Jong Kook Rho

Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2-Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain-Length

Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

Shifting of the Distribution of Aromatic Monomer-Units in Polyhydroxyalkanoic Acid to Longer Units by Salicylic Acid in Pseudomonas fluorescens BM07 Grown With Mixtures of Fructose and 11-Phenoxyundecanoic Acid

Medium‐chain‐length‐polyhydroxyalkanoic acids (MCL‐PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer‐units whose precursors are supplied via β‐oxidation degradation of MCL fatty acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl‐group transfer from 3‐hydroxyacyl‐ACPs derived from acetyl‐CoA to coenzyme A. It was found that salicylic acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer‐units derived from a MCL fatty acid co‐added as carbon source (e.g., 11‐phenoxyundecanoic acid (11‐POU)) to longer monomer‐units. Both SA and acrylic acid were found to induce high accumulations of 3‐ketohexanoic acid in BM07 wild‐type cells grown with n‐hexanoic acid as well as to inhibit the formation of acetyl‐CoA from acetoacetyl‐CoA by BM07 cell extract, suggesting that 3‐ketoacyl‐CoA thiolase is their common β‐oxidation target. The structural motif of acrylic acid present in the molecular structure of SA may self‐explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild‐type and ΔphaG mutant cells grown on the mixtures of fructose and 11‐POU revealed that both PhaG and β‐oxidation inhibitor may play a critical role in the synthesis of PHA with longer side‐chain ω‐functional substitutions. Biotechnol. Bioeng. 2009;102: 1209–1221.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with ω-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid β-oxidation inhibition.

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07.