Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones.

As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site

Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy

After 30 Years of Study, the Bacterial SOS Response Still Surprises Us

The bacterial SOS response kicks in when bacteria experience DNA damage, and helps the organisms correct and survive DNA damage events. This primer provides a foundation for understanding these events

The transcription elongation factor NusA is required for stressinduced mutagenesis in Escherichia coli

Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac+ revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac+ revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis.National Institutes of Health (U.S.) (Grant CA21615)National Institute of Environmental Health Sciences (Grant P30 ES002109

Key Role of Mfd in the Development of Fluoroquinolone Resistance in Campylobacter jejuni

Campylobacter jejuni is a major food-borne pathogen and a common causative agent of human enterocolitis. Fluoroquinolones are a key class of antibiotics prescribed for clinical treatment of enteric infections including campylobacteriosis, but fluoroquinolone-resistant Campylobacter readily emerges under the antibiotic selection pressure. To understand the mechanisms involved in the development of fluoroquinolone-resistant Campylobacter, we compared the gene expression profiles of C. jejuni in the presence and absence of ciprofloxacin using DNA microarray. Our analysis revealed that multiple genes showed significant changes in expression in the presence of a suprainhibitory concentration of ciprofloxacin. Most importantly, ciprofloxacin induced the expression of mfd, which encodes a transcription-repair coupling factor involved in strand-specific DNA repair. Mutation of the mfd gene resulted in an approximately 100-fold reduction in the rate of spontaneous mutation to ciprofloxacin resistance, while overexpression of mfd elevated the mutation frequency. In addition, loss of mfd in C. jejuni significantly reduced the development of fluoroquinolone-resistant Campylobacter in culture media or chickens treated with fluoroquinolones. These findings indicate that Mfd is important for the development of fluoroquinolone resistance in Campylobacter, reveal a previously unrecognized function of Mfd in promoting mutation frequencies, and identify a potential molecular target for reducing the emergence of fluoroquinolone-resistant Campylobacter

The σE stress response is required for stress-induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve

DNA Damage Triggers Genetic Exchange in Helicobacter pylori

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations

Mutability and Importance of a Hypermutable Cell Subpopulation that Produces Stress-Induced Mutants in Escherichia coli

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac+ mutants, with and without evidence of descent from the HMS, have similar Lac+ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac+ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones