Pseudomonas aeruginosa Directly Shunts β-Oxidation Degradation Intermediates into De Novo Fatty Acid Biosynthesis

Abstract

We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C 8 -CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed.

Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays

“…The deuterium-tracing results from this study help to clarify, and suggest specifically, that it is the C 8 -acyl-CoA ␤-oxidation intermediate that is utilized. This is supported by recent evidence showing that P. aeruginosa can directly shunt C 8 -acyl-CoA into the fatty acid synthesis pathway (39).…”

Glycerophospholipid synthesis and functions in Pseudomonas

“…This condensing enzyme performs a task usually attributed to FabH but uses a Cys-His-His active site of FabB/FabF enzymes (Yuan et al, 2012b). The fabY mutant is still viable indicating a possible alternative secondary mechanism of FAS in P. aeruginosa (Yuan et al, 2012a). It exhibits a growth defect and a decreased production of quorum sensing signaling molecules, rhamnolipids and siderophores (Yuan et al, 2012b).…”

Deletion of the β-Acetoacetyl Synthase FabY in Pseudomonas aeruginosa Induces Hypoacylation of Lipopolysaccharide and Increases Antimicrobial Susceptibility

“…The PA3286 shunt mechanism endows P. aeruginosa with a unique metabolic capacity among bacteria for FAS initiation using C8-CoA primer obtained directly from degradation of exogenous fatty acid (11,54). Deuterated fatty acid labeling studies have shown that all cellular demands for FAS can be met via the PA3286 shunt provided exogenous fatty acids C8 and longer are present in the medium (11), including for acylation of lipid A and hence the reestablishment of wild-type antibiotic susceptibility. There is a rich supply of long-chain fatty acids in human serum (55), thus the PA3286 shunting mechanism could bypass the function of FabY in vivo and prevent lipid A from being hypoacylated.…”

“…The ⌬fabY phenotype is characterized by muted quorum sensing and diminished siderophore secretion (8), a finding consistent with the need for FAS intermediates in the synthesis of the three major acylated quorum-sensing signal molecules [2-heptyl-3-hydroxy-4-quinolone (PQS), N-(3-oxododecanoyl)-L-homoserine lactone, and N-butanoyl-L-homoserine lactone] (9) and in siderophore assembly (10). Residual FAS initiation in the absence of fabY is thought to be due in part to the ␤-ketoacyl-ACP synthase PA3286, a KASIII family condensing enzyme with low catalytic activity when using short-chain acetyl-CoA as an initiating substrate (11). The main cellular role for PA3286 in wild-type P. aeruginosa, however, is in the shunting of exogenous fatty acidCoA degradation intermediates from the catabolic ␤-oxidation pathway into FAS at the octanoyl-CoA (C8-CoA) substrate chain length through condensation with malonyl-ACP to form decanoyl-ACP (11).…”

“…Residual FAS initiation in the absence of fabY is thought to be due in part to the ␤-ketoacyl-ACP synthase PA3286, a KASIII family condensing enzyme with low catalytic activity when using short-chain acetyl-CoA as an initiating substrate (11). The main cellular role for PA3286 in wild-type P. aeruginosa, however, is in the shunting of exogenous fatty acidCoA degradation intermediates from the catabolic ␤-oxidation pathway into FAS at the octanoyl-CoA (C8-CoA) substrate chain length through condensation with malonyl-ACP to form decanoyl-ACP (11). In addition, there may be yet a third uncharacterized mechanism for FabY-independent fatty acid initiation akin to that observed in E. coli lacking FabH (12).…”