Characterization of novel antibiotic resistance ... - Wiley Online Library

Environmental Microbiology (2011) 13(4), 1101–1114

doi:10.1111/j.1462-2920.2010.02422.x

Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples

Gloria Torres-Cortés,1 Vicenta Millán,1 Hugo C. Ramírez-Saad,2 Rafael Nisa-Martínez,1 Nicolás Toro1 and Francisco Martínez-Abarca1* 1 Genetic Ecology Group, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain. 2 Laboratorio de Ecología Molecular, Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana – Xochimilco, 04960 México. Summary

emi_2422

1101..1114

The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (g- and a-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (Km of 8.9 mM for NADPH and 3.7 mM for dihydrofolate and IC50 of 20 mM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences. Received 27 October, 2010; accepted 15 December, 2010. *For correspondence. E-mail [email protected]; Tel. (+34) 958181600; Fax (+34) 958129600.

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd

Introduction The diversity of the microbes hidden in soil (up to 109 microorganisms/g; Trevors, 2010) has been explored with a view to developing new clinical and medical applications (Daniel, 2004; Da Costa et al., 2007; Molinari, 2009). The most significant application to date has undoubtedly been the implementation of natural antibiotic products, which has revolutionized our approach to treating infectious diseases. More than 80% of the antibiotics in clinical use originate from soil bacteria, either directly, as natural products, or as their semisynthetic derivatives (Martin and Liras, 1989; Kieser et al., 2000). Thus, soil may serve as a hidden reservoir for antibiotic resistance that has already emerged or has the potential to emerge in clinically important bacteria (Riesenfeld et al., 2004; Da Costa et al., 2006; Allen et al., 2009; Donato et al., 2010). Consequently, an understanding of resistance determinants present in soil will provide information not only about antibiotic resistance frequencies, but also about novel mechanisms that may emerge as clinical problems and about the role of antibiotic resistance genes in natural environments (Wright, 2007). The success of antibiotics for treating infections and, conversely, the risk to human health posed by antibiotic resistance have focused research principally on the clinical setting (Aminov, 2009; Martinez, 2009). By contrast, the function of antibiotics in natural (non-clinical) environments has received relatively little attention. Do antibiotics and antibiotic resistance genes play the same role in non-clinical environments? This may be the case in some instances, but there is compelling evidence to indicate very different roles in mediating the interactions of microbial communities in natural environments (Fajardo et al., 2009; Martinez et al., 2009). In recent years, metagenomic tools have identified antibiotic resistance genes in DNA libraries from environmental samples (Riesenfeld et al., 2004; Allen et al., 2009; Mori et al., 2008; Kazimierczak et al., 2009; Donato et al., 2010). The analysis of metagenomic clones is often based on random sequencing (Venter et al., 2004; Tringe et al., 2005) or the PCR amplification of target genes (De la Torre et al., 2003; Henriques et al., 2006; Demaneche

1102 G. Torres-Cortés et al. et al., 2008). Alternatively, functional metagenomics, which involves the heterologous expression of metagenomic DNA in a surrogate host and activity-based screening, provides a means of discovering genes, the function of which may not be obvious from their sequence. The term ‘antibiotic resistome’ was proposed for the collection of all antibiotic resistance genes in microorganisms, including those from pathogenic and non-pathogenic bacteria. This term exploits the concept of a unique reservoir of antibiotic resistance genes in environmental samples (Wright, 2007). In this study, we aimed to discover new antibiotic resistance genes, using metagenomic libraries constructed from DNA extracted from three different soils, followed by heterologous expression and the screening of resistance activity against different antibiotics. We hypothesized that genes conferring resistance to antibiotics are present in the environment, even in the absence of strong selection pressure that would be associated with high antibiotic concentrations. The identification of new resistance genes, particularly those potentially present in nonculturable microbes, may facilitate prediction of the emergence of resistance. We constructed libraries from fragments of cloned soil DNA and selected clones expressing resistance to various antibiotics targeting different bacterial functions. We identified genes encoding proteins that inactivated the antibiotic (b-lactamases for ampicillin, Ap, and acetyltransferases for gentamicin, Gm), genes encoding multidrug pumps conferring resistant to chloramphenicol (Cm) and genes conferring resistance to the folate antagonist trimethoprim (Tmp). Trimethoprim competitively inhibits the enzyme dihydrofolate reductase (DHFR), which is responsible for reducing dihydrofolate to tetrahydrofolate (Huovinen et al., 1995; Sköld, 2001). Cells that cannot regenerate tetrahydrofolate suffer defective DNA synthesis, eventually leading to death. Acquired resistance to Tmp includes mutations in the promoter region or in the DHFR structural gene (dhfr; Huovinen et al., 1995; Sköld et al., 2001), but the most common mechanism of Tmp resistance is the complementation of a Tmp-sensitive DHFR with a Tmp resistance-mediating DHFR. In this study, we isolated a new Tmp-resistant gene encoding a 26.8 kDa protein closely resembling reductases not assigned to dhfr genes. This protein, Tm8-3, confers Tmp resistance to Escherichia coli and Sinorhizobium meliloti. We demonstrate that this gene encodes a bona fide DHFR enzyme. This study thus provides an additional example of the use of functional metagenomics for the identification of resistance genes encoding proteins with activities that cannot be predicted from their amino acid sequences. This may facilitate prediction of the emergence of new resistance determinants.

Results Construction of metagenomic libraries and isolation of clones expressing antibiotic resistance Three metagenomic libraries were constructed with DNA extracted from different soils: one from an agricultural soil close to our research centre in Granada, Spain (the EEZ sample) and two from Mexican soils in the vicinity of Mammillaria carnea plants from the Tehuacan-Cuicatlan biosphere collected at the end of the rainy and dry seasons and named R-Mex and D-Mex respectively. Using the lambdaZAP-expressing phagemid system, we obtained about 550 000 recombinant clones only about 1/10 of which corresponded to the EEZ library. Restriction analysis of 10 randomly selected clones from each library showed the mean insert size to be 6.5–7 kb. About 3.5 Gb of environmental DNA was cloned in these libraries. Clones were selected from the libraries on the basis of their ability to grow in the presence of each of six antibiotics: Ap, Gm, streptomycin, Cm, erythromycin and Tmp. We obtained no streptomycin-resistant clones and a large number of erythromycin-resistant clones, most of which were found to be false positives in a secondary screening test. We identified 11 clones containing metagenomic DNA conferring resistance to four of the six antibiotics used in E. coli (Fig. 1; Table 1). We measured the minimum inhibitory concentrations (MICs; Table 1) of the antibiotics used for selection of the 11 resistance clones. An analysis of the MIC values for the three clones resistant to Amp showed that pBKAp6-8 from the D-Mex library had an MIC twice that of pBKApE1 and pBKApE14 from the EEZ library. A similar situation was observed for the two clones resistant to the aminoglycoside Gm, with the MIC value of pBKGm8-3 from the R-Mex library being 2.5 times higher than that of pBKGmE1 from the EEZ library. This pattern was even more marked for clones conferring Tmp resistance, with the MIC value of pBKTm8-3 (R-Mex library) being 3.0 times higher than the three clones obtained from the EEZ library (pBKTmE1, pBKTmE5 and pBKTmE25; Table 1). In general, the clones obtained from the Mexican soil libraries conferred resistance at higher MIC values in E. coli than did those obtained from the EEZ soil. Although this feature can be due to position and orientation of the antibiotic-resistant open reading frame (ORF) respect to the Lac promoter (Fig. 1) Identification of antibiotic resistance genes and phylogenetic analyses The size, gene organization and genetic similarities of the inserts are summarized in Fig. 1 and Table 1. The inserts of resistant clones were between 4 and 10 kb in size. For identification of the ORFs responsible for the resistance

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 1101–1114

Novel antibiotic resistance genes from soil metagenome 1103

Fig. 1. Schematic diagram of the organization of the ORFs from the 11 inserts conferring antibiotic resistance in the metagenomic soil libraries. The size of the inserts is indicated, to scale, as a dashed line, and the sequence deposited in the database is indicated as a solid line. ORFs are represented by arrows, which are shaded in grey for ORFs involved in antibiotic resistance. Asterisks indicate incomplete ORFs. The percentage similarity to database entries (where appropriate) is shown in brackets below the ORFs.


62.1

63.5

65.6

Cm-resistant clones pBKCmE6 (4 kb/4003) EEZ

Tm-resistant clones pBKTm8-3 (7.5 kb/1490) R-Mex

66.6

52.7

Cm-resistant clones pBKCmE1 (9 kb/4428) EEZ

pBKGmE1 (7 kb/1775) EEZ

Gm-resistant clones pBKGm8-3 (8 kb/8046) R-Mex

GenBank Accession No. ORFd

150 (30¥)

50 (5¥)

50 (5¥)

20 (8¥)

50 (20¥)

2

FN640474 1*

2 3 4

FN640470 1*

5 6*

FN640469 1* 2 3 4

2

Hypothetical protein (457), YP_001942216.1 Hypothetical protein TPR repeat protein (SEL1 SUPERFAMILY) (327), YP_002149794.1 Unknown MOSC domain protein (263), EDX86104 Gentamicin 3⬘-N-acetyltransferase (157), YP_002513892 GCN5-related N-acetyltransferase (140), YP_563491 Glyoxalase/bleomycin resistance protein/dioxygenase (140), ZP_01108256 Tellurite resistance protein TehB (197), YP_392289 Glyoxalase/bleomycin resistance protein/dioxygenase (133), YP_001869281 Glycosyl transferase family protein (413),YP_771367

Dihydrodipicolinate synthase (321), YP_001370746 Adenylate/guanylate cyclase (468), YP_002972998

Transcriptional regulator, LysR family (292), YP_002282440.1

Lipid-A-disaccharide synthase (383), YP_001951013 b-lactamase class A (281), YP823559 N-acetylmuramyl-L-alanine amidase (320), ZP_03631130 b-lactamase class A (352), YP_002129833.1 Hypothetical protein (455), YP_674672.1 Hypothetical protein (177), YP_673334.1 b-lactamase (385), YP_002976497.1

Most similar protein (No. of encoded amino acids), GenBank Accession No. of similar proteinf

Tm8-3/249

> 204

CmE6/405 154 75

> 202

184 > 61

> 74 CmE1/401 215 247

Two-component response regulator protein (119) YP_002545888 3-oxoacyl-(acyl-carrier-protein) reductase (267) ZP_01470074

Bcr/CflA subfamily drug transporter (399), YP_001355014 Unknown XRE family transcriptional regulator (71), YP_001685144

Putative transcriptional regulator (229), TetR NP_103053

Putative reductase (194), ZP_01004299 Bcr/CflA subfamily drug transporter (401), NP_103052 Putative transcriptional regulator (229), TetR NP_103053 Pyridoxal phosphate biosynthetic protein PdxJ (247), ZP_02166111 Acetolactate synthase 3 regulatory subunit (190), NP_386219 Acetolactate synthase, large subunit, biosynthetic type (592), YP_002500595

GmE1/> 234 Aminoglycoside 3⬘-N-acetyltransferase 262), AF486581

> 286

196 > 92

8 9* FN640468 1*

120 265 Gm8-3/165 142 125

3 4 5 6 7

> 242 143

327 > 262

5 6*

FN640465 1* 2

289

391 Ap6-8/319 > 170 ApE1/345 > 62 188 ApE14/380

Protein name/length (aa)e

4

100 (12.5¥) FN640464 1 2 50 (6.25¥) FN640466 1* 2 50 (6.25¥) FN640467 1* 2 3

G+C MIC (%) (mg ml-1)c

Ap-resistant clones pBKAp6-8 (6.9 kb/2951) 56.1 D-Mex pBKApE1 (6.5 kb/3033) 56.8 EEZ pBKApE14 (10 kb /5579) 53.1 EEZ

Plasmid (insert size and bp sequenced)/Libraryb

2e-18 1e-135 6e-51 5e-87

8e-81

5e-77

1e-67 2e-12

6e-65 5e-52 4e-40 2e-22

9e-23 5e-13

2e-79 1e-140

8e-131

2e-65 1e-81 6e-40 4e-65 2e-21 5e-62 7e-139

E-valuef

Synechococcus sp. BL107

3e-40

1e-12

6e-23

Caulobacter sp. K31 Agrobacterium radiobacter K84

7e-116

3e-53 Mesorhizobium loti

Mesorhizobium loti MAFF303099

Sinorhizobium meliloti 1021 4e-73 Methylobacterium nodulans ORS 2060 7e-16

Loktanella vestfoldensis SKA53 Mesorhizobium loti MAFF303099 Mesorhizobium loti MAFF303099 Hoeflea phototrophica DFL-43

Rhizobium leguminosarum bv. viciae 3841 Uncultured bacterium

Thiomicrospira crunogena XCL-2 Nostoc punctiforme PCC 73102

Synechococcus sp. PCC 7335 Thioalkalivibrio sp. HL-EbGR7 Shewanella denitrificans OS217 Flavobacteriales bacterium HTCC2170

Chlorobium limicola DSM 245 Proteus mirabilis HI4320

Geobacter lovleyi SZ Solibacter usitatus Ellin6076 bacterium Ellin514 Phenylobacterium zucineum HLK1 Mesorhizobium sp. BNC1 Mesorhizobium sp. BNC1 Rhizobium leguminosarum bv. trifolii WSM1325 Rhizobium leguminosarum bv. trifolii WSM2304 Ochrobactrum anthropi ATCC 49188 Rhizobium leguminosarum bv. trifolii WSM1325

