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Title: Isolation and molecular genetic characterization of SecA, the central component of the general protein secretion pathway in mycobacteria
Authors: Brown, Amanda Maria
Keywords: Microbiology.
Molecular biology.
Issue Date: 1996
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 57-04, Section: B, page: 2321.;Advisors: William Richard Jacobs, Jr.
Abstract: This thesis work describes the cloning and molecular genetic characterization of SecA homologues from two different mycobacterial species, Mycobacterium smegmatis, and M. bovis BCG. Southern analysis demonstrated that secA is conserved among several species of both fast and slow growing mycobacteria. Sequence comparison against other known SecA proteins revealed 48-55% identity in the region of the protein that encodes the ATP-binding domains. There is 79%, identity over the entire length of the M. smegmatis and M. bovis BCG SecA proteins. Two distinct differences between the BCG and M. smegmatis SecA proteins were found near the carboxyl-terminal end: The M. smegmatis protein is 67 amino acids longer than the BCG protein and there is a stretch of 62 amino acids in which the two proteins share no homology.;Azide resistant mutants of M. smegmatis were sought as a method of directly isolating alleles of secA. One class of azide resistant mutants were not complemented by the wild-type M. smegmatis or BCG secA genes, while a second class was complemented. These results suggest that azide resistance in mycobacteria maps to at least two distinct loci. A specific allele of BCG secA in multi-copy conferred azide resistance in wild-type M. smegmatis. A single base pair mutation, resulting in a change from the amino acid threonine 129 to isoleucine in one of the ATP-binding domains of the BCG secA gene, is responsible for the azide resistant phenotype.;A counter-selectable marker system was used to test the essentiality of the secA gene product in M. smegmatis. Selection for a double crossover event that would leave the disrupted copy in the chromosome could not be obtained. The secA gene in M. smegmatis may be part of an operon encoding other essential genes.;Lastly western analyses with antibodies generated against the BCG SecA protein specifically recognized the native BCG SecA protein of {dollar}\sim{dollar}102 kDa expressed from a multi-copy plasmid in M. smegmatis.
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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