Altered swimming and swarming motility of <i>P. aeruginosa fadD</i> mutants.

<p>(A) Swimming motility of <i>fadD</i> mutants and their complements. (B) Swarming migration of <i>fadD</i> mutants and their complements. These figures are representative of multiple experiments. Strain designation is the same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013557#pone-0013557-t003" target="_blank">Table 3</a>: wildtype PAO1, P007; <i>fadD1</i>^-, P175; <i>fadD2</i>^-, P547; Δ<i>fadD2D1</i>, P177; <i>fadD1</i>^- complement, P541; <i>fadD2</i>^- complement, P549; and Δ<i>fadD2D1</i> complement, P543.</p

Analysis of protease, hemolysin, lipase, and rhamnolipid production by <i>P. aeruginosa fadD</i> mutants.

<p>The <i>fadD2</i> mutant displayed significantly decreased production of proteases (A), hemolysins (B), lipases (C), and rhamnolipids (D), while no growth defects in LB were observed (E). These assays were conducted in triplicate and are expressed as a percentage of the mean value of the wildtype PAO1 ± s.e.m.</p

Primers used in this study.

a<p>Restriction enzyme sites utilized in this study are underlined.</p>b<p>Primers synthesized RNase free and HPLC purified.</p

SMART mapping of the transcriptional start sites for <i>fadD2</i> and <i>fadD1</i>.

<p>(A) One SMART product was observed after PCR amplification of the cDNA with SMART and <i>fadD2</i> primers (oligonucleotides #798 and #373). Sequencing of the single band with a nested <i>fadD2</i> primer (oligonucleotide #374) displayed a reverse-complement sequence chromatogram, showing the <i>fadD2</i> transcriptional start site (indicated by +1 at the <b>C</b>TTCG sequence) and the underlined SMART primer sequence. (B) Likewise, the downstream <i>fadD1</i> transcriptional start site was mapped (at the <b>G</b> of the sequence <b>G</b>CCTA) by sequencing a single PCR product. (C) <i>fadD2</i> and <i>fadD1</i> coding sequences and the predicted −10 and −35 promoter regions are indicated (boxed). The intergenic region between <i>fadD2</i> and <i>fadD1</i> contains a potential transcriptional terminator or attenuator sequence (inverted arrows). For each gene, three black arrows indicate primers 1, 2 and 3 (#372/#375, #373/376, and #374/377) used for mapping <i>fadD2</i> and <i>fadD1</i>. Dashed lines indicate missing protein sequences and dots indicate stop codons.</p

Complementation of the <i>E. coli fadD</i> mutant with <i>P. aeruginosa fadD</i> homologues.

a<p>(-) denotes no growth on a patch; (+) denotes growth: (+1) is very little growth and (+6) is heavy growth after 3 days.</p

Kinetic properties of FadD1 and FadD2 with various substrates.

a<p>Kinetic constants (V_max and <i>K_m</i>) determined using Hanes-Woolf plot.</p>b<p>nmole of acyl-CoA formed/min/mg of protein.</p>c<p>s^-1; determined using MW of FadD1 (61,655) and FadD2 (61,373).</p>d<p>mM of ATP or FA.</p>e<p>mM^-1 s^-1; represents enzyme catalytic efficiency.</p

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

Purification and biochemical characterization of the two <i>P. aeruginosa</i> FadDs.

<p>(A) FadD1 (lane 1; MW = 61,655) and FadD2 (lane 2; MW = 61,737) were purified to near homogeneity from an <i>E. coli fadD</i>^- strain to prevent potential contamination of <i>E. coli</i> FadD. FadD1 activities (B) were higher for LCFAs (C_18:1^Δ9>C_16:0), while FadD2 (C) had higher activities for shorter chain FAs (C_8:0>C_10:0>C_4:0>C_6:0).</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

Growth analysis of <i>fadD</i> mutants using various FAs as sole carbon sources.

<p>Although <i>fadD</i> mutants showed various defects when grown with FAs of different chain-lengths (see top of graphs in B-I), no growth defects were observed for any of the mutants when grown with casamino acids (CAA) as a control (A). All three <i>P. aeruginosa</i> mutants were fully complemented by the respective missing gene(s) and grew as well as the wildtype PAO1 on all carbon sources. Not shown are the three control mutant strains (PAO1-<i>fadD1</i>::<i>FRT</i>/<i>attB</i>::miniCTX2, PAO1-<i>fadD2</i>::<i>FRT</i>/<i>attB</i>::miniCTX2, and PAO1-Δ<i>fadD2D1</i>::<i>FRT</i>/<i>attB</i>::miniCTX2) having the empty miniCTX2 integrated into their chromosomes, where all had similar growth characteristics to the non-complemented mutants.</p