Table S1. PhyloPythia classification results for recovered antibiotic resistant metagenomic clones.  Clone ID Domain Phylum Class kan1 Bacteria Proteobacteria Alphaproteobacteria kan2 Bacteria Actinobacteria kan3 Bacteria Proteobacteria Alphaproteobacteria kan4 Bacteria gent1 Bacteria Proteobacteria Alphaproteobacteria chlor1 Bacteria Proteobacteria Gammaproteobacteria chlor2 Bacteria Proteobacteria chlor3 Bacteria Proteobacteria Betaproteobacteria rif1 Bacteria Firmicutes rif2 Bacteria Proteobacteria rif3 Archaea rif4 Bacteria rif5 Bacteria rif6 Bacteria Proteobacteria Gammaproteobacteria rif7 Bacteria Actinobacteria Actinobacteria tri1 Bacteria Proteobacteria Betaproteobacteria tri2 Bacteria tri3 Bacteria Proteobacteria tri4 Bacteria Actinobacteria tri5 Bacteria tri6 Bacteria tri7 Bacteria Proteobacteria Alphaproteobacteria tri8 Bacteria Actinobacteria Actinobacteria tri9 Bacteria Actinobacteria Actinobacteria tri10 Bacteria Proteobacteria Betaproteobacteria tri11 Bacteria Actinobacteria Actinobacteria tri12 Bacteria Proteobacteria Betaproteobacteria tri13 Bacteria tri14 Bacteria Proteobacteria tri15 Bacteria Actinobacteria Actinobacteria tri16 Bacteria Proteobacteria tri17 Bacteria tri18 Bacteria Proteobacteria Betaproteobacteria tri19 Bacteria Proteobacteria Alphaproteobacteria tri20 Bacteria Proteobacteria Betaproteobacteria tet1 Bacteria Actinobacteria tet2 Bacteria Firmicutes Bacilli tet3 Bacteria Proteobacteria tet4 Bacteria  

Supplementary Figure 1

Chlor2

Kan3

Tri4

Tri8

Gent1

Tet4

Glutathione S-transferase Bleomycin resistance

Bleomycin resistance

6’-N-acetyltransferase 6’-N-acetyltransferase

6’-N-acetyltransferase

DHFR

DHFR DHFR

ABC Transporter

ABC Transporter ABC Transporter

A

vector

Supplementary Figure 1

kan3_aac1

kan3_aac2

Ampicillin Kanamycin

Ampicillin Gentamicinvector

gent1_transp

gent1_aac

vector

tri4_DHFR

tri4_bleo

Ampicillin Trimethoprim

vector

tri8_DHFR1

tri8_DHFR2

vector

tet4_transp1

tet4_transp2

Ampicillin Trimethoprim

Ampicillin Tetracycline

B

C

DD

E

F

10 20 30 40 50 60 70 80 90

R G D R V G V R L R G N L T E V R Q A T E D D V E L L V A W H A D P D V A R Y W D G E T F T P D D M R A R L A R L D V D A Y V V E A E G R P V G Y L Q A W R - - - - - - - - - - E G D A G G L D M F- G H R V G L R L R G S L T E V R R A T E D D I E L L V A W H A D P D V A R Y W D G E T F T P D D M R A R L A R L D V D A Y V V E A K G R P V G Y L Q A W R - - - - - - - - - - E G D A G G L D M F- - - - - M P S E R R H Q Y S F R P V T E Q D L P M L A S W L A E P H V A A WW N D S E V E L A D I R G H I D S I S V E P L I V E F S G R P I A Y L Q S Y D P H L E D G H P Y Q D Q P F G T L G I D- - - - - - M E L R G E R V V L R R L A E A D V P R L A E I A A E P E V V R WW G E Q S A E D L G R K A R G E E P D E T F F T I V V E G E L A G L I Q Y Y E E D - - - - - - E P D F R H A G I D V F- - - - - - - - - - - - - - - - - - - - - - M D A L L A M F G A A F E D V D T Y G K A R P G A A Y M K S L L G S D Y F I A L V A M K D N A I V G G L A A Y E L R - - - - - - K F E Q E R S E I Y I Y

100 110 120 130 140 150 160 170 180

L I P - - - E A R G R G L G P D A A R A L A T A L R D Q - G W T R I T V D P Y L W N E P A I E A W K R A G F E P V A R R - - - - S A D D E H T A D W L L M E L T - - - - -L I P - - - E A R G R G L G P D A A R A L A T A L R D E - G W T R I T V D P Y L W N E R A V E A W K R A G F E P V E R R - - - - S A D D E H T A D W L L M E L A - - - - -I S I G D A E L V G I G H G S S I V R Q F T D Q L F A E - G A P R V I I D P D P A N G R A I R A Y E K A G F K P L G E R - - - - T S E Y G H V I L M A R D A E E G E D C GL S A G - - - F Q G R G V G A D A V R T V A R H L I D D L G H H R L T I D P A A D N A Y A I R C Y E K V G F R P V G V M R K Y W R D P D G V W R D G L L L D L L A K E L TD L A V A A A H R R E G I A T A L I E G L K K I A A D R - G T Y V I Y V Q A D V G D S P A I A L Y S K L G A R - - - - - - - - - - - - - - - - E D V L H F D I I V G - - -

kan3_aackan4_aackan1_aackan2_aacgent1_aac

kan3_aackan4_aackan1_aackan2_aacgent1_aac

Supplementary Figure 2

A

B

kan1_aac kan2_aac kan3_aac kan4_aac gent1_aac

kan1_aac -

kan2_aac 27% -

kan3_aac 26% 31% -

kan4_aac 27% 30% 92% -

gent1_aac 12% 14% 16% 14% -

Q53CM2 Z58IZ0 B5WYT8 B7VER9 B7VES0 B0FMU1 Q79D79 Q1HVJ4 C5MRP4

tri14_DHFR 23% 24% 24% 23% 24% 22% 23% 24% 23%

Supplementary Figure 3

Supplementary Figure 4

A

B

20 30 40 50 60 70

H P F K A I A A M S E N R V I G R G N S I P W H L P E D F K W F K K M T T G - - - - - - - N T V V M G R K T F Q SK H F K A I A A M A E N R V I G N G K A I P W H L P E D F K W F K R M T T G - - - - - - - Q V V V M G R K T F E SM R I S L V V A F D R E R A I G V D N R L P W H L P D D L K R F K A L T L G - - - - - - - K P I L M G R L T A E SI L T L G P V A R D R E G V I G K D G T L P W R L S D D L K R F K A L T L G - - - - - - - K P V I M G R K T W D SL M I V V A V A Q N - - G V I G R D N Q L L W R L K T D L R R F K D L T W G - - - - - - - K P M I M G R K T F Q SM I L S A I V A V S D N D V M A K D G K I P W F V R G E Q L I F K R L T T G - - - - - - - P P I I M G R K T F E T

M V T M I A A V A E N N A L G K D N K L L W H L P D D F K R F K K L T T G - - - - - - - H H I I M G R K T F E SM I V S F I V A V S D N N A I G R K N T L P W H L P E D L K F F K R T T I G - - - - - - - K P V I M G R K T Y E SS R I S L I V A M A K N R V I G A G N K I P W H L P S E L K L F K S L T M G - - - - - - - H H I V M G R R T Y E S

M I S L I A A L A V D R V I G M E N A M P W N L P A D L A W F K R N T L N - - - - - - - K P V I M G R H T W E SM R L S I I V A M D R N R V I G R G G A L P W R L S A D L Q R F K A L T M G - - - - - - - H H L MM G R K T F E SM K V T L V A A V A R G G V I G R G N T I P W R I P E D M A R F R V L T T G - - - - - - - H P V V M G R R T W D S

I G L V W A Q A A N G V I G R D G T L P WQ L P E D L A R F K A L T T G - - - - - - - A T V V M G R A T W D SR R I V L V A A V A D N G V I G D G P N I P W H V P G E Q A G F K Q L T M G - - - - - - - R V L L M G R T T Y E TR R V V L V A A V A R N R V I G D G P D I P W A L P G E Q R L F K E L T L G - - - - - - - H T L L M G R T T Y E S

