A working model for <i>P. aeruginosa fabAB</i> regulation.

<p>A working model for <i>P. aeruginosa fabAB</i> regulation.</p

Construction of an <i>E. coli</i> host strain and a genomic library of <i>P. aeruginosa</i>.

<p><b>A</b>) A <i>fabA</i>′-′<i>lacZY</i> translational fusion was assembled on a mini-Tn<i>7</i> suicide delivery vector. <b>B</b>) The mini-Tn<i>7</i> vector was co-electroporated with the Tn<i>7</i> transposase expressing helper plasmid pTNS1 into an <i>E. coli</i> Δ<i>lac</i> strain. Since the suicide delivery vector cannot replicate in <i>E. coli</i> due to the presence of the conditional protein-dependent <i>ori</i>R6K, gentamycin-resistant (Gm^r) transformants will result from site- and orientation-specific integration at the chromosomal <i>att</i>Tn<i>7</i> site which is located immediately downstream of the <i>glmS</i> gene in the <i>glmS - pstS</i> intergenic region. <b>C</b>) A <i>Pst</i>I-<i>Eco</i>RI <i>P. aeruginosa</i> chromosomal DNA library was constructed by ligation of partially digested <i>Pst</i>I-<i>Eco</i>RI fragments into pUC18. <b>D</b>) The library was used to transform a <i>P. aeruginosa</i> strain harboring a chromosomally integrated <i>fabA</i>′-′<i>lacZY</i> fusion. Since <i>fabA</i>′-′<i>lacZY</i> is only expressed at low levels, the host strain will only form light blue colonies on X-Gal-containing indicator medium. Transformants expressing putative activating proteins indicated by “+” will appear as darker blue colonies. Abbreviations: Ap^r, ampicillin resistance; <i>FRT</i>, Flp-recombinase target; Tn<i>7</i>L and Tn<i>7</i>R, left and right end of Tn<i>7</i>, respectively.</p

Bacterial strains and plasmids used in this study.

a<p>Abbreviations: Ap, ampicillin; <i>att</i>, λ attachment site (s); <i>FRT</i>, Flp recombinase target site; Gm, gentamycin; Km, kanamycin; MCS, multiple cloning site; p, promoters; Sp, spectinomycin</p>b<p>see text for plasmid or strain construction details.</p

<i>mariner</i> insertions in genes causing unconditional UFA auxotrophy in <i>fabA</i>(Ts) PAO1.

<p><i>mariner</i> insertions in genes causing unconditional UFA auxotrophy in <i>fabA</i>(Ts) PAO1.</p

Effect of Anr in regulation of <i>fabAB</i> expression.

<p><b>A</b>) <i>anr</i> is responsible for activation of <i>fabA</i>′-′<i>lacZY</i> transcription in <i>E. coli</i>. The restriction maps shown here are from cloned chromosomal DNA fragments isolated from blue, putative activator-expressing colonies. Plasmids isolated from ten blue colonies were sequenced by using M13 forward and reverse primers to verify the presence of the indicated genes. <b>B</b>) Effects of <i>anr</i> on <i>fabA</i> expression in <i>P. aeruginosa</i>. β-galactosidase activities were measured in wild-type and Δ<i>anr</i> PAO mutants containing a chromosomally integrated p<i>fabA</i>′-<i>lacZ</i> fusion. Columns: 1, PAO1 (wild-type); 2, PAO1010 (PAO1 Δ<i>anr</i>); 3, PAO1010 with pPS1684 (pUCP20 with <i>anr</i>⁺); 4, PAO1010 with pPS1682 (pVLT35 with <i>anr</i>+) in the absence of IPTG; 5, PAO1010 with pPS1682 in the presence of IPTG.</p

<i>lacZ</i> expression in PAO1 containing chromosomally integrated <i>lacZ, fabA′-lacZ or fabA</i>Δ30<i>′-lacZ</i> transcriptional fusions.

