Elucidating the <i>Pseudomonas aeruginosa</i> Fatty Acid Degradation Pathway: Identification of Additional Fatty Acyl-CoA Synthetase Homologues

<div><p>The fatty acid (FA) degradation pathway of <i>Pseudomonas aeruginosa</i>, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that <i>P. aeruginosa</i> has more than two fatty acyl-CoA synthetase genes (<i>fadD</i>; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the <i>Escherichia coli</i> FadD in the present study. Four new homologues of <i>fadD</i> (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the <i>E. coli fadD</i> mutant on FA-containing media. Growth phenotypes of 17 combinatorial <i>fadD</i> mutants on different FAs, as sole carbon sources, indicated that the four new <i>fadD</i> homologues are involved in FA degradation, bringing the total number of <i>P. aeruginosa fadD</i> genes to six. Of the four new homologues, <i>fadD4</i> (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various <i>fadD</i> mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that <i>fadD4</i> is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple <i>fadD</i> mutant, relative to the Δ<i>fadD1D2</i> mutant, was only observed during BALB/c mouse lung infection at 24 h.</p></div

Growth phenotypes of various <i>fadD</i> homologues mutants on acyclic terpenes.

<p>Strains were grown in liquid 1x M9 medium +1% (w/v) Brij-58 supplemented with 20 mM glucose, 0.1% (w/v) of citronellic acid, or 0.1% (w/v) geranic acid at 30°C. Optical densities (ODs) of cultures were measured and compared to PAO1 at day one (A, C, and E). Growth of Δ<i>fadD4</i> mutant and Δ<i>fadD4</i>/<i>attB</i>::<i>fadD4</i> complement strain in different carbon source were compared to PAO1 and ODs from day six are presented (B, D, and F). Results shown are from representative experiments that were performed twice by measuring triplicate cultures.</p

Oligonucleotides primers utilized in this study.

a<p>Restriction enzyme sequences are underlined.</p>b<p>Single copy complementation in <i>E. coli.</i></p>c<p>Single copy complementation in <i>P. aeruginosa.</i></p>d<p><i>fadD</i> homologues cloning.</p

<i>P. aeruginosa</i> fatty acid degradation pathway (FA degradation).

<p>(A) <i>P. aeruginosa</i> FA degradation model was based on the <i>E. coli</i> β-oxidation pathway. Known <i>P. aeruginosa</i> FA degradation enzyme homologues are indicated by numbers: FadD1 (PA3299), FadD2 (PA3300), FadD3 (PA3860), FadD4 (PA1617), FadD5 (PA2893), FadD6 (PA3924), FadAB1 (PA1736–PA1737), and FadBA5 (PA3013–PA3014). Abbreviations: FadA, 3-ketoacyl-CoA thiolase; FadB, <i>cis</i>-Δ³-<i>trans</i>-Δ²-enoyl-CoA isomerase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 3-hydroxyacyl-CoA dehydrogenase; FadD, fatty acyl-CoA synthetase; FadE, acyl-CoA dehydrogenase; FadL, outer membrane long-chain fatty acid translocase; OM, outer membrane; IN, inner membrane. (B) Alignment of FadD homologues motifs with <i>E. coli</i> FadD motifs. Amino acids with similar properties are assigned the same colors using CLC Sequence Viewer 6 software (<a href="http://www.clcbio.com" target="_blank">www.clcbio.com</a>).</p

Growth of various <i>P. aeruginisa fadD</i> mutants on FAs after 24 h.

<p>Strains were grown on 1x M9 medium +1% (w/v) Brij-58 supplemented with 0.2% (w/v) fatty acids or 20 mM glucose (Glu).</p><p>– indicates no growth on a patch and+denotes growth:</p><p>+1 is very little growth.</p><p>+4 is a heavy growth comparable to PAO1 on glucose at 24 h.</p><p>+6 is a very heavy growth comparable to PAO1 on glucose at 96 h.</p

Single copy complementation of the <i>E.coli fadD</i> mutant with <i>P. aeruginosa fadD</i> homologues.

<p>Strains were grown on 1x M9 medium +1% (w/v) Brij-58 supplemented with 0.2% (w/v) fatty acids or 20 mM glucose (Glu) +0.25 mM IPTG for three days at 37°C.</p><p>– indicates no growth on a patch and+denotes growth.</p><p>+1 is very little growth whereas +6 is very heavy growth comparable to K12 on glucose at day 3.</p

Confirmation of the ability of four fosmid clones to individually confer natural competency or growth on DNA.

<p>(A) <i>gfp-</i>DNA uptake assay to assess natural competency. Individual fluorescent bacteria are visible in the representative images (green fluorescence and DIC overlay). The percentages of cells that fluoresce following <i>gfp-</i>DNA uptake are shown in (B). Numbers are from 3 representative images from duplicate experiments. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher %GFP positive comparing to the corresponding empty vector control in three fields (**, <i>P</i><0.005 based on unpaired <i>t</i>-test). (C) Growth of various fosmid-containing strains in 1x M9 with 0.25% DNA done in triplicate. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher growth in DNA comparing to the corresponding empty vector control (*, <i>P</i><0.05 based on unpaired <i>t</i>-test; **, <i>P</i><0.005 based on unpaired <i>t</i>-test).</p

DNA uptake and utilization.

<p>(A) Model of DNA uptake in Gram-negative bacteria (see text for detail). (B) Utilization of DNA as a sole carbon and energy source in selected <i>Burkholderia</i> species. <i>Bp</i> 1026b and <i>B</i>. <i>thiailandensis</i> E264 strains exhibited heavy growth; <i>Bp</i> K96243 showed intermediate growth, while <i>Bc</i> K56-2 and <i>B</i>. <i>mallei</i> ATCC23344 were unable to grow on DNA.</p

Plasmids used in this study.

<p>Abbreviations:</p><p>Ap^r, ampicillin resistance; <i>lac</i>, <i>E. coli</i> lactose operon; <i>rbs</i>, ribosomal binding site; Sp^r, streptomycin resistance.</p

Characterization of selected genes from fosmids.

<p>Characterization of selected genes from fosmids.</p