Organismf

Table 1. Annotation table of open reading frames (ORFs) predicted in the antibiotic-resistant clones from the soil environmental metagenome.a

41/58

42/57

75/85

57/74

55/70

71/82 62/77

58/73 63/76 50/64 65/76

69/76

54/65

61/76 46/59

44/65 69/78 55/76 43/65

32/48 47/69

55/71 90/95

80/87

40/57 57/70 59/74 45/58 79/87 65/76 63/79

Bacteria/Proteobacteria

Bacteria/Proteobacteria/aproteobacteria


Bacteria/Proteobacteria/bproteobacteria


Bacteria/Proteobacteria/aaproteobacteria

Bacteria


Aa identity/ Phylogenetic classification: similarity Domain/Phylum/Classg %f

1104 G. Torres-Cortés et al.


TmE5/174 > 119 TmE25/68 163 334 2 1* 2 3 4 61.1 pBKTmE25 (6.8 kb/3396) EEZ

50 (10¥)

FN640473

371 1 FN640472 50 (10¥) 59.8 pBKTmE5 (6.5 kb/1844) EEZ

a. Metagenomic soil libraries constructed in the lambda Zap System (Stratagene) were screened to find clones resistant to four antibiotics: Ap, ampicillin; Gm, gentamicin; Cm, chloramphenicol; Tmp, trimethoprim. b. Name of the plasmid (environmental DNA insert size of the antibiotic-resistant clones); D-Mex, soil sample from Mexico obtained in the dry season; R-Mex, soil sample from Mexico obtained in the rainy season; EEZ, soil sample from agricultural site in Granada Spain. c. Minimum inhibitory concentration. Measurements were made in metagenomic host cells (E. coli XLOLR strain) growing in LB broth supplemented with the corresponding antibiotic. Values are the median of three independent experiments. Numbers in parentheses indicates the fold difference with respect to empty vector control MIC values in the same host cells. d. ORFs involved in antibiotic resistance are shown in bold typeface and asterisks indicate incomplete ORFs. e. aa; amino acids. Proteins involved in antibiotic resistance are shown in bold typeface. f. Data obtained with BLASTp against NCBI database (November 2009). g. Phylogenetic classification of the DNA sequence fragments obtained with the PhyloPythia program (McHardy et al., 2007).

Bacteria/Proteobacteria/aproteobacteria 46/66 51/63 47/69 63/72 44/60 5e-41 5e-19 3e-41 3e-50 1e-74 Pseudomonas stutzeri A1501 Mesorhizobium loti MAFF303099 Pseudomonas syringae pv. oryzae str Phenylobacterium zucineum HLK1 Azorhizobium caulinodans ORS 571

Bacteria/Proteobacteria/ 52/65 1e-89 Mesorhizobium sp. BNC1

86/91 65/76 67/82 1e-136 2e-60 4e-59 264 TmE1/176 > 181 2 3 4*

Thymidylate synthase (264) NP_386233 Dhfr dihydrofolate reductase (175), NP_355014 HflK hydrolase serine protease transmembrane subunit K protein (361), YP_001979067 HflK hydrolase serine protease transmembrane subunit K protein (376), YP_674308 Dhfr dihydrofolate reductase (246), YP_001174482 protease subunit hflK (371), NP_103040 Dhfr dihydrofolate reductase (170), ZP_04590822 Acetyltransferase, GNAT family (160), YP_002131720 Hypothethical protein (350), YP_001525236.1

> 76 61.3 pBKTmE1 (4.5 kb/2336) EEZ

50 (10¥)

FN640471

1

UDP-glucose 4-epimerase (298), YP_002979022

Rhizobium leguminosarum bv. trifolii WSM1325 Sinorhizobium meliloti 1021 Agrobacterium tumefaciens str. C58 Rhizobium etli CIAT 652

5e-27

76/85


Novel antibiotic resistance genes from soil metagenome 1105 phenotype, clones were deleted with restriction enzymes and tested for maintenance of the resistance phenotype in E. coli. The sequences obtained from 11 plasmids showed 31 complete and 10 incomplete heterologous ORFs. The G+C content of the sequenced inserts varied from 52.7% to 66.6%, consistent with their diverse origins. The online program PhyloPythia (McHardy et al., 2007) was used to identify the possible origin of these metagenomic DNA fragments. It showed a predominance of the phylum Proteobacteria in the clones analysed (10 of the 11 clones; Table 1). The subphylum a-Proteobacteria was the most frequently represented among the isolated clones (four of the 11). The putative genes responsible for the resistance phenotypes were identified by sequencing of the full-length insert for the pBKGm8-3 and pBKCmE-6 clones, which confer resistance to Gm and Cm, respectively, in E. coli. We sequenced only derivative inserts that were smaller than the original plasmid for which the resistance phenotype was maintained, for the other nine clones (Fig. 1). Thus, ORFs encoding b-lactamases were found in the three clones conferring resistance to Ap on the E. coli host (Fig. 1, Table 1). These results suggest that the conferred resistance is due to inactivation of the Ap by disruption of the b-lactam ring. Clustering analysis of the amino acid sequence grouped the Ap6-8 and ApE1 proteins with class A b-lactamases. By contrast, the ApE14 protein was found to be related to class C b-lactamases. The conserved residues constituting the four elements surrounding the active site were present in all three clones (Ambler, 1980; Figs S1 and S2). The sequences of the three ORFs differed both from each other and from their best match (57%, 45% and 63% amino acid identity for Ap6-8, ApE1 and ApE14 respectively; Table 1). Phylogenetic analysis revealed that the clones from the EEZ library, ApE1 and ApE14, were closely related to the chromosomal b-lactamases of Phenylobacterium and Rhizobium respectively. However, the clone found in the D-Mex (Ap6-8) library belonged to a genus from a very different order: Solibacter (Table 1). Two clones conferred resistance to Gm in E. coli: one from the EEZ library (pBKGmE1) and the other from the R-Mex library (pBKGm8-3). Both clones contained ORFs likely to encode enzymes conferring resistance to aminoglycosides by 3′-N-acetylation (Fig. 1 and Table 1). The pBKGm8.3 clone conferring Gm resistance also contains putative genes related to tellurite and bleomycin resistance (Fig. 1 and Table 1). Both aminoglycoside 3′-Nacetyl transferases contain the ‘GNAT’ motif characteristic of GCN5-related enzymes (Dyda et al., 2000; Azucena and Mobashery, 2001). However, amino acids involved in the transfer of the acetyl group from acetyl-coenzyme A to the primary amine of the aminoglycoside were identified only in Gm8-3 (Fig. S4). Gm8-3 is related to an AAC(3)


