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

<i>pslA</i> transcription and translation in over-expression strains.

<p>(A) Fold change in the <i>pslA</i> transcriptional response to gene over-expression. (B) Fold change in the <i>pslA</i> translational response to gene over-expression. β-galactosidase activity was measured at both exponential (black bars) and stationary phase (white bars). Bars represent fold change in <i>pslA</i> transcription or translation compared to the empty vector control strain, PAO1 pJN105 (dashed line). While fold change in <i>pslA</i> transcription or translation is reported for clarity, statistics were performed on normalized luminesence values of empty vector control and over-expression strains assayed on the same day (see Materials and Methods). At least 3 biological replicates were performed in duplicate. Error bars represent standard error of the mean, n=3. *, <i>P</i> value < 0.05 by Student’s <i>t</i> test compared to the empty vector control strain.</p

Psl production in clinical isolates from 4 patients with CF.

<p>Strains were grown in SCFM for ~16 hours and Psl was extracted from whole cultures. Psl extracts were normalized to equal protein concentrations for each patient and subjected to immunoblot blot analyses as described in the Materials and Methods. Each isolate is centered on its corresponding number of <i>in </i><i>vivo</i> generations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082621#pone.0082621.s006" target="_blank">Table S3</a>). Isolates at time 0 are the ancestor. At least 3 biological replicates were performed, and red boxes indicate isolates that produced more Psl on average than the ancestor. SCV: small colony variant.</p

Biofilm formation and Psl production in over-expression strains.

<p>(A) Fold change in biofilm formation of wild-type PAO1 over-expression strains. Bars represent the average fold change in biofilm formation compared to the empty vector control strain, PAO1 pJN105 (dashed line). Δ<i>pslA</i> pJN105, a strain that cannot produce Psl, served as a negative control. While fold change in biofilm formation is reported for clarity, statistics were performed on absorbance values for empty vector control and over-expression strains assayed on the same day. At least 3 biological replicates were performed in octuplicate. *, <i>P</i> value < 0.001 by Student’s <i>t</i> test. Error bars represent standard error of the mean, n=3. (B) Fold change in biofilm formation of Δ<i>pslA</i> over-expression strains. Bars represent the average fold change in biofilm formation compared to the empty vector control strain, Δ<i>pslA</i> pJN105 (dashed line). Statistics and error bars are reported as in A. (C) Psl production in PAO1 pJN105 (top) and PAO1 over-expression strains (bottom). The top panels represent Psl production in the wild-type empty vector control strain, PAO1 pJN105, while the bottom panels represent Psl production in the over-expression strains, or as in the last bottom panel, the negative control strain Δ<i>pslA</i> pJN105. At least 3 biological replicates were performed.</p

Protein abundances are more conserved than mRNA abundances across diverse taxa

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression datasets from seven different species: two bacteria, yeast, nematode, fly, human, and plant. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge

Protein abundances are more conserved than mRNA abundances across diverse taxa.

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge

Protein abundances are more conserved than mRNA abundances across diverse taxa

Proteins play major roles in most biological processes as a consequence protein expression levels are highly regulated While extensive post transcriptional translational and protein degradation control clearly influence protein concentration and functionality it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs Hence surprisingly a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances We tested if this phenomenon is general by collecting and testing matching large scale protein and mRNA expression data sets from seven different species two bacteria yeast nematode fly human and rice We find that steady state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p = 0 0008 paired Wilcoxon) These data support the presence of strong selective pressure to maintain protein abundances during evolution even when mRNA abundances divergclose44404

Protein abundances are more conserved than mRNA abundances across diverse taxa.

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge