Phenylacetate-coenzyme A ligase is induced during growth on phenylacetic acid in different bacteria of several genera

FEMS Microbiology Letters 108 (1993) 1-6 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier FEMSLE 05328 Srdjan Vitovski School of Biological Sciences, University of Wales, Bangor, Gwynedd, UK (Received 20 October 1992; revision received 29 December 1992; accepted 4 January 1993) Abstract: Nine different bacterial strains that utilise phenylacetic acid as the only carbon and energy source were isolated from samples of different geographical origin. The isolates were characterised taxonomically and physiologically. Evidence is presented that in all the isolates as well as in four previously isolated control strains with the ability to utilize phenylacetic acid, the enzyme phenylacetate-CoA ligase is specifically induced during growth on phenylacetic acid. The Michaelis constant (K m) in one Pseudomonas strain was sufficiently low (-1 mM) to suggest that the enzyme may have a role in phenylacetic acid metabolism. Key words: Coenzyme A; Phenylacetic acid; Pseudomonas Introduction The pathways of aerobic catabolism of simple aromatic compounds by microbes have been well documented. With only one recently reported exception [19,20,26] these involve the enzymatic conversion of the compound into 1,2 or 1,4 dihydroxylated benzenes followed by the opening of the aromatic ring by ring-cleaving dioxygenases [1]. One compound for which there is some doubt as to whether such a route operates is phenylacetic acid (C6HsCH2COOH, PAA) in spite of Correspondence to: S. Vitovski, School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, UK. PAA being a fairly common source of carbon and energy for bacteria from a wide range of different genera. Pathways have been described for monohydroxylated derivatives of PAA which fit into the general pattern of aromatic catabolism outlined above [2-4], but none of the possible routes which might be expected for PAA catabolism by analogy with other aromatic catabolism appear to operate for many PAA-degrading bacteria [3,5,6]. Recently, however, a single report of a new enzyme, phenylacetate-CoA (PA-CoA) ligase, which catalyses the reaction: ChHsCHzCOOH + CoA + ATP = C6HsCH2COCoA + AMP + PPi has been described in the PAA-degrading strain Pseudomonas putida U [7] and may be the first step of what would be a very novel route for aromatic catabolism under aerobic conditions. Phenylacetate-coenzyme A ligase is induced during growth on phenylacetic acid in different bacteria of several genera The main aim of this work has been to investigate whether the involvement of PA-CoA ligase is general in the bacterial utilization of PAA. Materials and Methods Enrichment and isolation Soil samples were collected from different geographical areas and suspended in minimal medium supplemented with 2.5 mM PAA as the only source of carbon and energy. Batch enrichment and growth of bacteria were carried out in Erlenmeyer flasks at 30°C on a rotary shaker at 200 rpm. After two transfers of the enrichment cultures, pure cultures were obtained by streaking the cultures on minimal medium agar containing 2.5 mM PAA. Enzyme assays Enzyme assays were performed on cells grown to late exponential phase in 500 ml liquid minimal medium supplemented by the appropriate carbon source. Cells were harvested by centrifugation. Cell-free extracts were prepared and PACoA ligase was assayed by measuring the rate of production of phenylacetyl hydroxamate as previously described [7]. The kinetic measurements were made using the standard assay procedure keeping all concentrations constant except that of the PAA which varied between 0.1 mM and 22 mM. Aliquots were removed from each assay after 5, 10, 15 and 20 min to develop the colour and the rate of reaction were determined from a linear plot through the four time points. The Michaelis constant (K m) was determined using the program ENZPACK3 (Biosoft, Cambridge, UK). Protein estimation Protein concentration in cell-flee extracts was determined by method of Lowry et al. [11] using bovine serum albumin as a standard. Table 1 Taxonomic and growth characteristics of PAA-utilising strains used in this study Strain Generation time (min) PAA tolerance (mM) Source Alcaligenes denitrificans 31P Pseudomonas cepacia 54P Pseudomonas putida 63P Pseudomonas fluorescens 73P Pseudomonas cepacia 91P Pseudomonas cepacia 104P 2 700 220 85 130 480 175 140 420 85 460 1080 120 140 7 > 25 > 25 15 > 25 7 20 > 25 15 > 25 2.5 10 > 25 Greece Netherlands Netherlands Algeria Algeria Yugoslavia Yugoslavia Yugoslavia Algeria ATCC ATCC P.A. Williams P.A. Williams C D C group IV l12P Pseudomonas cepacia 132P Acinetobacter calcoaceticus E G B E. coli A T C C l l l 0 5 Providencia rettgeri ATCC31052 Pseudomonas sp. MT14 Pseudomonas putida U Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table 1. For isolation, growth test and induction experiments minimal medium [8] was used, supplemented by appropriate carbon sources in the following concentrations: PAA, 2.5 mM; benzoate, 5 mM; succinate, 10 mM. All strains were grown at 30°C. Growth of the culture was measured photometrically by determining the turbidity at 550 nm. Strain characterization Taxonomic identification was carried out based by published criteria [9,10]. Chemicals PAA, CoA and ATP were from Sigma. All other chemicals were of the highest purity commercially available. Results Growth of strains on PAA The capability of different strains to utilise PAA as the only source of carbon and energy for growth is shown in Table 1. The doubling time and the maximum concentration of PAA which still support growth varies to considerable extent. For example, for Providencia rettgeri the maximum tolerable concentration of PAA is 2.5 mM and corresponding doubling time is 2640 min. By contrast Pseudomonas putida 63P is able to grow on up to 25 mM PAA with a generation time of 85 min. There is considerable variation within the Table 2 Specific activities of P A - C o A ligase in different strains during growth on P A A Strain P A - C o A ligase activity ( n m o l / m i n / m g protein) 31P 54P 63P 73P 91P 104P 112P 132P EGB A T C C 11105 ATCC31052 MT 14 Strain U 14.2 21.1 16.7 19.8 11.7 32.5 20.8 12.6 19.6 26.9 23.5 30.4 24.5 Specific activities of cells grown on succinate or benzoate as sole source of carbon and energy were < 2 n m o l / m i n / m g protein in all cases. Enzyme activities in cell-free extracts Cells grown on PAA, benzoate and succinate were assayed for enzyme activity. In cell-free extracts induced levels of PA-CoA ligase activity could be detected only during growth on PAA (Table 2). Michaelis constant The Michaelis constant for the enzyme from strain 63P was calculated by the Wilkinson method of non-linear regression to be 0.92 mM with a standard error of _+0.18. Discussion The route of routes by which PAA is metabolised presents some problems. Earlier reports (...truncated)