1106 G. Torres-Cortés et al. cluster consisting of the six AAC (3)-I proteins (Fig. S4; Riccio et al., 2003; Ahmed et al., 2004). GmE1 was found to be the most similar (69% amino acid identity) to a 3′-N-acetyltransferase conferring resistance to the aminoglycoside kanamycin and identified by a metagenomic approach to the analysis of soil samples (Table 1; Courtois et al., 2003). GmE1 was found to be related to three AAC(3)-III enzymes, all of which have identical resistance profiles and the genes of which have been cloned from Pseudomonas spp. (Shaw et al., 1993). It was also found to be more distantly related to a cluster of four AAC(3) enzymes encoded by genes from actinomycetes. Phylogenetic analysis suggested that GmE1 may be a new AAC(3) III cluster gene (Fig. S4). Only the EEZ library contained clones conferring resistance to Cm (pBKCmE1 and pBKCmE6). The ORF2 of both clones was similar to proteins of the Bcr/CflA subfamily of drug transporters (Fig. 1 and Table 1). Phylogenetic analysis showed that these proteins, CmE1 and CmE6, were very similar to each other (65% amino acid identity) and to other drug transporters present in described soil proteobacteria (57% and 63% amino acid identity for CME6 and CME1 respectively; Fig. S5). CmE1 and CmE6 are located upstream from the gene encoding the predicted multidrug efflux pump and have a transcriptional regulator resembling the genome of Mesorhizobium loti (MAFF303099 strain), which belongs to the subphylum a-Proteobacteria. The proteins of the Bcr/CflA family are predicted to have 12 membrane-spanning alpha-helix regions, and these regions are present in the CME6 and CME1 proteins (Fig. S6). Furthermore, one of the best matches to both CmE1 and CmE6 was found to be an ORF encoding a multidrug transporter (n access: AAS90611) found in clone CR4 from an unculturable soil bacterium and conferring resistance to tetracycline (Fig. S5; Riesenfeld et al., 2004). Both the pBKCME1 and pBKCME6 clones were tested and found to confer resistance to tetracycline in E. coli Dh5a cells (data not shown). By contrast to previous reports (Donato et al., 2010), we identified no genes related to horizontal transfer or mobility, such as repeated elements, insertion sequences or transposons, in any of the 41 ORFs analysed (Table 1). Resistance to trimethoprim pBKTmE1, pBKTmE5 and pBKTmE25 (found in EEZ soil library) and pBKTm8-3 (found in R-Mex soil library) conferred resistance to Tmp in E. coli. They have inserts of 4.5, 6.5, 6.8 and 7.5 kb respectively. The three clones found in EEZ soil contained ORFs resembling a type I dhfr (Table 1 and Fig. 2). A gene encoding a subunit of the HflK protease was found downstream from the dhfr genes. This gene is normally related to chromosome-encoded dhfr genes. Phylogenetic analysis showed TmE1 to be

closely related to other DHFR proteins found principally in the bacteria of the order Rhizobiales and more distantly related to DHFRs from b- and g-proteobacteria. TmE5 and TmE25 are closely related proteins forming a separate cluster (Fig. 2). All three proteins contain conserved amino acid residues that interact with the folate and NADP groups of the active centre and variations of these positions involved in Tmp binding in the Tmp-resistant DHFRs of S. haemolyticus (Fig. 3A, Sekiguchi et al., 2005). TmE1, 5 and 25 were identified as chromosomal type I dhfr genes distantly related to the plasmid-encoded type II DHFRs (Fig. 2). The pBKTm8-3 clone conferring resistance to Tmp was found in the R-Mex library. By using restriction enzymes to make derivatives of the original 7.5 kb clone, we were able to restrict the DNA fragment conferring the phenotype of resistance to Tmp to as little as 1.8 kb of metagenomic DNA (Fig. 4A; pBKTm8-3X). None of the predicted ORFs within this 1.8 kb DNA fragment resembled a putative DHFR-encoding gene. ORF1 encodes a protein displaying 42% amino acid identity to some two-component response regulators (Accession No.: YP_002545888.1) and ORF2 encodes a protein displaying 41% amino acid identity to an 3-oxoacyl-(acyl-carrier-protein) reductase (Accession No.: ZP_01470074.1). We generated a frameshift mutation in each ORF to determine which of these proteins was responsible for resistance (Fig. 4). ORF2 disruption resulted in sensitivity to Tmp. Furthermore, a plasmid carrying only the coding sequence of ORF2 under the control of a constitutive promoter conferred resistance to Tmp in E. coli and S. meliloti host cells (Fig. 4B). The gene was named Tm8-3. Identification and biochemical characterization of a new Tmpr gene, Tm8-3, in a soil metagenomic library Tm8-3 corresponds to a 749 nt ORF encoding a putative 249-amino-acid protein. This protein confers resistance to Tmp, but its sequence very different from those of the DHFR proteins described to date (Fig. 2). It displays similarity to other reductases involved in lipid metabolism (3-oxoacyl-(acyl-carrier-protein) reductase). A Rossmannfold NAD(P)H/NAD(P)(+) binding domain found in numerous dehydrogenases and many other redox enzymes can be identified in Tm8-3. Furthermore, some specific conserved residues of the OAR proteins are also conserved in this protein (Fig. 3B). Two of the three active-site residues conserved in all bacterial and plant OARs are conserved in Tm8-3 (Y160 and K164), and the group of two arginine residues playing a key role in ACP binding (Wickramasinghe et al., 2006) is partially conserved in Tm8-3 (R135 and H181; Fig. 3B). For functional analysis of the ORF2 of the pBKTm8-3 gene, we overproduced and purified recombinant. After


Novel antibiotic resistance genes from soil metagenome 1107

Fig. 2. Neighbour-joining tree of aligned amino acid sequences of the Tm resistance genes found in this study. The alignment was performed with type I and II dhfr genes. Tm8-3 and its closest relative, the oxo-acyl carrier protein reductase (EAU70455), were used for tree rooting. Genera, species and strains are followed by their GenBank accession numbers. Some of the branches were grouped to improve the visibility of the tree (the number of sequences grouped is indicated in parentheses). Bootstrap values were calculated as a percentage of 1000 replicates and those above 70% are shown at the branching points. The sequences obtained in this study are depicted in bold. See Table S4 for amino acid sequence accession numbers. Scale bar = 0.2 changes/site.

loading the soluble crude extract on a Mono Q column, we recovered 75% of the initial DHFR activity in a single fraction in which the protein was 95% pure (Fig. 5B lane 3). SDS-PAGE of the crude extract and this fraction showed to be enriched in a 26.8 kDa protein. For identification of the principal active form of Tm8-3, the 95% pure fraction was subjected to gel filtration column chromatog-

raphy. Two fractions with DHFR activity were obtained (D and M; Fig. 5), containing proteins with estimated molecular weights of 60 and 28 kDa, respectively, consistent with the calculated expected molecular weights of dimeric and monomeric forms respectively. As most of the protein was eluted in fraction D, we can conclude that Tm8-3 is found mostly as a dimer in its native form.


