title: Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins.
abstract: To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a beta-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional beta-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a beta-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.
authors.affiliation: Yale University, Department of Molecular, Cellular, and Developmental Biology, P.O. Box 208103, New Haven, CT 06520-8103, USA.
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Donato
authors.forename: Luke A
authors.initials: LA
authors.lastname: Moe
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Converse
authors.forename: Keith D
authors.initials: KD
authors.lastname: Smart
authors.forename: Flora C
authors.initials: FC
authors.lastname: Berklein
authors.forename: Patricia S
authors.initials: PS
authors.lastname: McManus
authors.forename: Jo
authors.initials: J
authors.lastname: Handelsman
mesh_terms: D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007612:Kanamycin / Q000494:pharmacology; D027845:Malus / Q000254:growth & development*; D056186:Metagenomics / Q000379:methods*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012987:Soil / Q000032:analysis*; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D011506:Proteins; D012987:Soil; D007612:Kanamycin; D002701:Chloramphenicol; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase; D019895:Transposases; D001618:beta-Lactamases
references.citation: Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):277-82
references.pmid: 8990199
references.citation: J Bacteriol. 1993 Feb;175(3):732-40
references.pmid: 8380801
references.citation: Curr Opin Pharmacol. 2003 Oct;3(5):474-9
references.pmid: 14559091
references.citation: Antimicrob Agents Chemother. 2002 May;46(5):1402-9
references.pmid: 11959575
references.citation: Appl Environ Microbiol. 2008 May;74(10):3302-5
references.pmid: 18359830
references.citation: ISME J. 2009 Feb;3(2):243-51
references.pmid: 18843302
references.citation: Am J Respir Med. 2003;2(4):321-32
references.pmid: 14719998
references.citation: FEMS Microbiol Ecol. 2001 Mar;35(1):37-48
references.pmid: 11248388
references.citation: Science. 2003 Nov 28;302(5650):1569-71
references.pmid: 14645850
references.citation: Appl Environ Microbiol. 1997 Jul;63(7):2647-53
references.pmid: 9212415
references.citation: BMC Genomics. 2008 Feb 08;9:75
references.pmid: 18261238
references.citation: Science. 2006 Jan 20;311(5759):374-7
references.pmid: 16424339
references.citation: FEMS Microbiol Ecol. 2002 Nov 1;42(2):277-88
references.pmid: 19709288
references.citation: Appl Environ Microbiol. 2002 Apr;68(4):1854-63
references.pmid: 11916705
references.citation: Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321-31
references.pmid: 6109327
references.citation: Gene. 1995 Jan 11;152(1):47-51
references.pmid: 7828927
references.citation: Appl Environ Microbiol. 2003 May;69(5):2684-91
references.pmid: 12732537
references.citation: Antimicrob Agents Chemother. 2005 May;49(5):1708-13
references.pmid: 15855485
references.citation: Antimicrob Agents Chemother. 2007 Jul;51(7):2359-65
references.pmid: 17438050
references.citation: Biochemistry. 2007 May 1;46(17):5270-82
references.pmid: 17417880
references.citation: Clin Infect Dis. 2009 Jan 1;48(1):1-12
references.pmid: 19035777
references.citation: Antimicrob Agents Chemother. 2005 Aug;49(8):3289-96
references.pmid: 16048938
references.citation: Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):1152-6
references.pmid: 9927709
references.citation: Clin Microbiol Infect. 2006 Mar;12(3):270-8
references.pmid: 16451415
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Appl Environ Microbiol. 1996 Jun;62(6):1935-43
references.pmid: 8787391
references.citation: Curr Microbiol. 2002 Jul;45(1):63-9
references.pmid: 12029529
references.citation: J Biol Chem. 2000 Sep 8;275(36):28075-82
references.pmid: 10825176
references.citation: Environ Microbiol. 2004 Sep;6(9):981-9
references.pmid: 15305923
references.citation: Environ Microbiol. 2007 Jan;9(1):197-205
references.pmid: 17227424