1887

Abstract

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the genome contains four genes (, , and ) coding for the large catalytic subunit of AHAS, whereas only one gene () 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 and , we inactivated the gene of . 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 or C carbon sources, suggesting that the gene product is the major AHAS in . Depletion of these branched chain amino acids in the medium led to loss of viability of the Δ strain , 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 Δ strain in mice indicated that although this mutant strain showed defective growth , it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

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2009-09-01
2019-12-15
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Genomic organization of genes coding for large and small subunits of AHAS in , , , and . [PDF](15 kb) Amino acid alignments of full-length AHAS large subunits, regions involved in binding to TPP and FAD, and active site residues of and . Percentage similarity and identity amongst various regions of and AHAS large subunits is shown in tables adjacent to the amino acid alignments. [PDF](84 kb) Analysis of SCO recombinants of by PCR. [PDF](45 kb)

PDF

Genomic organization of genes coding for large and small subunits of AHAS in , , , and . [PDF](15 kb) Amino acid alignments of full-length AHAS large subunits, regions involved in binding to TPP and FAD, and active site residues of and . Percentage similarity and identity amongst various regions of and AHAS large subunits is shown in tables adjacent to the amino acid alignments. [PDF](84 kb) Analysis of SCO recombinants of by PCR. [PDF](45 kb)

PDF

Genomic organization of genes coding for large and small subunits of AHAS in , , , and . [PDF](15 kb) Amino acid alignments of full-length AHAS large subunits, regions involved in binding to TPP and FAD, and active site residues of and . Percentage similarity and identity amongst various regions of and AHAS large subunits is shown in tables adjacent to the amino acid alignments. [PDF](84 kb) Analysis of SCO recombinants of by PCR. [PDF](45 kb)

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