1108 G. Torres-Cortés et al.

Fig. 3. Alignments of the trimethoprim-resistant proteins analysed in this study. Multiple-sequence alignments were carried out with the ClustalW online tool (http://align.bmr.kyushu-u.ac.jp/mafft/software/) for protein sequences. The degree of conservation of residues is indicated by shading, running from dark grey for the most strongly conserved residues to no shading for non-conserved residues (Clamp et al., 2004). A. Partial alignment and identification of conserved motifs for type I dihydrofolate reductases and the TmE1, TmE5 and Tm25 proteins. The figure shows the amino acid residues interacting with the NADP cofactor (closed triangles), with the folate substrate (closed circles) and with trimethoprim (stars), based on studies of the E. coli chromosomal DHFR (Howell, 2005; Sekiguchi et al., 2005). B. Partial alignment of the Tm8-3 clone with other 3′-oxoacyl-acyl carrier protein reductases (OARs). Active-site residues conserved in all bacterial and plant OARs are indicated by open triangles. Open circles indicate the two arginine residues playing a key role in ACP binding (Wickramasinghe et al., 2006). See Table S4 for amino acid sequence accession numbers.

Some of the kinetic parameters of the purified Tm8-3 are presented in Table 2. The Michaelis constant (Km) values of Tm8-3 for DHF and NADPH substrates were 3.7 and 8.9 mM respectively. These values are similar to those reported for other type I and II DHFRs. The 50% inhibitory concentration of Tmp for Tm8-3 was 150 times higher than that for chromosomal E. coli DHFR (Sekiguchi et al., 2005), but only one-fifth that of for the S2DHFR described in Staphylococcus haemolyticus (Table 2). In summary, our results identify Tm8-3 as a previously undescribed, new type of DHFR. Table 2. Enzyme kinetics, inhibition and structural properties of purified recombinant DHFRs.

DHFR

NADPH

DHF

IC50 of Tmp (mM)

S. pneumoniae chr DHFR (I) S. aureus S1DHFR(I) S. haemolyticus S2DHFR (I) E. coli chr DHFR (I) R67 DHFR (II) Tm8-3

15.7a

4.4a

0.48a

–

12.4b 1.7b

6.6b 5.1b

9.8b 127b

– –

4.8b 3c 8.9

0.7b 5.8c 3.7

0.13b – 20

Monomer 18 kDac Tetramer 34 kDac Dimer 54 kDa

Km (mM)

a. Lee and colleagues (2010). b. Sekiguchi and colleagues (2005). c. Howell (2005).

Enzyme form

Discussion Metagenomic technology is a powerful tool that has been applied to the discovery of novel natural products and enzymes of biotechnological interest. In this work, we identified 11 clones conferring resistance to various types of antibiotics in E. coli, through the screening of functional metagenomic libraries. Eight of those clones were found in the library of the disturbed soil sample (EEZ), indicating a major incidence of anthropogenic activity as the main origin of these resistant clones as has been described in other soil samples (Nwosu, 2001; Aminov and Mackie, 2007; Yang et al., 2010). The genetic determinants of antibiotic resistance within these clones displayed 41–63% amino acid identity to known functional proteins (Table 1). These resistance genes encoded b-lactamases, aminoglycoside acetyltransferases, multidrug efflux pumps and DHFRs. One of the genes identified, Tm8-3, belongs to a new group of antibiotic resistance genes never before described. As in a previous study (Donato et al., 2010), we found no streptomycin-resistant clones in our functional screens. The most likely explanation for this is that streptomycin-resistant genes are regulated by nonfunctional genetic elements in the E. coli host. Only three of the 11 clones found were closely related to resistance proteins identified in analogous metagenomic


Novel antibiotic resistance genes from soil metagenome 1109 samples belonged to class A (ApE1 and Ap6-8) and class C (ApE14). Both clones with Gm resistance phenotypes encoded N-acetyltransferase enzymes. Gm8-3 is related to the proteins of the g-proteobacteria AAC (3′)-I family (Riccio et al., 2003; Ahmed et al., 2004), and GmE1 is related to the AAC (3′)-III family, although it could also be assigned to a new subclass (Figs S3 and S4). The absence of other aminoglycoside-modifying enzymes (such as phosphotransferases or adenyl transferases) is consistent with the findings of previous clinical studies that the use of gentamicin is correlated with the occurrence of AAC3 genes (Vakulenko and Mobashery, 2003). All the isolated clones conferring Cm resistance were found to encode putative extrusion pumps. We identified no Cm resistance genes

Fig. 4. Characteristics of the pBKTm8-3 trimetroprim-resistant clone. A. Schematic diagram of the organization of the ORFs in plasmid pBKTm8-3. Black bars indicate the extent of the pBKTm8-3 clone, pBKTm8-3A and pBKTm8-3X derivatives. Significant restriction sites and their locations are indicated. Arrows indicate the locations and the directions of transcription of the ORFs. The ORF involved in trimetroprim resistance is indicated by a grey arrow. B. Drop assay of pBKTm8-3(7.5 kb) and derivative plasmids on LB plates containing 20 mg l-1 trimetroprim, carried out with serial dilutions (1 to 10-4) of overnight cultures. pBKTm8-3A (3 kb) and pBKTm8-3X (1.8 kb) are deletion-derived plasmids generated by digestion with Asp718 and XhoI respectively. pBKTm8-3XE and pBKTm8-3XB are derivatives of pBKTm8-3X in which ORF1 or ORF2 have been mutated by frameshift, with EcoRI or BamHI sites, respectively, filled in with the Klenow fragment of DNA polymerase I. We assessed the resistance phenotype based on Tm8-3 (ORF2) in a-proteobacteria, such as S. meliloti, by expressing this gene under the control of a constitutive promoter in pBBCMS vectors (Kovach et al., 1994; pBBSyn:Tm8-3). Empty vector (pBBSyn) was used as a control. Each assay was performed at least three times, with independent cultures.

approaches. CmE1 and CmE6, both of which confer Cm resistance, display 55% and 65% amino acid identity, respectively, to tcaB of the CR4 clone identified as a tetracycline resistance gene in another soil library (Accession No.: AY566822; Riesenfeld et al., 2004). The Gm-resistant gene GmE1 encodes a protein with an amino acid sequence 69% identical to that of another kanamycin-resistant clone also isolated from a metagenomic soil cosmid library (Courtois et al., 2003). The Ap resistance genes identified in this study encode b-lactamase enzymes. By contrast to previous studies, in which class B b-lactamases were found to be abundant in Alaska soils (Allen et al., 2009), the clones present in our

