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. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of ß-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.

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. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an anti-mycobacterial target.

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). Although 6S RNA and (p)ppGpp were known in literature for a long time, their role in transcriptional switching has been understood only in recent years. With the elucidation of function of DksA, a new dimension has been added to the phenomenon of stringent response. As the final outcome of actions of (p)ppGpp, DksA, 6S RNA and Rsd is similar, there is a need to analyse these mechanisms in a collective manner. We review the recent trends in understanding the regulation of sigma(70) by (p)ppGpp, DksA, Rsd and 6S RNA and present a case for evolving a unified model of RNAP redistribution during starvation by modulation of sigma(70) activity in E. coli.

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. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to sigma(70) with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

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. Binding of AsiA to sigma(70) in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of sigma(70) binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of sigma(70) has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4.2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of sigma(70) showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in sigma(70) fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only sigma(70) fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to sigma(70), in vivo, requires the presence of both sub-domains of region 4 of sigma(70).

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. These mutants were tested for their ability to bind to sigma(70) in vivo in E. coli, monitored as a relative toxicity assay, co-purification of sigma(70), inhibition of [3H-uridine] incorporation and also in the yeast two hybrid system. A good correlation was found between the loss of toxicity of AsiA to E. coli cells and the inability of mutant AsiAs to bind to sigma(70) It was observed that deletion of C-terminal 17 amino acid residues of AsiA did not affect the activity whereas a mutant asiA lacking C-terminal 28 amino acid residues had the toxicity reduced to a large extent, suggesting that amino acid residues between 64 and 73 played a role in binding to AsiA. A mutant with a deletion of 34 amino acids in the C-terminus did not show any toxicity to E. coli cells. In the N-terminal region, deletion of five amino acid residues was tolerated but extending the deletion to ten amino acids abolished the AsiA activity completely. The conversion of glutamic acid (E10) to either leucine, serine, glutamine, tyrosine or alanine did not affect the toxicity to a great extent suggesting that a negative charge at E10 is not critical for interaction with sigma(70). The results of our in vivo studies suggest that the primary sigma(70) binding site of AsiA is in N-terminus, but, it requires the presence of C-terminal 64-73 amino acid residues for effective binding in vivo.