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 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

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 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

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

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

Blocking Phosphatidylcholine Utilization in <i>Pseudomonas aeruginosa</i>, via Mutagenesis of Fatty Acid, Glycerol and Choline Degradation Pathways, Confirms the Importance of This Nutrient Source <i>In Vivo</i>

<div><p><i>Pseudomonas aeruginosa</i> can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of <i>P. aeruginosa in vivo.</i> The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related <i>fadBA</i>-operons (<i>fadBA1-5</i>, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (<i>fadBA145</i>) of the five <i>fadBA</i>-operons are dominant in medium-chain and long-chain Fad. The triple <i>fadBA145</i> mutant also showed reduced ability to degrade PC <i>in vitro</i>. We have previously shown that by partially blocking Fad, via mutagenesis of <i>fadBA5</i> and <i>fadD</i>s, we could significantly reduce the ability of <i>P. aeruginosa</i> to replicate on FA and PC <i>in vitro</i>, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or <i>in vivo</i>. Hence, we constructed additional mutants (Δ<i>fadBA145</i>Δ<i>glpD</i>, Δ<i>fadBA145</i>Δ<i>betAB</i>, and Δ<i>fadBA145</i>Δ<i>betAB</i>Δ<i>glpD</i>) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC <i>in vitro</i>, resulted in decreased replication fitness and competitiveness <i>in vivo</i> compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for <i>P. aeruginosa</i> during CF lung infection.</p></div

Bacterial strains used in this study^a.

a<p>For strains constructed in this study, please see text for further details.</p>b<p>Please use Lab ID for requesting strains.</p

Plasmids used in this study^a.

a<p>For plasmids constructed in this study, please see text for further details.</p>b<p>Please use Lab ID for requesting plasmids.</p