Kun Zhu

RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa

The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A ΔfabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild‐type cells produced 18:1Δ11 as the principal UFA, whereas the ΔfabA strain produced only 16:1Δ9. The double bond in the 16:1Δ9 was introduced after phospholipid formation and was localized in the sn‐2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The ΔfabAΔdesA double mutant required exogenous fatty acids for growth but the ΔfabAdesB double mutant did not. Exogenous stearate was converted to 18:1Δ9 and supported the growth of ΔfabAΔdesA double mutant. A ΔfabAΔdesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5‐fold increase in desA expression in ΔfabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2‐position phospholipid Δ9‐desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl‐CoA Δ9‐desaturase whose expression is repressed by DesT.

PqsD Is Responsible for the Synthesis of 2,4-Dihydroxyquinoline, an Extracellular Metabolite Produced by Pseudomonas aeruginosa

2,4-Dihydroxyquinoline (DHQ) is an abundant extracellular metabolite of the opportunistic pathogen Pseudomonas aeruginosa that is secreted into growth medium in stationary phase to concentrations comparable with those of the Pseudomonas quinolone signal. Using a combination of biochemical and genetic approaches, we show that PqsD, a condensing enzyme in the pqs operon that is essential for Pseudomonas quinolone signal synthesis, accounts for DHQ formation in vivo. First, the anthraniloyl moiety is transferred to the active-site Cys of PqsD to form an anthraniloyl-PqsD intermediate, which then condenses with either malonyl-CoA or malonyl-acyl carrier protein to produce 3-(2-aminophenyl)-3-oxopropanoyl-CoA. This short-lived intermediate undergoes an intramolecular rearrangement to form DHQ. DHQ was produced by Escherichia coli coexpressing PqsA and PqsD, illustrating that these two proteins are the only factors necessary for DHQ synthesis. Thus, PqsD is responsible for the production of DHQ in P. aeruginosa.

Exogenous Isoleucine and Fatty Acid Shortening Ensure the High Content of Anteiso-C15:0 Fatty Acid Required for Low-Temperature Growth of Listeria monocytogenes

ABSTRACT Previous studies have demonstrated that the branched-chain fatty acid anteiso-C15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C15:0 solely at the expense of anteiso-C17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10°C. Under this condition, the increase of anteiso-C15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain α-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of β-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C15:0 at the expense of anteiso-C17:0.

Transcriptional Regulation of Membrane Lipid Homeostasis in Escherichia coli

The biophysical properties of membrane phospholipids are controlled by the composition of their constituent fatty acids and are tightly regulated in Escherichia coli. The FabR (fatty acid biosynthesis repressor) transcriptional repressor controls the proportion of unsaturated fatty acids in the membrane by regulating the expression of the fabB (β-ketoacyl-ACP synthase I) and fabA (β-hydroxydecanoyl-ACP dehydratase/isomerase) genes. FabR binding to a DNA palindrome located within the promoters of the fabB and fabA genes required the presence of an unsaturated acyl-acyl carrier protein (ACP) or acyl-CoA and was antagonized by saturated acyl-ACP or acyl-CoA. The FabR-dependent repression of fabB and fabA by exogenous unsaturated fatty acids confirmed the role for FabR in responding to the acyl-CoA pool composition, and the perturbation of the unsaturated:saturated acyl-ACP ratio using a specific inhibitor of lipid A formation verified FabR-dependent regulation of fabB by the acyl-ACP composition in vivo. Thus, FabR plays a key role in controlling the membrane biophysical properties by regulating gene expression in response to the composition of the long-chain acyl-thioester pool. This mechanism ensures that a balanced composition of fatty acids is available for incorporation into the membrane via the PlsB/PlsC acyltransferases.

A Pseudomonas aeruginosa transcription factor that senses fatty acid structure.

Cells regulate their membrane phospholipid biophysical properties by the co‐ordinated synthesis of saturated and unsaturated fatty acids. In bacteria, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalysed by desaturases. A transcriptional repressor in Pseudomonas aeruginosa, DesT (PA4890), regulates the expression of an acyl‐CoA desaturase operon (desCB, PA4889 and PA4888). The desCB operon is located adjacent to desT and is transcribed in the opposite direction. The expression level of desCB is strongly and selectively upregulated in a ΔdesT‐deletion strain. Both electrophoresis mobility shift assay and DNase I footprinting analysis demonstrated the existence of two DesT binding sites in the desT–desCB promoter region, P1 and P2. The binding of purified DesT to P2 was enhanced by unsaturated acyl‐CoAs, whereas saturated acyl‐CoAs prevented DesT interaction with P2. The biological importance of this interaction was verified by the upregulation of desCB and desT in cells grown in the presence of stearate and their repression when oleate was present. This unique ligand selectivity allows DesT to sense the physical properties of the cellular acyl‐CoA pool and modulate the expression of the acyl‐CoA Δ9‐desaturase system to adjust fatty acid desaturation activity accordingly.

FabH selectivity for anteiso branched-chain fatty acid precursors in low-temperature adaptation in Listeria monocytogenes.

Gram-positive bacteria, including Listeria monocytogenes, adjust membrane fluidity by shortening the fatty acid chain length and increasing the proportional production of anteiso fatty acids at lower growth temperatures. The first condensation reaction in fatty acid biosynthesis is carried out by beta-ketoacyl-acyl carrier protein synthase III (FabH), which determines the type of fatty acid produced in bacteria. Here, we measured the initial rates of FabH-catalyzed condensation of malonyl-acyl carrier protein and alternate branched-chain precursor acyl-CoAs utilizing affinity-purified His-tagged L. monocytogenes FabH heterologously expressed in Escherichia coli. Listeria monocytogenes FabH showed a preference for 2-methylbutyryl-CoA, the precursor of odd-numbered anteiso fatty acids, at 30 degrees C, which was further increased at a low temperature (10 degrees C), suggesting that temperature-dependent substrate selectivity of FabH underlies the increased formation of anteiso branched-chain fatty acids during low-temperature adaptation. The increased FabH preferential condensation of 2-methylbutyryl-CoA could not be attributed to a significantly higher availability of this fatty acid precursor as acyl-CoA pool levels were reduced similarly for all fatty acid precursors at low temperatures.