An IPTG Inducible Conditional Expression System for Mycobacteria.

Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout.

Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target.

DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms.

Transcriptional switching in Escherichia coli during stress and starvation by modulation of sigma activity.

During active growth of Escherichia coli, majority of the transcriptional activity is carried out by the housekeeping sigma factor (sigma(70)), whose association with core RNAP is generally favoured because of its higher intracellular level and higher affinity to core RNAP. In order to facilitate transcription by alternative sigma factors during nutrient starvation, the bacterial cell uses multiple strategies by which the transcriptional ability of sigma(70) is diminished in a reversible manner. The facilitators of shifting the balance in favour of alternative sigma factors happen to be as diverse as a small molecule (p)ppGpp (represents ppGpp or pppGpp), proteins (DksA, Rsd) and a species of RNA (6S RNA).

Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA to E. coli RNA polymerase lead to diverse physiological consequences.

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different.

Both regions 4.1 and 4.2 of E. coli sigma(70) are together required for binding to bacteriophage T4 AsiA in vivo.

The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing -10 and -35 consensus sequence by binding to sigma(70) of E. coli.

Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to sigma(70) of Escherichia coli in vivo.

The T4 AsiA is an anti-sigma factor encoded by an early gene of bacteriophage T4. AsiA has been shown to inhibit T4 early promoters in vitro and expression of this protein from a plasmid causes transcriptional shut off in the host cells leading to cell death. By reasoning that mutant AsiA expression in Escherichia coli will not inhibit the host transcription and hence lead to healthy colony formation, a strategy was developed wherein inactive or partially active mutants of AsiA could be isolated.