The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis

Abstract

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent beta-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-beta-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase absolutely required for the synthesis of the beta-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.

FIG. 1

Schematic representation of the fatty acid biosynthetic pathway showing the deduced role of the RhlG protein in the production of rhamnolipids and PHAs. Initiation of the fatty acid biosynthetic cycle, catalyzed by FabH, requires acetyl-CoA and malonyl-ACP to form aceto-acetyl-ACP. Subsequent cycles are initiated by condensation of malonyl-ACP with acyl-ACP, catalyzed by FabB and FabF. In the second step, the resulting β-ketoester is reduced to a β-hydroxyacyl-ACP by FabG. The third step in the cycle is catalyzed by either FabA or FabZ. The fourth and final step is the conversion of trans-2-enoyl-ACP to acyl-ACP, a reaction catalyzed by FabI. TDP-r, thymidine-diphospho-l-rhamnose; PhaC, PHA synthase; rhl 1, monorhamnolipid; rhl 2, dirhamnolipid; β-hdd, β-hydroxydecanoyl-β-hydroxydecanoate.

FIG. 2

Characterization of the transcription arrangement of the P. aeruginosa PAO1 rhlG and rcsF genes. (A) Nucleotide sequence of the genes and regulatory sequences. The sequence and position of the oligonucleotides used during this work are shown in the figure and identified as Ln or Rn, depending on their polarity (L oligonucleotides amplify the sequence from 5′ to 3′, and R oligonucleotides have the opposite polarity). The sequence corresponding to the lux box is double underlined. Arrows indicate the two transcription start sites detected (P1 and P2). SD (Shine-Dalgarno) indicates the ribosome binding site sequence for mRNA translation. The sequences corresponding to putative transcriptional termination sites (term) are shown. (B) Primer extension analysis of the rhlG gene with two different oligonucleotides as primers. In panel BI, the primer extension analysis was done with oligonucleotide R3 and revealed the existence of the mRNA secondary structures shown. In panel BII, the oligonucleotide R4 was used, and two transcription start points indicated as P1 and P2 were found.

FIG. 3

Multiple alignment of the RhlG deduced amino acid sequence with different NADPH-dependent β-ketoacyl-ACP reductase (FabG) and NADPH-dependent ketoacyl-CoA reductase (PhaB) proteins. Residues within rectangles correspond to identical amino acids, and those shaded are conserved among most of the proteins analyzed. The percentage of identity of the different proteins with PAO1 RhlG is shown in the bottom right column of the figure. Asterisks mark the residues which form the NADPH binding signature, and circles show the amino acids conserved in dehydrogenases. FabGBjap, FabG from Bradyrhizobium japonicum; FabGMsmeg, FabG from Mycobacterium smegmatis; FabGMtub, FabG from Mycobacterium tuberculosis; FabGAact, FabG from Actinobacillus actinomycetemcomitans; FabGAtha, FabG from Arabidopsis thaliana; FabGClan, FabG from Cuphea lanceolata; FabGBsub, FabG from Bacillus subtilis; FabGEcoli, FabG from E. coli; FabGVharv, FabG from Vibrio harveyi; FabGHinf, FabG from Haemophilus influenzae; FabGPaer, FabG from P. aeruginosa (GenBank database accession no. U91631); FabGPAO1, FabG from P. aeruginosa PAO1 (contig 1761); PhaBAsp, PhaB from Alcaligenes sp. strain SH69; PhaBAcsp, PhaB from Acinetobacter sp. strain RA3849; RhlGPAO1, RhlG from P. aeruginosa PAO1 (contig 1780); RhlGW51D, RhlG from P. aeruginosa W51D (GenBank database accession no. AF052586).

FIG. 4

Molecular characterization of the PAO1 rhlG mutant ACP5. (A) Schematic representation of the strategy to construct the rhlG mutants (ACP5 and W51D-10). (B) Southern blotting hybridization with the 600-bp insert of plasmid pJC1 (I) and the 1.4-kb Tc^r resistance cassette (II) used as probes. Lanes correspond to DNA samples digested with PstI endonuclease from the PAO1 genome (lane 1), the ACP5 genome (lane 2), the Tc^r cassette (lane 3), and the PCR product of the amplification of the W51D genome with oligonucleotides L2′ and R2′ (lane 4). (C) Amplification by PCR with different oligonucleotides specific for the rhlG and rcsF genes. Lanes correspond to the following DNA samples: 1, λ phage genome digested with HindIII; 2, PCR product with W51D DNA as a template and the L2′ and R2 oligonucleotides as primers; 3, PCR product with PAO1 DNA as a template and the L2 and R2 oligonucleotides as primers; 4, PCR product with ACP5 DNA as a template and the L2 and R2 oligonucleotides as primers; 5, PCR product with W51D DNA as a template and the L2′ and R1 oligonucleotides as primers; 6, PCR product with PAO1 DNA as a template and the L2 and R1 oligonucleotides as primers; 7, PCR product with W51D DNA as a template and the L3 and R1 oligonucleotides as primers; and 8, PCR product with PAO1 DNA as a template and the L3 and R1 oligonucleotides as primers.

FIG. 5

Electron micrographs of the P. aeruginosa strains PAO1 (A), ACP5 (B), and ACP5/pJC4 (C) grown for 24 h on MM + gluconate. Some of the PHA granules are pointed out. Micrographs were taken at a ×20,000 magnification.