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

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

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

Characterization of selected genes from fosmids.

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

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

Gene content of four fosmid clones conferring natural competency and/or DNA catabolism.

<p>(A) The fosmid backbone is indicated in black and the red region depicts the extent of <i>Bp</i> 1026b genomic DNA inserted in each fosmid. Gene products with known predicted functions are indicated by gene names and those with unknown functions are labeled with gene identifications. (B) Synteny map comparing fosmid regions of <i>Bp</i> K96243 and 1026b. Red regions indicate the high level of similarity between fosmid regions in strain K96243 (non-competent) versus 1026b (competent) aligned using Artemis webACT. Identical regions are indicated in red, non-identical regions in white, and blue indicates a region that is inverted in the two strains.</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

<i>Burkholderia pseudomallei</i> natural competency and DNA catabolism: Identification and characterization of relevant genes from a constructed fosmid library

<div><p><i>Burkholderia</i> spp. are genetically and physiologically diverse. Some strains are naturally transformable and capable of DNA catabolism. <i>Burkholderia pseudomallei</i> (<i>Bp</i>) strains 1026b and K96243 and <i>B</i>. <i>thailandensis</i> strain E264 are able to utilize DNA as a sole carbon source for growth, while only strains 1026b and E264 are naturally transformable. In this study, we constructed low-copy broad-host-range fosmid library, containing <i>Bp</i> strain 1026b chromosomal DNA fragments, and employed a novel positive selection approach to identify genes responsible for DNA uptake and DNA catabolism. The library was transferred to non-competent <i>Bp</i> K96243 and <i>B</i>. <i>cenocepacia</i> (<i>Bc</i>) K56-2, harboring chromosomally-inserted <i>FRT</i>-flanked <i>sacB</i> and <i>pheS</i> counter-selection markers. The library was incubated with DNA encoding Flp recombinase, followed by counter-selection on sucrose and chlorinated phenylalanine, to select for clones that took up <i>flp</i>-DNA, transiently expressed Flp, and excised the <i>sacB-pheS</i> cassette. Putative clones that survived the counter-selection were subsequently incubated with <i>gfp</i>-DNA and bacteria were visualized via fluorescent microscopy to confirm natural competency. Fosmid sequencing identified several 1026b genes implicated in DNA uptake, which were validated using chromosomal mutants. One of the naturally competent clones selected in <i>Bc</i> K56-2 enabled <i>Bc</i>, <i>Bp</i> and <i>B</i>. <i>mallei</i> to utilize DNA as a sole carbon source, and all fosmids were used to successfully create mutations in non-naturally-competent <i>B</i>. <i>mallei</i> and <i>Bp</i> strains.</p></div