In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients.

Mutational analysis of the RecJ exonuclease of Escherichia coli: identification of phosphoesterase motifs.

The recJ gene, identified in Escherichia coli, encodes a Mg(+2)-dependent 5'-to-3' exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including Bacillus and Helicobacter, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of E. coli RecJ. These motifs may define interactions with Mg(2+) ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of Mycoplasma, a domain of putative polyA polymerases in Synechocystis and Aquifex, PRUNE of Drosophila, and an exopolyphosphatase (PPX1) of Saccharomyces cereviseae. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.

Suppression of recJ exonuclease mutants of Escherichia coli by alterations in DNA helicases II (uvrD) and IV (helD).

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, "uvrD517am," at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517am mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517am as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517am derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor "srjD" is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism.

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 x 10(-5) per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.

A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli.

In the genomes of many organisms, deletions arise between tandemly repeated DNA sequences of lengths ranging from several kilobases to only a few nucleotides. Using a plasmid-based assay for deletion of a 787-bp tandem repeat, we have found that a recA-independent mechanism contributes substantially to the deletion process of even this large region of homology. No Escherichia coli recombination gene tested, including recA, had greater than a fivefold effect on deletion rates. The recA-independence of deletion formation is also observed with constructions present on the chromosome. RecA promotes synapsis and transfer of homologous DNA strands in vitro and is indispensable for intermolecular recombination events in vivo measured after conjugation. Because deletion formation in E. coli shows little or no dependence on recA, it has been assumed that homologous recombination contributes little to the deletion process. However, we have found recA-independent deletion products suggestive of reciprocal crossovers when branch migration in the cell is inhibited by a ruvA mutation. We propose a model for recA-independent crossovers between replicating sister strands, which can also explain deletion or amplification of repeated sequences. We suggest that this process may be initiated as post-replicational DNA repair; subsequent strand misalignment at repeated sequences leads to genetic rearrangements.