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Applied and Environmental Microbiology, August 2009, p. 5100-5110, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00133-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Worldwide Prevalence of Class 2 Integrases outside the Clinical Setting Is Associated with Human Impact{triangledown} ,{dagger}

Carlos M. Rodríguez-Minguela,1,{ddagger} Juha H. A. Apajalahti,2 Benli Chai,1,3 James R. Cole,1,3 and James M. Tiedje1*

Center for Microbial Ecology, Michigan State University, 540 Plant and Soil Sciences Building, East Lansing, Michigan 48824-1325,1 Alimetrics Ltd., Koskelontie 19 B, 02920 Espoo, Finland 02460,2 Ribosomal Database Project, Michigan State University, 2225A Biomedical and Physical Sciences Building, East Lansing, Michigan 48824-43203

Received 19 January 2009/ Accepted 1 June 2009

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (≤98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised ~70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction (~40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (≤98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.

* Corresponding author. Mailing address: Center for Microbial Ecology, Michigan State University, 540 Plant and Soil Sciences Building, East Lansing, MI 48824-1325. Phone: (517) 353-9021. Fax: (517) 353-2917. E-mail: tiedjej{at}msu.edu

{triangledown} Published ahead of print on 5 June 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: University of Puerto Rico, Mayagüez Campus, Biology Department, P.O. Box 9012, 00681-9012, Mayagüez, PR.

Applied and Environmental Microbiology, August 2009, p. 5100-5110, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00133-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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