Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung

The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.open6

Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human respiratory samples and the rhizosphere

A

Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions-4

of possible homologues of the genes. The organisation of the locus is shown in the top left panel followed by the three sets of putative homologues; two of which are on chromosome two and one on chromosome three. The grey bars indicate the reading frame with the top being +/- 1, middle +/- 2 and bottom +/- 3; the arrows indicate if the genes are on the coding (left to right) or complement (right to left) strands. The percentage identity of the product of each putative gene to their HcnABChomologue is shown in labels underneath each panel. The Putative Bcc cyanide synthase proteins have conserved Fe-S (HcnA) and ADP binding motifs (HcnB and HcnC). ., . and . ClustalX alignments of putative J2315 cyanide synthase protein sequences with hydrogen cyanide synthases from (PAO1) and (PF-5). . Sequence of HcnA C-terminal from PAO1 and PF-5 aligned with putative proteins, BCAM01268a, BCAM0669 and BCAS0164. Diamonds indicate a potential Fe-S binding site at 4 cysteine residues in HcnA. . Sequence of the start of HcnB from PAO1 and PF-5 aligned with putative proteins BCAM01269, BCAM0668 and BCAS0163. . Sequence from the start of HcnC sequence from PAO1 and PF-5 aligned with putative proteins BCAM01268, BCAM0667 and BCAS0165. Arrows indicate eleven amino acid ADP-binding motif in HcnB and HcnC.<p><b>Copyright information:</b></p><p>Taken from "Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions"</p><p>http://www.biomedcentral.com/1471-2180/8/108</p><p>BMC Microbiology 2008;8():108-108.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2504479.</p><p></p

Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions-3

Rapped in 1 ml 4 M NaOH. ., . and . cyanide concentration trapped in NaOH for PAO1, J2315 and K56-2 respectively. Solid bars: with glass beads, diagonal striped bars: without glass beads. Measurements are averages of 3 independent replicates with SE error bars.<p><b>Copyright information:</b></p><p>Taken from "Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions"</p><p>http://www.biomedcentral.com/1471-2180/8/108</p><p>BMC Microbiology 2008;8():108-108.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2504479.</p><p></p

Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions-0

of possible homologues of the genes. The organisation of the locus is shown in the top left panel followed by the three sets of putative homologues; two of which are on chromosome two and one on chromosome three. The grey bars indicate the reading frame with the top being +/- 1, middle +/- 2 and bottom +/- 3; the arrows indicate if the genes are on the coding (left to right) or complement (right to left) strands. The percentage identity of the product of each putative gene to their HcnABChomologue is shown in labels underneath each panel. The Putative Bcc cyanide synthase proteins have conserved Fe-S (HcnA) and ADP binding motifs (HcnB and HcnC). ., . and . ClustalX alignments of putative J2315 cyanide synthase protein sequences with hydrogen cyanide synthases from (PAO1) and (PF-5). . Sequence of HcnA C-terminal from PAO1 and PF-5 aligned with putative proteins, BCAM01268a, BCAM0669 and BCAS0164. Diamonds indicate a potential Fe-S binding site at 4 cysteine residues in HcnA. . Sequence of the start of HcnB from PAO1 and PF-5 aligned with putative proteins BCAM01269, BCAM0668 and BCAS0163. . Sequence from the start of HcnC sequence from PAO1 and PF-5 aligned with putative proteins BCAM01268, BCAM0667 and BCAS0165. Arrows indicate eleven amino acid ADP-binding motif in HcnB and HcnC.<p><b>Copyright information:</b></p><p>Taken from "Bacteria of the complex are cyanogenic under biofilm and colonial growth conditions"</p><p>http://www.biomedcentral.com/1471-2180/8/108</p><p>BMC Microbiology 2008;8():108-108.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2504479.</p><p></p