Mutations in the <i>mucA</i> gene of mucoid isolates from strains MPA, MPA-T1, MPA-T2 and MPA-T3.

a<p>ΔG at 426 corresponds to a −1 bp deletion within G⁵SSR₄₂₆; ΔC at 362 corresponds to a −1 bp deletion within a mononucleotide SSR of four Cs from 362 to 365 (C⁴SSR₃₆₂); ΔC at 431 corresponds to a −1 bp deletion within C⁶SSR₄₃₁. “None” refers to conversion to mucoidy occurring in the absence of <i>mucA</i> mutations. The nature of these non-<i>mucA</i> alterations leading to a mucoid phenotype was not investigated in this work.</p>b<p>Stop codon produced at the site of the mutation by substitutions or placed in frame by frameshift mutations.</p

Site directed mutagenesis on the <i>mucA</i> gene.

<p>A fragment of the <i>P. aeruginosa mucA</i> gene where the site directed mutagenesis was performed is shown. Base changes (bold face) were designed in order to maintain the amino acidic sequence unaltered (shown below), and codons were chosen that are commonly used by <i>P. aeruginosa</i>. The premature stop codon generated by a hypothetical −1 bp deletion between positions 426 and 436 of <i>mucA</i> is underlined. Wild type <i>mucA</i> allele from MPA strain with G⁵SSR₄₂₆ is highlighted in yellow. The <i>mucAT1</i> allele from strain MPA-T1 was generated by replacing G-to-A at 426 and G-to-T at 429, thus eliminating G⁵SSR₄₂₆. The <i>mucAT2</i> allele from strain MPA-T2 was generated by replacing G-to-A at 426 and G-to-T at 429 (eliminating G⁵SSR₄₂₆), and G-to-C at 432 and 435 to generate C⁶SSR₄₃₁ (highlighted in blue). The <i>mucAT3</i> allele from strain MPA-T3 was generated by replacing G-to-C at 432 and 435 to generate C⁶SSR₄₃₁ (highlighted in blue), and maintaining G⁵SSR₄₂₆ (highlighted in yellow) unaltered.</p

Classification of <i>mucA</i> mutations reported in different studies for <i>P. aeruginosa</i> CF isolates.

a<p>For each study, only the isolates for which the <i>mucA</i> mutations were reported were considered in the analysis.</p>b<p>Insertion or deletion mutations different from −1 bp deletions.</p

Bacterial strains, plasmids and oligonucleotides used in this study.

a<p>Resistance markers: Tc, tetracycline; Rf, rifampin; Sm, streptomycin; Ap, ampicillin; Suc, sucrose.</p

Role of SSRs in <i>P. aeruginosa</i> mucoid conversion.

<p>(A) Mucoid variants, visualized as mucous droplet-like colonies emerging from MPA, MPA-T1, MPA-T2 and MPA-T3 strains plated onto MMA plates. (B) Relative emergence of mucoid variants obtained from MPA-T1, MPA-T2 and MPA-T3 strains. The values were compared to those obtained for the MPA strain. *Values for MPA-T1 and MPA-T3 were significantly different (p<0.05, as determined by the t-test) from that of MPA. (C) Spectrum of <i>mucA</i> mutations observed in mucoid variants obtained from MPA, MPA-T1, MPA-T2 and MPA-T3 strains. Bars indicate the observed percentage for each kind of mutation of the total number of mucoid clones analyzed in each strain (total clones analyzed: MPA, 11; MPA-T1, 13; MPA-T2, 13; and MPA-T3, 15). Bars in yellow indicate those mucoid variants that occurred in the absence of <i>mucA</i> alterations. The observed mutations included base substitutions (gray) and −1 bp deletions in mononucleotide G:C SSRs (blue).</p

Mononucleotide SSRs in the <i>P. aeruginosa</i> PAO1 genome.

<p>The plots show the counts for mononucleotide SSRs (red circles) in the whole <i>P. aeruginosa</i> PAO1 genome and in random sequences generated by various predictive models (black symbols). b and m1: homogeneous models (Bernoulli and first-order Markov); b-b, b-bp, m1-m1, m1-m1p, m1-c and m1-c1: heterogeneous models (see Methods). Counts are shown of mononucleotide G:C and A:T SSRs in the coding and non-coding regions of the genome.</p

Genome-wide effect of MRS-deficient hypermutability on the mutagenesis of mononucleotide SSRs.

<p>The bars show the fold increase in the mutations per year that occurred in mononucleotide G:C SSRs and A:T SSRs, and in other types of mutation after <i>P. aeruginosa</i> PACS2 became mutator<b>.</b></p

Involvement of mononucleotide SSRs in genes involved in <i>P. aeruginosa</i> adaptation during CF chronic infection.

<p>The bar graphs show (i) the percentages of the 60 genes mutated during CF lung chronic infection as reported by Smith <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080514#pone.0080514-Smith1" target="_blank">[1]</a> that harbor mononucleotide G:C SSRs (A) and A:T SSRs (B), relative to the length of the SSR (black bars); (ii) the percentages of those genes carrying mononucleotide SSRs that were mutated during the process of chronic infection, relative to the length of the SSR (gray bars).</p

Average total catabolic function of isolates from CFA and CFD lineages.

<p>Total catabolic function was calculated relative to CFA_2004/01 and CFD_1991/01 as a weighted average across all substrates for each CFA (A) and CFD (B) isolate. Total catabolic function was defined as 1 for the reference levels (CFA_2004/01 and CFD_1991/01). Lower values indicate decay. Isolates CFA_2010/01, CFA_2010/11, and CFA_2010/31 were excluded from the analysis because significant dispersion was observed in the duplicates.</p

Minimum spanning trees (MSTs) of genomes among CFA and CFD lineages.

<p>MSTs for CFA (A) and CFD (B) were constructed based on the total number of genes altered by nonsynonymous SNPs and indel mutations in the respective genomes. Links between nodes represents the minimum distance in terms of mutated genes. Numbers above each link indicate the total amount of mutated genes between the two connected nodes (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004651#pgen.1004651.s008" target="_blank">Table S6</a>). For tree construction, ancestors CFA_2004/01 and CFD_1991/01 were considered as origins.</p