<p>Strains were grown to mid-log phase in LB medium with or without oleate (OA) supplementation and β-galactosidase activities were measured. Activities are expressed in Miller Units.</p

Characterization of Molecular Mechanisms Controlling <em>fabAB</em> Transcription in <em>Pseudomonas aeruginosa</em>

<div><h3>Background</h3><p>The FabAB pathway is one of the unsaturated fatty acid (UFA) synthesis pathways for <em>Pseudomonas aeruginosa</em>. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt <em>fabA</em> upstream sequence, which is conserved in <em>P. aeruginosa</em>, <em>P. putida</em>, and <em>P. syringae</em>, led to a significant decrease in <em>fabA</em> transcription, suggesting positive regulation by an unknown positive regulatory mechanism.</p> <h3>Methods/Principal Findings</h3><p>Here, genetic and biochemical approaches were employed to identify a potential <em>fabAB</em> activator. Deletion of candidate genes such as <em>PA1611</em> or <em>PA1627</em> was performed to determine if any of these gene products act as a <em>fabAB</em> activator. However, none of these genes were involved in the regulation of <em>fabAB</em> transcription. Use of <em>mariner</em>-based random mutagenesis to screen for <em>fabA</em> activator(s) showed that several genes encoding unknown functions, <em>rpoN</em> and DesA may be involved in <em>fabA</em> regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of <em>fabAB</em> operon.</p> <h3>Conclusion/Significance</h3><p>The data suggest that <em>fabA</em> expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism.</p> </div

Effects of FabR homologs on <i>fabA</i>′-<i>lacZ</i> expression.

<p>β-galactosidase activities were measured in wild-type PAO1 and Δ<i>PA4890</i> and Δ<i>PA1539</i> mutant strains containing a chromosomal <i>fabA</i>′-<i>lacZ</i> fusion. Cells were grown in LB medium. Where indicated, 0.05% oleic acid (OA) was added to cells with 0.05% Brij-58.</p

Characterization of <i>fabA</i> regulatory region.

<p><b>A</b>) Positions of primer-binding sites in the <i>fabA-PA1611</i> intergenic region. Each primer is symbolized as follows: P0, fabA0; P1-P11, fabA1-attB2 through fabA11-attB2; R, fabA-attB1. Primer P11 is placed at the 124^th–144^th nucleotide from the first nucleotide of the <i>fabA</i> coding region. The sequence shaded and boxed in the gray box indicates the putative 30 bp regulatory element. Vertical arrow heads indicate the end points of sequences present in the <i>lacZ</i> fusion constructs analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045646#pone-0045646-g003" target="_blank">Fig. 3</a>B. <b>B</b>) The 30 bp sequence is important for <i>fabAB</i> expression. PAO1 contained <i>fabA′-lacZ</i> vectors with <i>fabA</i> upstream regions amplified with primers 1 through 11. The 5 bp addition in primer 4a, which restores a complete 30 bp sequence, recovered <i>lacZ</i> expression indicating that it is important for <i>fabA</i> transcription. Cells were grown and β-galactosidase activities were measured as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045646#pone-0045646-g002" target="_blank">Fig. 2</a> with and without oleate (OA) supplementation. <b>C</b>) Characterization of the promoter region of <i>fabA</i> using RT-PCR analysis of <i>fabA</i> expression. RNA was extracted from PAO1 grown at 37°C using the hot phenol extraction method. cDNA was synthesized using the Superscript III First-strand kit (Invitrogen) and primer R. Resulting cDNAs were used as templates for PCR amplification utilizing primer R and the indicated primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045646#pone-0045646-g003" target="_blank">Fig. 3</a>A for location of primer-binding sites). <b>D</b>) β-galactosidase activities in PAO1 containing <i>lacZ</i> fusions with various <i>fabA</i> upstream fragments. The upstream fragments were amplified with primers pPA1612, and primers p1 and p2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045646#pone-0045646-g003" target="_blank">Fig. 3</a>A for primer-binding sites).</p

LPS types present in <i>B</i>. <i>pseudomallei</i> strains used in this study.

<p>The O-antigen structures shown here are based on previously published structures in the case of Type A and Type A_V and unpublished data in the cases of Type B and B2. Inner core is based on structures found in <i>Burkholderia cenocepacia</i> and lipid A is a composite of structures found in this work and others referenced in the text. <i>B</i>. <i>mallei</i> is used as an example of the Type A_V LPS and was not investigated in this study. Red circles indicate variable methylation and acetylation of the L-6dTal<i>p</i> residues of Type A_V LPS.</p