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A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway.

ABSTRACT: The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacyl-ACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida ΔfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the ΔfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida β-oxidation pathway gave increased accumulation. The ΔfabB ΔfabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the ΔfabF2 ΔPput_2425 double-mutant strain still accumulated suppressors at low UFA concentrations.

SUBMITTER: Dong H

PROVIDER: S-EPMC8319022 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from β-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

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A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway.

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