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Identification and characterization of response regulators in mycobacteria

Posted on:2001-11-01Degree:Ph.DType:Dissertation
University:The University of Alabama at BirminghamCandidate:Harris, Randall HerbertFull Text:PDF
GTID:1464390014455509Subject:Biology
Abstract/Summary:
Bacteria sense and respond to changes in their environment, often via two component regulatory systems. This dissertation describes the characterization of mutations associated with two such regulatory systems in mycobacteria. A homolog of the Mycobacterium tuberculosis senX3regX3 two component system was cloned from Mycobacterium smegmatis. A null mutation in regX3 resulted in an M. smegmatis strain with a growth defect on certain media that was not caused by an auxotrophy. Putative extragenic suppressor mutations restored normal growth of the regX3 mutant. This growth defect was complemented by senX3regX3 from M. smegmatis and M. tuberculosis but not M. leprae. regX3 in Mycobacterium leprae has a frameshift mutation that has made the gene nonfucntional. The M. smegmatis regX3 mutant overproduced a lipooligosaccharide in which the sugar moiety was composed of a pyruvylated, methlylated trehalose-containing pentaglucoside. The acyl groups of the glycolipid are attached to the trehalose unit. The regX3 mutant was considerably more sensitive to 5 mM hydrogen peroxide and 0.1% sodium dodecyl sulfate. The sodium dodecyl sulfate sensitivity was partially and fully complemented by senX3regX3 from M. smegmatis and M. tuberculosis, respectively. The lipooligosaccharide and the hydrogen peroxide sensitivity were not complemented by senX3regX3 from M. smegmatis or M. tuberculosis. The incomplete complementation was apparently to a secondary mutation unrelated to the suppressor mutation alleviating the growth defect.;The effect of a naturally occurring IS6110 insertion upstream of the trcRS two component system in an M. tuberculosis strain was also investigated. Transcriptional fusions of the trcR promoter containing IS6110 to lacZ revealed that the IS6110 insertion resulted in 30-fold more activity in M. smegmatis and 20-fold more activity in M. bovis Bacille Calmette-Guerin than wild-type trcR promoter fused to lacZ. The IS6110 was found to contain an internal promoter that mapped to the final 100 bp of the right side of the element. TrcR is able to regulate its own promoter in Escherichia coli. The IS6110 insertion had no effect on autoregulation by TrcR in E. coli. The effect of the IS6110 insertion upstream of trcRS on the virulence of M. tuberculosis is under investigation.
Keywords/Search Tags:IS6110 insertion, Two component, Tuberculosis, Trcr
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