Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Transposon site hybridization in Mycobacterium tuberculosis.

Microarray mapping of transposon insertions can be used to quantify the relative abundance of different transposon mutants within a complex pool after exposure to selective pressure. The transposon site hybridization (TraSH) method applies this strategy to the study of Mycobacterium tuberculosis and can be adapted to the study of other microorganisms. This chapter describes the methods used to mutagenize mycobacteria with transposons, extract genomic DNA, amplify genomic DNA adjacent to transposon ends using polymerase chain reaction and T7 transcription, and synthesize labeled cDNA. It also describes methods used to construct an appropriate microarray, hybridize labeled cDNA, and analyze the microarray data. Important considerations involved in the experimental design of the selective pressure, the design of the microarray, and the statistical analysis of collected data are discussed.

Scaling down: a PCR-based method to efficiently screen for desired knockouts in a high density Mycobacterium tuberculosis picked mutant library.

Transposon mutagenesis produces random mycobacterial mutants at high frequency. Because they are random, however, it is difficult to isolate mutations in particular target genes. Here we describe the use of an arrayed library of Mycobacterium tuberculosis together with a PCR screening strategy to rapidly identify strains with defined insertion mutations. This method is useful for many genetic applications.