1. ¹Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA
2. ²Department of Microbiology, Universidade de Sao Paulo, Instituto de Ciencias Biomedicas II, Sao Paulo, Brazil
3. ³Department of Natural Products, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
4. ⁴Department of Zoology, Michigan State University, East Lansing, MI, USA
5. ⁵Institute for Environmental Genomics, Department of Botany and Microbiology, University of Oklahoma, Norman, OK, USA

Correspondence: Dr MB Leigh, Institute of Arctic Biology, University of Alaska Fairbanks, PO Box 757000, Fairbanks, AK 99775-7000, USA. E-mail: mb.leigh@uaf.edu

Received 23 January 2007; Revised 21 March 2007; Accepted 21 March 2007; Published online 24 May 2007.

Abstract

Bacteria and functional genes associated with biphenyl (BP) degradation in the root zone of an Austrian pine (Pinus nigra L.) growing naturally in polychlorinated-BP (PCB)-contaminated soil were identified using stable isotope probing (SIP) integrated with comprehensive functional gene analyses. SIP revealed 75 different genera that derived carbon from ¹³C-BP, with Pseudonocardia, Kribella, Nocardiodes and Sphingomonas predominating carbon acquisition. Rhodococcus spp. were not detected with SIP, despite being the most abundant BP utilizers isolated from agar plates. Only one organism, an Arthrobacter spp., was detected as a BP utilizer by both cultivation and SIP methods. Time-course SIP analyses indicated that secondary carbon flow from BP-utilizing bacteria into other soil organisms may have occurred largely between 4 and 14 days incubation. Functional gene contents of the BP-utilizing metagenome (¹³C-DNA) were explored using the GeoChip, a functional gene array containing 6465 probes targeting aromatic degradative genes. The GeoChip detected 27 genes, including several associated with catabolism of BP, benzoate and a variety of aromatic ring hydroxylating dioygenase (ARHD) subunits. Genes associated with the -ketoadipate pathway were also detected, suggesting a potential role for this plant aromatic catabolic pathway in PCB degradation. Further ARHD analyses using targeted polymerase chain reaction primers and sequence analyses revealed novel dioxygenase sequences in ¹³C-DNA, including several sequences that clustered distantly from all known ARHDs and others that resembled known Rhodococcus ARHDs. The findings improve our understanding of BP degradation and carbon flow in soil, reveal the extent of culture bias, and may benefit bioremediation research by facilitating the development of molecular tools to detect, quantify and monitor populations involved in degradative processes.