Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil
Introduction
Phenylacetic acid (PA) and hydroxylated derivatives of PA are produced as catabolic intermediates by both bacteria and fungi growing on a wide variety of different naturally occurring aromatic compounds such as aromatic amino acids or lignin as well as various synthetic aromatic compounds. Several bacteria and fungi utilize aromatic amino acids as carbon sources either aerobically (Sariaslani et al., 1974) or anaerobically (Harwood et al., 1999). Aerobic metabolism of PA by fungi such as Exophiala jeanselmei (Cox et al., 1996) and Aspergillus nidulans (Mingot et al., 1999) typically involves the sequential introduction of hydroxyl groups in reactions catalysed by monooxygenases, generating homogentistate as a ring cleavage intermediate. The evidence for direct oxygenation of PA by bacteria is much less clear. It has been proposed that many Pseudomonads convert PA to 4-hydroxyphenylacetate, which is further oxidised to 3,4-dihydroxyphenylacetate (Dagley et al., 1952, Agrawal et al., 1996), while in Flavobacterium sp., PA is believed to be converted to 3-hydroxyphenylacetate and subsequently to 2,5-dihydroxyphenylacetate (Van den Tweel et al., 1988). In Nocardia salmonicolor, 2-hydroxyphenylacetate has been reported as the initial product of PA metabolism (Sariaslani et al., 1974). Recently, a new pathway for aerobic PA metabolism involving the activation of PA to PA-CoA has been reported in different bacterial species including Pseudomonas putida U (Olivera et al., 1998), Escherichia coli (Ferrández et al., 1998) and Azoarcus evansii (Mohamed et al., 2002). Interestingly A. evansii utilizes two different pathways for PA degradation (Mohamed et al., 2002, Rost et al., 2002). Furthermore, microbial genome projects have uncovered homologous pathways in bacteria not previously considered to be PA utilisers, for example, Bordetella sp., Deinococcus radiodurans, Ralstonia metallidurans and Streptomyces coelicolor (Mohamed et al., 2002), expanding our knowledge regarding the diversity of organisms able to degrade PA.
The PA catabolic pathway in P. putida strain U, the so called phenylacetyl-CoA catabolon (Olivera et al., 1998), is believed to be a convergent route responsible for the catabolism of structurally related compounds and much interest has centred on PA metabolism from a biotechnological standpoint (Luengo et al., 2001) particularly in relation to its potential use as a starting material for the biosynthesis of natural penicillins (Luengo et al., 1986, Luengo et al., 2001, Ferrández et al., 1998).
In an attempt to assess the biodiversity, which exists with respect to PA metabolism within a population of soil microorganisms we screened for new isolates, which were capable of growth with PA as the sole source of carbon and energy. In addition we performed preliminary biochemical analysis on these strains in an attempt to determine the pathway for PA metabolism in operation across the various genera which we isolated.
Section snippets
Chemicals
Phenylacetic acid (PA), ortho-hydroxyphenylacetic acid, meta-hydroxyphenylacetic acid, para-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid were purchased at the highest available purity from Fluka Chemie AG, Buchs Switzerland.
Media and buffers
Mineral salts medium (Evans) was made as previously described (Evans et al., 1970). All sterilised growth substrates were added to the medium post-autoclaving to a concentration of 5 mM.
Microbial strains
All PA degrading strains, forming the PA
16S rDNA gene sequence analysis
Phylogenetic analysis of 16S rDNA sequences from isolates capable of utilizing PA as a sole source of carbon and energy is shown in Fig. 1. The 18 representatives from the RFLP analysis did not cluster into a monophyletic group. They were distributed between the Gram-positive and Gram-negative divisions (Table 1). The isolates that clustered in the Gram-positive division were divided between two phyla, the Firmicutes and Actinobacteria (Brosius et al., 1978). The 16S rDNA sequence identity
Discussion
Molecular analysis was performed on a group of soil isolates which were selected based on their ability to utilize PA as a sole source of carbon in an attempt to assess the biodiversity which exists with respect to the metabolism of PA within typical populations of soil microorganisms. 16S rDNA gene analysis reveals a wide diversity of organisms capable of growth on PA (Table 1). Further investigation of the phylogenetic distribution of the isolates shows that these organisms displayed 16S rRNA
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