OBJECTIVES Pentapeptide repeat proteins (PRPs) QnrA, QnrB and QnrS confer reduced susceptibility to quinolones. This study presents an in vitro analysis of the genetic evolution of quinolone resistance mediated by changes in the coding sequences and promoter regions of qnrA1, qnrS1 and qnrB1 genes. METHODS A random mutagenesis technique was used to predict the evolutionary potential of these ...

Biologists use random transposon mutagenesis to construct knockout libraries for bacteria. Random mutagenesis offers cost and efficiency benefits over the standard site directed mutagenesis,[8] but one can no longer ensure that all the nonessential genes will appear in the library. In random libraries for haploid organisms, there is always a class of genes for which knockout clones have not bee...

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons...

Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and man...

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophil...

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be...

We thank John Connolly for helpful discussions and critical reading of the manuscript. We are very grateful to Fabrice Chareyre, Laetitia Gressin, Catherine Giudicelli, Jean Christophe Beaudoin and Isabelle Legall for help in the BAC screening, sequencing and genotyping. This work was supported by a grant from the Association pour la Recherche sur le Cancer (ARC) and the Ministère de l’Educatio...

A new analytical mutagenesis technique is described that involves randomizing the DNA sequence of a short stretch of a gene (3-6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structur...

Procedures to introduce point mutations into specific DNA fragments are important tools to study gene function (2,10). UV light and chemical mutagens have been used in vivo to increase the frequency of random mutagenesis for specific DNA targets carried on plasmid or phage vectors being propagated in growing cells (2,11). Both methods induced mutations in the target DNA as well as in vector and...

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good sta...