Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase-peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG-defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase-peroxidase which reduce catalase activity also decrease peroxidase activity.

Transcription of downstream genes in the early operons of phage A requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of A N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.The positive control of genes that facilitate the bimodal development of A and related phages in Escherichia coli depends on two distinct operon-specific antiterminators (1). The N antiterminator activates the early operons, whereas the Q antiterminator activates the late operon. Both proteins function by a common mechanism: they capture RNA polymerase (RNAP) during early phases of transcription and mask RNAP's response to the downstream terminators (2-8). However, each antiterminator recognizes the respective genetic signal and captures RNAP by distinct mechanisms. The signals for Q action span the late promoter and the early transcribed region. Q binds to a DNA sequence within the late promoter and acts upon RNAP paused at a defined site (9). Specific nucleotides in the nontemplate strand of this region interact with RNAP not only to induce pausing but also to endow upon RNAP the conformation that is essential for engagement by Q (10). In contrast, the nut site, required for N action, functions in the form of . It can facilitate the productive interaction between N and RNAP at remote sites, suggesting that nut RNA may act similarly to DNA enhancers, binding N and delivering N to RNAP through RNA looping (11). Finally, while a single host factor (NusA) appears to be sufficient for Q activity, processive antitermination by N demands three additional factors: NusB, S10 ribosomal protein (NusE), and NusG (2, 14-16).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §173...

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Delafloxacin demonstrates excellent antibacterial potency and exhibits a low probability for the selection of resistant mutants in MRSA. Although mutants can be selected at low frequencies in vitro from quinolone-resistant isolates, delafloxacin MICs and MPCs remain low and a fitness cost can be observed. Consequently delafloxacin warrants further investigation for the potential treatment of drug-resistant MRSA infections.