Analysis of catabolic genes for naphthalene and phenanthrene degradation in contaminated New Zealand soils Gareth Lloyd-Jones a; *, Andrew D. Laurie a;b , David W.F. Hunter a , Rhonda Fraser a a Landcare Research, Private Bag 3127, Hamilton, New Zealand b Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand Received 30 August 1998; received in revised form 4 December 1998; accepted 6 December 1998 Abstract Culture-dependent and culture-independent methods were used to investigate the diversity of three polycyclic aromatic hydrocarbon (PAH) catabolic genes in contaminated soils. PAH-degrading bacteria were isolated based on growth at the expense of naphthalene (44 isolates) or phenanthrene (35 isolates). Of these 79 PAH-degraders, 53% (42 isolates) failed to hybridise with three gene probes specific for PAH degradation. The gene for the naphthalene dioxygenase iron^sulfur protein (nahAc) from Pseudomonas putida G7 hybridised to 45% (20/44) of the culturable naphthalene-degrading bacteria of the `classical' nah-type, whilst analogues of the bacterial glutathione S-transferase (GST) encoding gene of Sphingomonas paucimobilis EPA505 were associated with culturable phenanthrene-degrading isolates and hybridised to 29% (10/35) of these isolates. Apart from the host strain Burkholderia RP007, we were not able to detect the phnAc gene amongst cultured isolates by hybridisation or PCR, though could directly amplify this gene from contaminated soils. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Naphthalene ; Phenanthrene ; Polycyclic aromatic hydrocarbon ; nah-like; phn ; Glutathione S-transferase 1. Introduction Enumeration of the PAH-degrading bacterial pop- ulation in contaminated soils using traditional micro- biological methods can take an inordinate length of time, and often underestimates numbers as a result of our inability to cultivate the majority of soil or- ganisms. Molecular biological approaches using hy- bridisation or polymerase chain reaction (PCR) methods are valid alternatives. An impediment to these approaches is the available database of appro- priate gene sequences, which for PAH-degrading bacteria is currently limited. Another obstacle arises from extrapolating sequence data originally obtained from readily cultivated bacterial groups which can bias our perception of the importance of readily iso- lated genotypes in soil microbial ecology, and does not provide for an adequate description of natural populations. A prime example of a readily isolated group of soil bacteria are Pseudomonas and Pseudomonas-like strains isolated for their ability to degrade naphtha- lene and its close analogues. Numerous examples of 0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII:S0168-6496(98)00131-7 * Corresponding author. Tel.: +64 (7) 858-3700; Fax: +64 (7) 858-4164; E-mail:lloyd-jonesg@landcare.cri.nz FEMS Microbiology Ecology 29 (1999) 69^79 Downloaded from https://academic.oup.com/femsec/article/29/1/69/766604 by guest on 07 January 2024