Search

Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a Δ phoQ mutant, as did individual or tandem deletion of cprRS . Individual deletion of colR or colS in a Δ phoQ mutant also suppressed 4-amino- l -arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the Δ phoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino- l -arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa . Episomal expression of colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the Δ phoQ mutant, while episomal expression of colR , colS , or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS ; when expressed in a Δ phoQ background, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa , indicate that addition of 4-amino- l -arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance.

The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa . The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa . There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R′, previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY , a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is of increasing importance as an opportunistic human pathogen. P. aeruginosa infections are common in patients with compromised antibacterial defenses. A particular niche for infections that has been created by advances in pediatric care is the lungs of cystic fibrosis (CF) patients. As effective management of other clinical complications has been introduced and life expectancy has increased, chronic pulmonary infections with P. aeruginosa have emerged as the leading cause of morbidity and mortality in CF (11, 17). Despite steady improvements in treatment, CF pulmonary infections resist eradication with antibiotics and lead, over a period of many years, to irreversible tissue destruction (11). Available data suggest that most CF patients are infected by unique strains of P. aeruginosa acquired from environmental reservoirs (15, 30, 31) and that the infections are frequently clonal (5, 29, 36, 39). During the time that a particular strain is resident in the airways of a CF patient, considerable genetic adaptation occurs. A well-known example of this process is the conversion of strains from nonmucoid to mucoid phenotypes due to mutations in mucA (11, 21, 22), a gene whose product is a negative regulator of the biosynthesis of the secreted polysaccharide alginate. Another example is the frequent loss of O antigen (8, 14, 18, 28, 29), a change that also appears to be due to mutation. Restriction mapping of P. aeruginosa strains from diverse backgrounds indicates that a significant proportion of the variation among isolates is due to insertions and deletions of up to 500 kbp of genomic material (30, 34). In this respect, P. aeruginosa is superficially similar to Escherichia coli. Comparison of the pathogenic O157 and the nonpathogenic K-12 E. coli strains has revealed that much of their difference can be attributed to blocks of DNA that are strain specific (26). These so-called K islands and O islands comprise 12% of the genome of K-12 and 26% of the genome of O157, respectively, and are interspersed in a conserved backbone sequence with relatively little polymorphism (26). In P. aeruginosa, few strain-specific regions have been well characterized. One example is the 50-kbp P. aeruginosa genomic island 1, PAGI-1, found in many clinical isolates of P. aeruginosa in place of the 7-kbp of PAO1 sequence and hypothesized to play a role in evading the host immune response (20). Other examples include a 20-kbp island found in P. aeruginosa strain PAK, containing genes involved in glycosylation of a-type flagellin (2), and the recently described 11 groups of gene clusters at the O-antigen biosynthetic locus (27). However, despite the evidence indicating that genomic islands may play an important role in P. aeruginosa biology, little is known about the sequence and location of other islands. Sequence-based studies of genetic variation in P. aeruginosa appear to support the presence of conserved backbone sequences, similar to the E. coli model. Sequencing of six housekeeping genes in 19 environmental and clinical isolates revealed levels of genetic diversity even lower than in the E. coli comparisons, with average pairwise nucleotide substitution rates on the order of a few tenths of a percentage point (15). These data, along with those from restriction-mapping experiments, suggest that P. aeruginosa genomes have numerous strain-specific regions interspersed in a well-conserved backbone. More detailed studies of DNA sequence variation in P. aeruginosa are needed to define these backbone and strain-specific sequences, which may promote our understanding of the genetic determinants of pathogenicity in CF lung infections. The recent sequencing of the genome of the standard laboratory strain of P. aeruginosa, PAO1 (37), has laid the groundwork for more extensive studies of genomic variation in clinical and environmental isolates of this bacterium. We have carried out whole-genome-sample sequencing of two P. aeruginosa strains isolated from late-stage CF-related infections and a strain from an environmental source. These data provide a detailed view of the pattern of sequence variation among the three new strains relative to the PAO1 reference.

The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium .

Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.