No abstract

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.

The transcription termination factor Rho of Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure–function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain (G51V, G53V, and Y80C) and in the C-terminal ATP binding domain (Y274D, P279S, P279L, G324D, N340S, I382N) including the two important structural elements, the Q-loop (P279S, P279L) and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants (Y274D, G324D and N340S), which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step.

Aconitases (Acns), the iron-sulfur proteins, are considered ancient enzymes in the evolution of metabolic pathways (5). The iron-sulfur clusters of these proteins not only participate in electron transport during reversible isomerization of citrate and isocitrate in citric acid cycle (7) but also serve as iron and oxygen sensors of the cell (6, 23). The binary activities are exerted through the assembly and disassembly of iron and sulfur clusters (6, 23). The protein with an intact 4Fe-4S cluster functions as Acn, whereas the protein with 3Fe-4S is an RNAbinding translational regulator (21,29). The stability and functionality of Acns as translation regulators are affected by not only iron levels but also oxidative stress, which induces these iron-regulatory proteins (IRPs) to bind to iron-responsive elements (IREs) and maintain iron homeostasis (15). IRPs maintain iron homeostasis by posttranscriptionally binding to conserved RNA stem-loop structures or IREs present at either the 5Ј or 3Ј ends of untranslated regions (UTRs) of mRNA. Depending on whether the IRE is present on the 3Ј or 5Ј end, binding of IRPs to IREs either protects the mRNA from degradation or inhibits its translation (1, 24). IRE-like sequences are present in the UTRs of at least two enzymes of the citric acid cycle, Acn and succinate dehydrogenase in mammals, implying that IRPs play an important role in mediating iron regulation of mitochondrial energy production (20). IRPs are also activated by both hydrogen peroxide and iron-mediated oxidative stress (27). The reactivity of H 2 O 2 with iron (Fenton reaction) intimately connects oxidative stress and cellular iron metabolism (28). Thus, recruitment of IRPs constitutes a highly effective strategy employed by pathogens for survival.Acns and IRPs are related with respect to the conserved amino acid residues across the family. This became evident when active-site residues identified in the pig heart mitochondria Acn crystal structure were found to be conserved across mammalian IRPs (16). It is worth mentioning that most of the knowledge on IRP binding to the IRE and the regulatory consequences has been collected from eukaryotic systems where partitioning between cytosolic Acn (IRPs) and mitochondrial Acn exists (references 14, 19, 26, 30, 31 and the references therein). However, only a few Acns have been reported so far from prokaryotes. Based on primary structure similarity, all bacterial Acns, including the ␣-proteobacterial Acns, are categorized mainly either into the Acn group similar to eukaryotic IRP or cytosolic Acn (AcnA/IRP group) or into the Acn group found only in bacteria (AcnB) (4, 37). Several bacteria, such as Escherichia coli, have two isoforms of Acn, AcnA and AcnB, with different physiological properties and expression profiles (22,34,36), while prokaryotes like Bacillus or Xanthomonas (1, 33, 38) have only one Acn. Bacillus Acn has been reported to bind to IRE-like sequences and therefore displays IRP properties (1). The M. tuberculosis energy cycle has separate oxidative and ...