Escherichia coli Frameshift Mutation Rate Depends on the Chromosomal Context but Not on the GATC Content Near the Mutation Site

Different studies have suggested that mutation rate varies at different positions in the genome. In this work we analyzed if the chromosomal context and/or the presence of GATC sites can affect the frameshift mutation rate in the Escherichia coli genome. We show that in a mismatch repair deficient background, a condition where the mutation rate reflects the fidelity of the DNA polymerization process, the frameshift mutation rate could vary up to four times among different chromosomal contexts. Furthermore, the mismatch repair efficiency could vary up to eight times when compared at different chromosomal locations, indicating that detection and/or repair of frameshift events also depends on the chromosomal context. Also, GATC sequences have been proved to be essential for the correct functioning of the E. coli mismatch repair system. Using bacteriophage heteroduplexes molecules it has been shown that GATC influence the mismatch repair efficiency in a distance- and number-dependent manner, being almost nonfunctional when GATC sequences are located at 1 kb or more from the mutation site. Interestingly, we found that in E. coli genomic DNA the mismatch repair system can efficiently function even if the nearest GATC sequence is located more than 2 kb away from the mutation site. The results presented in this work show that even though frameshift mutations can be efficiently generated and/or repaired anywhere in the genome, these processes can be modulated by the chromosomal context that surrounds the mutation site

Mucoidy, Quorum Sensing, Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung

Sistema para la cría de larvas de Haematobia irritans (Diptera: Muscidae) a partir de ejemplares salvajes de Argentina

Se describe un sistema simple para sostener el desarrollo larval de moscas de los cuernos salvajes, Haematobia irritans (L.) en condiciones controla- das de laboratorio. Dicho sistema consiste en un medio de cultivo y una cámara de cría. El medio de cultivo se basa en una suspensión bacteriana obtenida a partir de materia fecal bovina. Además, se establecen las condiciones óptimas para la extracción y conservación de la suspensión bacteriana. Dada una concentración óptima de suspensión bacteriana que permite el desarrollo de un número estable- cido de larvas, se determinó que la duración de la etapa larval fue de 96 ± 4 h. El porcentaje de larvas vivas a las 48 h fue del 86,5%, mientras que el 68,5% (96 h) logró iniciar la metamorfosis y alcanzar el estado adulto. Utilizando la materia fecal bovina (MFB) fresca como medio de cultivo, se obtuvieron resultados comparables. Además de su fácil preparación, prolongado almacenamiento y alta reproducibili- dad, la utilización de la suspensión bacteriana como medio de cultivo permite la inspección visual permanente de las larvas y puede ser empleada para realizar bioensayos sobre larvas de mosca de los cuernos con compuestos insecticidas solubles. Por otra parte, en este trabajo se describen las características y paráme- tros de funcionamiento de una cámara de cría simple y económica, que permite la incubación de los medios de cultivo a una temperatura y humedad controlada

nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa.

nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved

Mutation rates to ciprofloxacin resistance^a.

a<p>Mutation rates (<i>µ</i>) per replication and 95% confidence limits (CL) were calculated as described in Material and Methods. Mutation rates for the WT and mutator (<i>mutS</i>, <i>mutT</i>, <i>mutY</i> and <i>mutM</i>) strains were calculated at 1.0 µg/ml Cip. Assays with 2-aminopurine (2AP), cisplatin (CPT) and hydrogen peroxide (HP) were performed at 0.5 µg/ml Cip. ^bMutation rates calculated for the WT strain at each set of experiment (mutator strain and mutagen assessment) were used for data normalization.</p

Cm^R emergence rates.

<p>Cm^R emergence rates of wild-type derivative strains relative to Wt-X1 strain (blue bars) and of <i>mutS</i> derivative strains relative to <i>mutS</i>-X1 strain (red bars). The mutation rate value of Wt-X1 strain was 5.38 (95% CI, 4.93–5.83)×10⁻⁸ and that of <i>mutS</i>-X1 strain was 8.14 (95% CI, 7.74–8.51)×10⁻⁶. Error bars indicate the 95% confidence limits on the mutation rates.</p