Fig. 5. Purification and characterization of the Tm8-3 protein. A. Results obtained by gel filtration column chromatography as a final step in the purification of Tm8-3 extracts. The loading material was the peak of DHFR activity obtained on Mono Q column chromatography. Absorbance at 280 nm (A280) was monitored continuously during protein elution. The molecular masses of the protein species were obtained by comparison with standards (indicated in kDa above the figure), using the same column and elution conditions. Arrows indicate the fractions (D and M) in which DHFR activity was detected. B. SDS gel electrophoresis of samples obtained during Tm8-3 purification. The gel contained 12% (w/v) polyacrylamide and was run under reducing conditions. Lane 1, molecular mass standards in kDa; lane 2, crude extract; lane 3, concentrated fraction sample after MonoQ column chromatography; lane 4, concentrated fraction sample D corresponding to the Tm8-3 dimer; lane 5, concentrated fraction sample M corresponding to the Tm8-3 monomer.


1110 G. Torres-Cortés et al. encoding enzymes, such as Cm acetyltransferases. This may reflect poor representation of these types of resistance determinants in the study population or the limitations of the E. coli-based screening. The two isolated clones conferring Cm resistance were very similar to each other and to tetracycline resistance genes. Both clones conferred tetracycline resistance in E. coli (data not shown). Trimethoprim resistance in bacteria has often been associated with changes in the sensitivity of DHFR activity in resistant isolates (Dale et al., 1997). Only two types of dhfr genes have been described to date (Howell, 2005): dhfr type I, generally present on the bacterial chromosome, and dhfr type II genes, the location of which is unknown (Howell, 2005). Three of the four identified Tmpresistant clones (pBKTmE1, pBKTmE5 and pBKTmE25) encoded a gene meeting the consensus criteria for a dhfr type I gene. However, the gene in clone pBKTm8-3 was found to encode a reductase very similar to a group of enzymes involved in lipid metabolism [3-oxoacyl-(acylcarrier-protein) reductase], but with low levels of amino acid identity (41%). We identified the conserved residues involved in NADPH binding, but not the binding site for dihydrofolate. The differences with respect to the DHFRs described below (Figs 1 and 2) led us to purify and characterize this enzyme biochemically. Tm8-3 has been shown to reduce dihydrofolate, but not acetoacetyl CoA (data not shown), the substrate of 3-oxoacyl-(acyl-carrierprotein) reductase enzymes. The specificities of Tm8-3 for the NADPH and dihydrofolate substrates were very similar to those of other DHFR enzymes (Fierke et al., 1987; Lee et al., 2010). The IC50 of Tm showed Tm8-3 to be a DHFR conferring Tm resistance, and that the resistance conferred to E. coli was not purely due to overproduction of the enzyme by the recombinant system. The native form of Tm8-3 appears to be a dimer (Fig. 5). This contrasts with other known DHFRs, as the chromosomal DHFR enzymes (Type I) are usually monomers, whereas the plasmid enzymes (type II) are active in the tetrameric form (Howell, 2005). Our findings highlight the benefit of functional metagenomics, as we were able to identify novel enzymes without limitation to genes recognizable as resistance determinants on the basis of sequence. We identified various antibiotic resistance mechanisms and a novel resistance determinant. This finding could explain noncanonical resistance mechanisms found in clinical settings. The low level of sequence similarity between the proteins conferring antibiotic resistance characterized in this study and those deposited in databases supports the hypotheses developed over the last few years, according to which these proteins may play a very different role in natural conditions. It seems plausible that molecules selected by pharmaceutical companies because of their

antibiotic properties at therapeutic concentrations would have also distinct functions at naturally occurring lower concentrations (Martinez, 2008). Thus, it is widely accepted that the enzymes deactivating aminoglycoside antibiotics may play a metabolic role in the cell (Miller et al., 1967) or be involved in the acetylation of peptidoglycan (Macinga et al., 1999). Similarities in structure between the antibiotic and the substrate of the enzyme may result in the development of resistance, even if the molecules concerned have very different roles. The characterized enzyme conferring Tmp resistance, Tm8-3, may constitute a new example supporting this hypothesis. Experimental procedures Soil description Three soils were used to construct the metagenomic libraries described in this work. Soils were collected, sieved (2 mm pores) and stored at 4°C. DNA was extracted within 1 week of collection. The EEZ soil (May, 2005) was a loamy soil from an agricultural field with a long history of cropping (> 20 years) located at the Zaidín Experimental Station (Granada, Spain 37°9.957′N, 3°35.493′W). This soil had a pH of 8.1 (soil to deionized water, 1:1 ratio); and 1.81% organic matter; its characteristics are described elsewhere (Ruiz-Lozano et al., 2001). Mexican soil samples were taken in the vicinity of Mammillaria carnea plants collected from the TehuacanCuicatlan nature reserve in Oaxaca, Mexico, from a site located at 17°44.675′N, 96°58.506′W with little human influence. The Mexican samples were collected at the end of the rainy season (R-Mex, late August 2005), and during the dry season (D-Mex, March 2006). These soils were coarse loamy arid soils with a pH of 7.3 (soil to deionized water, 1:1 ratio), poor in nitrogen and organic matter (1.8%), with water content differing by a factor of five to six between the rainy and dry samples (J.F. Aguirre-Garrido, D. Montiel-Lugo, C.H. Hernández-Rodríguez, G. Torres-Cortés, V. Millán, N. Toro et al., in preparation).

Bacterial strains and culture conditions All chemicals and antibiotics were purchased from SigmaAldrich. Escherichia coli strain XL1-Blue MRF’ was used for library construction and strain XLOLR was used for expression of the antibiotic resistance determinants from phagemids (Stratagene, La Jolla, CA, USA). Escherichia coli cells were routinely cultured in LB medium at 37°C, and plating media contained 1.5% Bacto Agar. When appropriate, media were amended with 10 mg l-1 tetracycline and 50 mg l-1 kanamycin for strain and plasmid maintenance respectively. We used pBK-CMV and the ZAP Express vector digested with BamHI (Stratagene) to construct the soil metagenomic libraries.

DNA isolation and metagenomic library construction Total DNA was extracted from 5 g soil samples, with a CETAB-based extraction protocol (Porteous et al., 1997).


