystallin

Regulation of theMycobacterium tuberculosishypoxic response gene encoding α-crystallin

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

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