Effect of different classes of inhibitors on DNA gyrase from Mycobacterium smegmatis.

Quinolones, coumarins, cyclothialidines, CcdB and microcin B17 inhibit DNA gyrase. Information regarding these various inhibitors comes from studies performed with the enzyme from Escherichia coli, and subsequent analyses have also primarily been confined to this system. We have carried out a detailed analysis of the effect of various groups of inhibitors on Mycobacterium smegmatis gyrase and demonstrate differential susceptibility of the E. coli and M. smegmatis gyrases. Interestingly, M. smegmatis gyrase was refractory to the plasmid-borne proteinaceous inhibitors CcdB and microcin B17. Ciprofloxacin, a fluoroquinolone, showed a 10-fold reduction in efficacy against M. smegmatis compared with E. coli gyrase. We have also shown that etoposide, an antineoplastic drug, inhibits DNA gyrase activity by trapping the gyrase-DNA complex. DNA gyrases from both E. coli and M. smegmatis were susceptible to etoposide at comparable levels.

Alternate paradigm for intrinsic transcription termination in eubacteria.

Intrinsic transcription terminators are functionally defined as sites that bring about termination in vitro with purified RNA polymerase alone. Based on studies in Escherichia coli, intrinsic termination requires a palindromic stretch followed by a trail of T (or U) residues in the coding strand. We have developed a highly efficient algorithm to identify hairpin potential sequences in bacterial genomes in order to build a general model for intrinsic transcription termination. The algorithm was applied to analyze the Mycobacterium tuberculosis genome. We find that hairpin potential sequences are concentrated in the immediate downstream of stop codons. However, most of these structures either lack the U trail entirely or have a mixed A/U trail reflecting an evolutionarily relaxed requirement for the U trail in the mycobacterial genome. Predicted atypical structures were shown to work efficiently as terminators both inside the mycobacterial cell and in vitro with purified RNA polymerase. The results are discussed in light of the kinetic competition models for transcription termination. The algorithm identifies >90% of experimentally tested terminators in bacteria and is an invaluable tool in identifying transcription units in whole genomes.

Functional cooperation between topoisomerase I and single strand DNA-binding protein.

Protein-protein interactions play important role in cell biochemistry by favorably or adversely influencing major molecular events. In most documented cases, the interaction is direct between the partner molecules. Influence of activity in the absence of direct physical interaction between DNA transaction proteins is another important means of modulation. We show here that single strand binding protein stimulates DNA topoisomerase I activity without direct protein-protein interactions. The stimulation is specific to topoisomerase I, as DNA gyrase activity is unaffected by SSB. We propose that such cases of functional collaboration between DNA transaction proteins play important roles in vivo.

Axial distortion as a sensor of supercoil changes: a molecular model for the homeostatic regulation of DNA gyrase.

Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the -10 region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the -10 region in the gyrA promoter. On the basis of analysis of various mutants in the -10 region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA anmd gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling.

The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding.

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A(2)B(2) subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBDelta160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBDelta160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E. coli gyrase.

Optimum DNA curvature using a hybrid approach involving an artificial neural network and genetic algorithm.

In the present paper, a hybrid technique involving artificial neural network (ANN) and genetic algorithm (GA) has been proposed for performing modeling and optimization of complex biological systems. In this approach, first an ANN approximates (models) the nonlinear relationship(s) existing between its input and output example data sets. Next, the GA, which is a stochastic optimization technique, searches the input space of the ANN with a view to optimize the ANN output. The efficacy of this formalism has been tested by conducting a case study involving optimization of DNA curvature characterized in terms of the RL value. Using the ANN-GA methodology, a number of sequences possessing high RL values have been obtained and analyzed to verify the existence of features known to be responsible for the occurrence of curvature. A couple of sequences have also been tested experimentally. The experimental results validate qualitatively and also near-quantitatively, the solutions obtained using the hybrid formalism. The ANN-GA technique is a useful tool to obtain, ahead of experimentation, sequences that yield high RL values. The methodology is a general one and can be suitably employed for optimizing any other biological feature.

Regulation of DNA gyrase operon in Mycobacterium smegmatis: a distinct mechanism of relaxation stimulated transcription.

BACKGROUND: The topological state of DNA is a result of the diverse influences of various topoisomerases present in the cell. Amongst these, DNA gyrase is the only enzyme that is capable of supercoiling DNA. In all the eubacterial cells tested so far, DNA gyrase has proved to be essential for survival. We have earlier cloned gyr genes from Mycobacterium smegmatis. Unlike the situation in Escherichia coli, genes encoding the two subunits of gyrase are present as a contiguous stretch in the M. smegmatis genome. RESULTS: We have demonstrated that the two subunits are encoded by a single dicistronic message, with the transcriptional start site mapping 57 base pairs upstream of the putative translational start of the gyrB ORF. The gyr promoter is specific to M. smegmatis and does not function in E. coli. We have shown that the synthesis of DNA gyrase in M. smegmatis is induced by novobiocin-a known inhibitor of gyrase. Short fragments encompassing the promoter region, when cloned in a promoter selection vector, do not show any response to changes in supercoil levels. Larger fragments show a supercoil sensitive behaviour, as seen in the genomic context. CONCLUSIONS: The gene structure and the transcriptional organization of the gyr operon suggest an overall regulatory scheme that is unique to mycobacteria. In contrast to E. coli, promoter and regions in its vicinity are not sufficient to confer supercoil sensitivity. Promoter distal regions- 600 bp downstream of the promoter-appear to be necessary for relaxation-stimulated transcription in M. smegmatis.