The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation
Pseudomonas aeruginosa is capable of synthesizing polyhydroxyalkanoic acids (PHAs) and rhamnolipids, both of which are composed of 3-hydroxydecanoic acids connected by ester bonds, as well as synthesizing the biofilm matrix polymer alginate. In order to study the influence of PHA biosynthesis on rhamnolipid and alginate biosynthesis, as well as stress tolerance and biofilm formation, isogenic knock-out mutants deficient in PHA biosynthesis were generated for P. aeruginosa PAO1 and the alginate-overproducing P. aeruginosa FRD1. A gentamicin-resistance cassette was inserted replacing the 3′ region of phaC1, the whole of phaZ and the 5′ region of phaC2. Gas chromatography/mass spectrometry analysis showed that PHA accumulation was completely abolished in both strains. Interestingly, this gene replacement did not abolish rhamnolipid production. Thus, as previously suggested, the PHA synthase is not directly involved in rhamnolipid biosynthesis. In the PHA-negative mutant of mucoid FRD1 alginate biosynthesis was not affected, whereas in the PHA-negative PAO1 mutant an almost threefold increase in biosynthesis was observed compared to the wild-type. Consistently, PHA accumulation in FRD1 contributed only 4·7 % of cell dry weight, which is fourfold less than in PAO1. These data suggest that PHA biosynthesis and alginate biosynthesis are in competition with respect to a common precursor. The surface attachment and biofilm development of the PHA-negative mutants were also compared to those of wild-type strains in glass flow-cell reactors. PHA-negative mutants of P. aeruginosa PAO1 and FRD1 showed reduced attachment to glass. However, the PAO1 PHA-negative mutant, in contrast to the wild-type, formed a stable biofilm with large, distinct and differentiated microcolonies characteristic of alginate-overproducing strains of P. aeruginosa. The stress tolerance of PHA-negative mutants with respect to elevated temperature was strongly impaired. These data indicated a functional role for PHA in stress response and tolerance.
Amara, A. A. & Rehm, B. H. A.(2003). Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa mediated synthesis of a new polyester, identification of catalytic residues. Biochem J374, 413–421.[CrossRef][Google Scholar]
Blumenkrantz, N. & Asboe-Hansen, G.(1973). New method for quantitative determination of uronic acids. Anal Biochem54, 484–489.[CrossRef][Google Scholar]
Brandl, H., Gross, R. A., Lenz, R. W. & Fuller, R. C.(1988).Pseudomonas oleovorans as a source of poly(3-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol54, 1977–1982.
[Google Scholar]
Campos-Garcia, J., Caro, A. D., Najera, R., Miller-Maier, R. M., Al Tahhan, R. A. & Soberon-Chavez, G.(1998). The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis. J Bacteriol180, 4442–4451.
[Google Scholar]
Chandrasekan, E. V. & Bemiller, J. N.(1980). Constituent analyses of glycosaminoglycans. Methods Carbohydr Chem8, 89–96.
[Google Scholar]
Cochran, W. L., Suh, S. J., McFeters, G. A. & Stewart, P. S.(2000). Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine. J Appl Microbiol88, 546–553.[CrossRef][Google Scholar]
Davey, M. E., Caiazza, N. C. & O'Toole, G. A.(2003). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol185, 1027–1036.[CrossRef][Google Scholar]
Deziel, E., Lepine, F., Milot, S. & Villemur, R.(2003).rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa, 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. Microbiology149, 2005–2013.[CrossRef][Google Scholar]
Fiedler, S., Steinbüchel, A. & Rehm, B. H. A.(2000). PhaG-mediated synthesis of poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from non-related carbon sources in recombinant Pseudomonas fragi. Appl Environ Microbiol66, 2117–2124.[CrossRef][Google Scholar]
Gentry, D. R., Hernandez, V. J., Nguyen, L. H., Jensen, D. B. & Cashel, M.(1993). Synthesis of the stationary-phase sigma factor σs is positively regulated by ppGpp. J Bacteriol175, 7982–7989.