Novel antibiotic resistance genes from soil metagenome 1111 Expression libraries were constructed from the extracted soil DNA with the ZAP Express BamHI Predigested Vector Kit (Stratagene). The extracted metagenomic DNA was partially digested with Sau3a and subjected to electrophoresis. DNA fragments of 9–10 kb in size were isolated from the gel and concentrated on Microcon filters (Millipore, Billerica, MA, USA). We ligated 50–150 ng of DNA to lambda vector, according to the recommended protocols. Library titre was determined by mixing various dilutions of the packaged ligation product with E. coli XL1-Blue cells, according to the manufacturer’s instructions, and counted the plaques formed. The ZAP Express vector is designed to allow simple, efficient in vivo excision and recircularization of any cloned insert contained within the lambda phage vector, to form a phagemid (pBCK-CMV plasmid). Thus, it is possible to convert a lambda phage DNA library into a phagemid library by using the mass excision protocol described by the manufacturer (Stratagene). Library size was estimated by multiplying the number of clones by mean insert size for 10 clones of each library.

Isolation of clones expressing antibiotic resistance Pools of phagemids (106 phagemids per plate) from each library were plated on LB plates supplemented with inhibitory concentrations of Ap (50 mg l-1), Gm (20 mg l-1), Cm (50 mg l-1), erythromycin (60 mg l-1), streptomycin (50 mg l-1) and Tmp (50 mg l-1). Plates were incubated for 24 or 48 h at 37°C. Plasmids were isolated from antibiotic-resistant colonies and analysed by DNA restriction. We transferred E. coli DH5a with the plasmids, and the resistance phenotype was checked by plating the transformed cells on appropriate selective media. Minimum inhibitory concentrations were determined by serial dilution assays of the corresponding antibiotic in LB broth, with approximately 1 ¥ 105 cells ml-1. Experiments were done in triplicate.

Identification of antibiotic resistance genes The full-length sequences of the metagenomic DNA inserts were obtained by primer walking with universal primers. In some cases, the size of the inserts of plasmids containing the resistance genes was reduced with a different set of restriction enzymes. This strategy made it possible to decrease the amount of sequencing required to identify the gene responsible for the resistance phenotype. The putative ORFs were annotated with BLAST (Basic Local Alignment Search Tool, Altschul et al., 1990). Predicted ORFs within the DNA inserts of the various clones are listed in Table 1.

Phylogenetic analysis The 10 top hits for each ORF encoding the antibiotic resistance protein were identified and collected with Blastp (Altschul et al., 1990). Where appropriate, reference sequences from the corresponding protein families were also included (Tables S1 and S4). The amino acid sequences of all nonredundant enzymes were downloaded and aligned, with MAFT version 6 (Katoh and Toh, 2008), which offers multiple

alignment strategies; we used ClustalW, a simple progressive method. Dendrograms were constructed by the neighbourjoining method, and bootstrap analyses of the respective data sets were performed with the MEGA 4.1 software package (Kumar et al., 2008).

Expression and purification of the Tm8-3 protein The Tm8-3 gene was amplified from phagemid pBKTm8-3 by PCR with the Pfu polymerase (Promega, Madison, MA, USA), in 28 cycles, with primers 678Tm8-3f (5′-GCGGATCCtcaTCATCTGAGGTGCCGGCCAC-3′) and 1427Tm8-3r (5′-GCGCATATGATGACGACCACCTTGGC GG-3′). Primers were designed to introduce BamHI and NdeI sites (underlined) and an extra stop codon (in small letters) into the cloned fragment. The PCR product was inserted into pGEMTeasy (Promega), to generate pGEMT:Tm8-3. We ensured that no unintentional changes were introduced by PCR, by sequencing the 0.8 kb fragment, which includes the Tm8-3 ORF gene. pGEMT:Tm8-3 was digested with NdeI and BamHI and the 0.8 kb fragment was inserted between the corresponding sites of pET-3a (Novagen, UK). Freshly transformed E. coli BL21(DE3) cells carrying pET3a-Tm8-3 were used to overproduce the corresponding protein. Cultures were grown at 37°C to an OD600 of 0.5, and production of the recombinant protein was induced by the addition of 1 mM IPTG. After culture for 12 h at 28°C, the cells were harvested by centrifugation (5000 g, 15 min), suspended in 50 mM sodium phosphate buffer (pH 8) and disrupted by sonication. Cell debris was removed by centrifugation at 15 000 g for 15 min, and the protein was purified by ion chromatography. The soluble fraction was applied to a Mono-Q HR 5/5 column (Pharmacia Biotech), equilibrated with Tris-HCl pH 8 buffer. The eluted fractions were collected, concentrated and further purified by size exclusion chromatography (Superdex 75 HR; Pharmacia Biothech). Fractions containing the purified protein were finally resolved by SDS-PAGE, in 12% polyacrylamide gels, and visualized by Coomassie Brilliant Blue staining. We tested whether this ORF conferred Tmp resistance to other bacterial species, such as Sinorhizobium meliloti, by inserting the 0.8 kb fragment into a broad-host range cloning vector, pBBSyn. The Psyn promoter (Giacomini et al., 1994) was amplified with the PsynFw (5′-CAGGTACCTATAAA AATAATTCTTGAC-3′) with a KpnI site and PsynRv (5′CGGGATCCTTAATGGCGCATATTATAC-3′) with a BamHI site primers and inserted into the pGEMT Easy Vector System (Promega), to generate pGEMSyn. This plasmid was digested with KpnI and BamHI. The fragment containing the Psyn promoter was introduced into pBBR1MCS-2 (Kovach et al., 1994) as a KpnI–BamHI fragment, to generate pBBSyn. pBBSyn:Tm8-3 contains the 0.8 kb Tm8-3 gene inserted into pBBSyn. It was constructed by ligating the 0.9 kb NcoI/ SacI fragment of pGEMT:Tm8-3 between the XbaI/SacI sites of pBBSyn, after the filling in of the 5′ overhangs with the Klenow fragment of DNA polymerase I.

DHFR assays Dihydrofolic acid (Sigma-Aldrich) and NADPH (SigmaAldrich) were prepared as previously described (Maskell


1112 G. Torres-Cortés et al. et al., 2001), with adjustment of the final concentration of the stock solution to 100 mM. They were stored at -70°C, in 50 ml aliquots, and their concentration was confirmed by reading the absorbance of a diluted solution at 282 and 340 nm respectively. Trimethoprim lactate (Sigma-Aldrich) solutions were prepared in 0.1 M imidazole-HCl buffer (pH 7), at concentrations of 10 and 100 mM, and were stored at -70°C. Km values for each substrate (DHF and NADPH) and 50% inhibitory concentrations (IC50) for Tmp were determined as described by Baccanari and Joyner (1981). A Shimadzu UV-1800 series spectrophotometer (Shimadzu Corp., Kyoto, Japan) with a heated cuvette carriage and an inbuilt program for the integrated determination of initial reaction rates was used. Reactions were performed in semi-micro plastic cuvettes in a final volume of 1 ml at 30°C. For determinations of Km values for each substrate, the concentration of the other substrate was held at a constant at 100 mM. Non-specific NADPH-oxidase activity was determined for each enzyme preparation, by determining rates for each NADPH concentration in the absence of DHF, and then subtracting these values from the results obtained in the presence of DHF. Initial rates for each concentration of NADPH and DHF were used to determine Km and Vmax values, according to Michaelis–Menten kinetics. The standard reaction mixture for the determination of IC50 values was 100 mM NADPH, 100 mM DHF, 75 mM 2-mercaptoethanol, 50 mM TES buffer (pH 7.0), Tmp and enzyme extract in a final volume of 1 ml. IC50 values were determined by plotting initial reaction rates against each Tmp concentration.

