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 β-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.
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Acknowledgements
This work benefited from the technical assistance of Anthony Gaca, Cleopatra Mwansa and Najibah Rehman. Thanks to Mike Manefield and Mark J Bailey for training in SIP techniques. We thank Liyou Wu for providing GeoChips, Gene Wickham for training in hybridization methods and Ye Deng and Dan Cardin for bioinformatic support. T-RF prediction and Rieske center HMM analyses were performed by Benli Chai. SIP incubation methods were developed by Joonhong Park, Debora Rodrigues provided real-time PCR protocols and Stephan Gantner contributed to soil DNA extraction method development. Field site access was provided generously by Colorlak, logistical support provided by Vítek Matĕjů, Martina Macková and Tomáš Macek and Nathan Stewart assisted with field work. This work was supported by David L Boren (NSEP) Graduate International Fellowships and an NSF Postdoctoral Fellowship to MBL, NIEHS grant 2P42ES04911 under the Superfund Basic Science Program, and The Center for Microbial Ecology.
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Leigh, M., Pellizari, V., Uhlík, O. et al. Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs). ISME J 1, 134–148 (2007). https://doi.org/10.1038/ismej.2007.26
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DOI: https://doi.org/10.1038/ismej.2007.26
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