[Google Scholar]
Griebel, R., Smith, Z. & Merrick, J. M.(1968). Metabolism of poly-beta-hydroxybutyrate. I. Purification, composition and properties of native poly-beta-hydroxybutyrate granules from Bacillus megaterium. Biochemistry7, 3676–3681.[CrossRef][Google Scholar]
Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M. & Parsek, M. R.(2001). Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J Bacteriol183, 5395–5401.[CrossRef][Google Scholar]
Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P.(1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences, application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene212, 77–86.[CrossRef][Google Scholar]
Hoffmann, N. & Rehm, B. H. A.(2004). Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa. FEMS Microbiol Lett237, 1–7.[CrossRef][Google Scholar]
Hoffmann, N., Steinbüchel, A. & Rehm, B. H. A.(2000a). Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway. Appl Microbiol Biotechnol54, 665–670.[CrossRef][Google Scholar]
Hoffmann, N., Steinbüchel, A. & Rehm, B. H. A.(2000b). The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources. FEMS Microbiol Lett184, 253–259.[CrossRef][Google Scholar]
Hoffmann, N., Amara, A. A., Beermann, B. B., Qi, Q., Hinz, H. J. & Rehm, B. H. A.(2002). Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP : CoA transacylase, which diverts intermediates of fatty acid de novo biosynthesis. J Biol Chem277, 42926–42936.[CrossRef][Google Scholar]
Huisman, G. W., de Leeuw, O., Eggink, G. & Witholt, B.(1989). Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol55, 1949–1954.
[Google Scholar]
Kessler, B., Kraak, M. N., Ren, Q., Klinke, S., Prieto, M. & Witholt, B.(1998). Enzymology and molecular genetics of PHAmcl biosynthesis. In Biochemical Principles and Mechanisms of Biosynthesis and Degradation of Polymers, pp. 48–56. Edited by A. Steinbüchel. Weinheim, Germany: Wiley-VCH.
Maier, R. M. & Soberon-Chavez, G.(2000).Pseudomonas aeruginosa rhamnolipids, biosynthesis and potential applications. Appl Microbiol Biotechnol54, 625–633.[CrossRef][Google Scholar]
Moller, S., Sternberg, C., Andersen, J. B., Christensen, B. B., Ramos, J. L., Givskov, M. & Molin, S.(1998).In situ gene expression in mixed-culture biofilms, evidence of metabolic interactions between community members. Appl Environ Microbiol64, 721–732.
[Google Scholar]
Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J.(2001). Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol183, 1047–1057.[CrossRef][Google Scholar]
Ochsner, U. A. & Reiser, J.(1995). Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A92, 6424–6428.[CrossRef][Google Scholar]
Ohman, D. E. & Chakrabarty, A. M.(1981). Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun33, 142–148.
[Google Scholar]
Qi, Q., Steinbüchel, A. & Rehm, B. H. A.(2000).In vitro synthesis of poly(3-hydroxydecanoate), purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa. Appl Microbiol Biotechnol54, 37–43.[CrossRef][Google Scholar]
Rahim, R., Ochsner, U. A., Olvera, C., Graninger, M., Messner, P., Lam, J. S. & Soberon-Chavez, G.(2001). Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis. Mol Microbiol40, 708–718.[CrossRef][Google Scholar]
Rehm, B. H. A.(2003). Polyester synthases, catalysts for plastics. Biochem J376, 15–33.[CrossRef][Google Scholar]
Rehm, B. H. A. & Steinbüchel, A.(1999). Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol25, 3–19.[CrossRef][Google Scholar]
Rehm, B. H. A., Krüger, N. & Steinbüchel, A.(1998). A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis – the phaG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein coenzyme A transferase. J Biol Chem273, 24044–24051.[CrossRef][Google Scholar]
Rehm, B. H. A., Mitsky, T. A. & Steinbüchel, A.(2001). Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads, establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli. Appl Environ Microbiol67, 3102–3109.[CrossRef][Google Scholar]
Ren, Q., de Roo, G., Kessler, B. & Witholt, B.(2000). Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin. Biochem J349, 599–604.[CrossRef][Google Scholar]
Ruiz, J. A., Lopez, N. I., Fernandez, R. O. & Mendez, B. S.(2001). Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms. Appl Environ Microbiol67, 225–230.[CrossRef][Google Scholar]
Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schweizer, H. P. & Hoang, T. T.(1995). An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene158, 15–22.[CrossRef][Google Scholar]
Simon, R., Priefer, U. & Pühler, A.(1983). A broad host range mobilization system for in vivo genetic engineering, transposon mutagenesis in Gram-negative bacteria. Bio/Technology1, 784–791.[CrossRef][Google Scholar]
Webb, J. S., Givskov, M. & Kjelleberg, S.(2003). Bacterial biofilms, prokaryotic adventures in multicellularity. Curr Opin Microbiol6, 578–585.[CrossRef][Google Scholar]
Zhang, Y. & Miller, R. M.(1992). Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl Environ Microbiol58, 3276–3282.
[Google Scholar]
The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation