Fig 1.
The 3-ketoacyl-ACP synthase III reaction, organization of the R. solanacearum fatty acid biosynthesis gene clusters and alignment of R. solanacearum FabH and RSp0194 with E. coli FabH or P. aeruginosa PA3286.
(A) The reaction catalyzed by 3-ketoacyl-ACP synthase III. (B) Organization of the E.coli and R. solanacearum fatty acid biosynthesis gene clusters. The thick arrows indicate the relative sizes of the genes. The numbers below the arrows indicate the gene designations in the E. coli or R. solanacearum GeneBank database. (C) and (D) Alignments of R. solanacearum FabH with E. coli FabH (C) or R. solanacearum RSp0194 with P. aeruginosa PA3286 (D). Rs, Ec, and Pa are denoted as R. solanacearum, E. coli, and P. aeruginosa, respectively. The catalytic triad (Cys-His-Asn) are highlighted by asterisks. The alignment was done with Clustal W, based on identical residues.
Fig 2.
Growth of R. solanacearum fabH or fabW mutants on BG medium and fatty acid synthesis of R. solanacearum fabH.
(A) Growth of R. solanacearum fabH or fabW mutants on BG medium in the absence or presence of octanoic acid. (B) Argentation thin-layer chromatographic analysis of sodium [1-14C] acetate-labeled R. solanacearum fabH mutant as described in Materials and Methods. Sat, saturated fatty acid esters; Δ9C16: 1, methyl ester of cis-9-hexadecenoic acid; Δ11C18: 1, methyl ester of cis-11-octadecenoic acid. Lane 1 is the methyl esters of wild-type R. solanacearum GMI1000, lane 2 is the methyl esters of R. solanacearum fabH mutant strain RsmH, lane 3 is the methyl esters of R. solanacearum fabH mutant strain RsmH carrying R. solanacearum fabH encoded plasmid pYH3, and lane 4 is the methyl esters of R. solanacearum fabH mutant strain RsmH carrying E. coli fabH encoded plasmid pYH4.
Fig 3.
Characterization of R. solanacearum fabH mutant RsmH and fabH fabW double mutant RsmD on BG medium in the presence of various fatty acid species.
(A) The growth of mutants on BG medium in the presence of butanoic, hexanoic, or octanoic acid. (B) The growth of mutants on BG medium in the presence decanoic, dodecanoic, or tetradecanoic acid. −IPTG indicates no addition of IPTG to induce the expression of V.harveyi aasS; +IPTG indicates addition of IPTG to induce the expression of V.harveyi aasS.
Fig 4.
Condensation of malonyl-ACP with acyl-CoAs (C2-CoA to C10-CoA) or acyl-ACPs (C4-ACP to C10-ACP) by purified R. solanacearum FabW.
(A) The cycle of fatty acid synthesis was reconstructed in vitro using a combination of E. coli FabA, FabG, FabI, and R. solanacearum FabW with NADH, and NADPH as cofactors, and malonyl-ACP plus acyl-CoAs (C4-CoA to C10-CoA) as substrates. (B) The reaction mixture of fatty acid synthesis contained E. coli FabA, FabG, FabI, and R. solanacearum FabW or E. coli FabB, NADH, and NADPH as cofactors, and malonyl-ACP plus acyl-ACPs (C6-ACP to C10-ACP) as substrates. (C) Condensation of malonyl-ACP with acetyl-CoA or butyryl-ACP catalyzed by R. solanacearum FabW. The initial reaction of fatty acid synthesis was reconstructed using a combination of E. coli FabD, FabA, FabG, FabI, and R. solanacearum FabH (lane 2) or FabW (lane 3) with NADH, and NADPH as cofactors and malonyl-CoA and acetyl-CoA as substrates. The elongation reaction of fatty acid synthesis was reconstructed by addition of E. coli FabB (lane 4) or R. solanacearum FabW (lane 5) to the initial reaction catalyzed by R. solanacearum FabH.
Fig 5.
MALDI-TOF-MS of the products of the RsFabW reaction.
(A) Mass spectrum of the reaction mixture not containing RsFabW. (B) Mass spectrum of the reaction mixture containing RsFabW.
Table 1.
Substrate specificities of Ralstonia solanacearum FabH and FabW.
Fig 6.
Model of the fatty acid biosynthesis pathway in R. solanacearum.
Abbreviations: Acc, acetyl-CoA carboxylase; FabD, malonyl-CoA: ACP transacylase; RsFabH, 3-ketoacyl-ACP synthase III; RsFabW, 3-ketoacyl-ACP synthase III; FabG, 3-ketoacyl-ACP reductase; FabZ, 3-hydroxyacyl-ACP dehydratase; RsFabF, 3-ketoacyl-ACP synthase II; FabI, enoyl-ACP reductase. The full line indicates RsFabH uses acetyl-CoA substrate to initiate the fatty acid synthesis. The dotted line indicates RsFabW uses acyl-CoAs as substrates to initiate the fatty acid synthesis. The dashed line indicates RsFabW uses acyl-ACPs as substrates to initiate the fatty acid synthesis.