Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance

The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination. A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination. Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription.

Section: Phage Techniquesmentioning

confidence: 99%

The alpha subunit of RNA polymerase and transcription antitermination

Schauer

Cheng

Zheng

et al. 1996

“…In addition, gyrase is thought to intervene in the correction of topological perturbations associated with transcription (Liu and Wang, 1987;Figueroa and Bossi, 1988), and in the unlinking of parental strands during DNA replication (Baker et al, 1986;Hiasa et al, 1994). Inhibiting gyrase by mutation or by drugs leads to a panoply of functional defects in site-specific recombination (Friedman et al, 1984;Dove and Dorman, 1994), transcriptional regulation (reviewed in Menzel and Gellert, 1994), and DNA replication (Fairweather et al, 1980;Filutowicz and Jonczyk, 1981;Mirkin and Shmerling, 1982;Orr and Staudenbauer, 1981;Snyder and Drlica, 1979).…”

Section: Introductionmentioning

confidence: 99%

**A class of gyrase mutants of Salmonella typhimurium show quinolone‐like lethality and require Rec functions for viability**

Garí

Figueroa‐Bossi

Blanc‐Potard

et al. 1996

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37 degrees C. Selection of suppressors that concomitantly restore growth at 37 degrees C and SOS induction in a recB- background yielded mutations that relleve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone-treated gyr+ recB- strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37 degrees C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of "cleavable' gyrase-DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA-cells at restrictive temperatures. Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.

“…The conclusion that these conditions should block supercoiling of Mu DNA during infection is supported by the observation that supercoiling of infecting Mu DNA was severely diminished under gyrase inhibition by the drugs nalidixic acid and novobiocin (Puspurs et al, 1983). Likewise, infecting lambda phage DNA is not supercoiled after infection into a gyrB-himts host (Friedman et al, 1984), and DNA circles generated by site-specific recombination after norfloxacin treatment fail to become supercoiled, as expected if gyrase is completely inhibited by this treatment (Bliska and Cozzarelli, 1987). Therefore, even if a small amount of the Mu DNA were to become supercoiled under our conditions, a phage-encoded protein would have to be responsible.…”

Section: Gyrase and Integrative Transpositionmentioning

confidence: 84%

DNA gyrase requirements distinguish the alternate pathways of Mu transposition

Sokolsky

Baker

2003

SummaryThe MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA/ architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.