GenBank accessions The nucleotide sequences described in this study have been deposited in the GenBank database under accession numbers: FN640464–FN640474.

Acknowledgements This work was supported by the research projects BIO200302473, BIO2008-00740 and CSD 2009–0006 of the Consolider-Ingenio 2010 program from the Ministerio de Ciencia e Innovación. We would also like to thank the BBVA Foundation (BBVA BIOCON 04–084 Project). G.T.C and R.N.M. were supported by a CSIC predoctoral fellowship. V.M. was supported by a grant from the abovementioned BBVA Project. We are particularly grateful to M. Ferrer for invaluable assistance in the construction of the metagenomic libraries.

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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. Neighbour-joining tree showing the phylogenetic relations of the Ap resistance gene sequences found in this study. Species, strains or clone names are followed by their gene bank accession numbers. Some of the branches were grouped in order to improve the visibility of the tree (No. sequences grouped are indicated in parentheses number). Bootstrap values were calculated as percentage of 1000 replicates ad those above 70% are shown at the branching points. Sequences of this study are depicted in bold. See Table S1 for amino acid sequence accession numbers. Scale bar = 0.2 changes/site. Fig. S2. Full-sequence alignments and identification of conserved motifs for the beta-lactamases ApE1, ApE14 and Ap6-8 found in the metagenomics libraries. A. Multiple-sequence alignments of Ap6-8 and ApE1 with sequences closely related homologues that were identified from NCBI non-redundant database using BLAST searches. These proteins belong to class A of b-lactamases. B. Multiple-sequence alignments of ApE14 with b-lactamases of class C. Elements 1 to 4 (marked by closed circle) are conserved residues which surround the active site (Joris et al, 1991). Alignment of protein sequences was performed by the web http://align.bmr.kyushu-u.ac.jp/mafft/software/. The bacterial species and the corresponding accession numbers of the aligned sequences are: Candidatus koribacter (Ck, YP_590683); Solibacter usitatus (Su, YP_823589); Caulobacter sp (C sp, YP_001685148); Stappia agregata (Sa, ZP_01547832); Rhizobium etli CIAT 652 (Re; YP_001979560); Rhizobium leguminosarum bv. viciae 3841 (Rlv; YP_768739); Burkholderia sp. H160 (B sp; ZP_03269139). Fig. S3. Neighbour-joining tree showing the phylogenetic relations of the Gentamicin resistance gene sequences found in this study with AAC(3′) enzymes. Gene abbreviations and clade definition were shown according to elsewhere (Shaw et al., 1993). Bootstrap values were calculated as percentage of 1000 replicates ad those above 70% are shown at the branching points. AAC6′ ORFs were used as rooting outgroup. AAC(3′) enzymes are followed by their gene bank accession numbers. Sequences of this study are depicted in bold. See Table S2 for aminoacid sequence accession numbers. Scale bar = 0.2 changes/site. Fig. S4. Multiple-sequence alignment of the two enzymes identified in the metagenomics libraries that confers resistance to gentamicin.

A. Gm8.3 enzyme aligned with proteins of AAC(3)-I family [AAC(3)-Ia, AAC(3)-Ib, AAC(3)-Id and AAC(3)-Ie] (Vakulenko and Mobashery, 2003). B. GmE1 enzyme aligned with AAC(3)-III family. Alignment of protein sequences was performed by the web http://align. bmr.kyushu-u.ac.jp/mafft/software/. The figure shows with a closed circle the conserved amino acids that catalyse the transfer of an acetyl group form acetyl-coenzyme A to the primary amine of aminoglycoside (Dyda et al., 2000). The bacterial species and the corresponding accession numbers of the aligned sequences are: Pseudomonas aeuruginosa (Pa-Ib, AAA88422; Pa-IIIc AAA25683; Pa-IIIb PSEAAC3B; Pa-IIIa CAA39184); Plasmid R1033 (P R1033, CAA33850); Salmonella enterica (Se-Id, AAR21614, Se-Ie, AAR21853); Vibrio fluviales (Vf, BADOO738). Fig. S5. Neighbour-joining tree showing the phylogenetic relations of the chloramphenicol resistance gene sequences found in this study. Species, strains or clone names are followed by their gene bank accession numbers. Some of the branches were grouped (in parentheses number of sequences grouped are indicated) in order to improve the visibility of the tree. Bootstrap values were calculated as percentage of 1000 replicates ad those above 70% are shown at the branching points. Sequences of this study are depicted in bold. See Table S4 for amino acid sequence accession numbers. Scale bar = 0.1 changes/site. Fig. S6. Multiple-sequence alignment of CmE1 and CmE6, the two proteins that confers resistance to chloramphenicol. Full-sequence alignments with proteins of the Bcr/CflA family drug resistance transporter. The figure shows the secondary structure (made using: http://bioinf.cs.ucl.ac.uk/psipred/ psiform.html)of the proteins with twelve a-helix that corresponds with 12 transmembrane domains. The bacterial species and the corresponding accession numbers of the aligned sequences are: Mesorhizobium loti MAFF303099 (Ml, NP_103052); Uncultured bacterium (Ua, AAS90611); Rhizobium etli CFN 42 (Re, YP_472181); Agrobacterium radiobacter (Ar, YP_002545454). Table S1. Closely related sequences identified in NCBI database using the ampicillin resistance protein sequence found in the clones ApE1, ApE14 and Ap6-8 using in this study for the construction of the ampicillin phylogenetic tree and sequence alignments. Table S2. Sequences using in this study for the construction of the gentamicin resistance phylogenetic tree and sequence alignment (based in Shaw et al., 1993). Table S3. Closely related sequences identified in NCBI database using the Chloramphenicol resistance protein sequence found in this study (CmE1 and CmE6) for the construction of the chloramphenicol resistance phylogenetic tree and sequence alignments. Table S4. Closely related and reference sequences identified in NCBI database using the trimethoprim resistance protein sequence found in the clones TmE1, TmE5, TmE25 and Tm8-3. Additional protein sequences of the Type I DHFR using in this study for the construction of the trimethoprim phylogenetic tree and sequence alignment. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
