The epidemiology of chronic colonization of airways with Pseudomonas aeruginosa was monitored in 44 patients with cystic fibrosis (CF) by DraI/SpeI macrorestriction analyses of 489 isolates. Sequential P. aeruginosa isolates (144) that had been collected from 32 CF patients over < or = 2.5 years were investigated, and 12 patients were followed for 8 years after onset of colonization. Forty-eight different genotypes were uncovered from 481 typeable isolates. Ten genotypes were found in > 1 unrelated CF patient. The 6 most frequent clones were identified in 58% of isolates. Ten of the 12 patients monitored for 8 years were harboring their initially acquired P. aeruginosa clone at all times, with subtle shifts of fragment patterns indicating subclonal variation. During colonization, the bacteria gradually lost pyocin and phage typing responses, supporting the view that genotypically discordant P. aeruginosa strains develop a common phenotype.

We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.

BackgroundThe cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes.MethodsPatients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed.ResultsVariants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01

No abstract