R I V L V A A V A E N G V I G A A G G I P W H L P E D F K H F K A T T L G - - - - - - - H T L V M G R A T Y D SM T I S L I A A V A R N G V I G S D G Q I P W R L P G E L P R F K A L T T G - - - - - - - H V L V M G R K T F D SG E R G I M V A M S P E G V I G L E G K I P W H Y R G D L R R V K R L T L G - - - - - - - T T L I M G R V T W E S

M S I K I I V A Y G R N H E I G Y E N G M P W H L P N E L R W V S K V T R H T T D P Q R R N A I V M G R R T W E S

tri2_DHFRtri16_DHFR

tri19_DHFR

tri1_DHFRtri7_DHFR

tri3_DHFRtri12_DHFRtri17_DHFRtri10_DHFREcoli_DHFRtri13_DHFR

tri4_DHFRtri9_DHFRtri8_DHFR

tri15_DHFRtri11_DHFRtri5_DHFR

tri18_DHFRtri6_DHFR

tri4_DHFR tri5_DHFR tri13_DHFR !tri14_DHFR tri19_DHFR

tri4_DHFR -

tri5_DHFR 44% -

tri13_DHFR 39% 38% -

tri14_DHFR 7% 9% 9% -

tri19_DHFR 42% 41% 40% 7% -

10 20 30 40 50 60

M E F D P R N N V I K L C V Q G M D A E G K G E P G E A R R I F L Q A W N E S T N D F E K Y I A A Y Y V A R H Q D S V Q D K L RM E F N P S N P V I Q L C L Q G M D L E E K G K P E D A N K L F L Q A W N E A K N D F E K F I A A H Y V A R H Q K D V S T K L EM E F S P F N N V V K L C MQ G M D M E D K S K P D E A G R Q Y L Q A W N E A T D D F E K F L A A Y Y L A R L Q K D V S D K L QM E F S P N N N I V K L C L Q G M D M E A K G N H E E A G K I F L Q A W N E A T N D F E K Y L A A Y Y L A R Q Q T D V S A K L KM E F N P N N K I I Q L C V K G MM L E E K G E Q V E A G A I F H Q A W N E A S N Y F E K Y L S A Y F I A K H Q T N V S G K L N

70 80 90 100 110 120

W L E T S L Q F A L A I N D L T V R S A F P S L Y S N I A K C H E E L N D S D N A K K N H E L A S S F A D N P S DW L R L A L A L G Q K I N S D Q V N S A F P S L Y T S I A K C Y E E S N D G D N A K K N Q D L A A S F A S K P S DWQ Q T A L Q F A L R I N N D S V K G A F P S L Y L D I A K S Y E D L G D V D N A N R N H E L A I L F R D N P S DW F E T A L R F A S K T D N H S T N T A L P S I Y S D I A R C Y E S L N D V D S A E K Y S E L G I S A R S N P I D

W F E T A L Q F A L K T D S Y S V K S A M Y S L Y L N I A K C Y E D L N D N D N A K K N F G L A D S F K N I E Y D

Supplementary Figure 5

A

rif4_ARTrif5_ARTrif3_ARTrif2_ARTrif1_ART

rif4_ARTrif5_ARTrif3_ARTrif2_ARTrif1_ART

Supplementary Figure 6

rif2_ARTrif4_ARTrif6_ARTrif1_ART

rif2_ARTrif4_ARTrif6_ARTrif1_ART

170 180 190 200 210 220 230 240

N G A G L A A E L A P G D G Q A R V Y I V E P T G S F E S D P N V T D K K F P G N P T R S Y R S A A P L K I V G E A T D W V R L T P E E L K S W R E K L A K I E N S K G K I I NN G A G L A A E L S K G E G R E R V Y I V Q P T G E F E D D P N V T D K K F P G N P T R S Y R S A A P L K I L G E V T D W E R L T P E A L A E W R N R L A N I - - - K G D I I N

NG A G L A A A L A N S A G R E R V Y I V E P T G P F E N D P N V T D K K F P G N P T R S Y R S E A P L K I I G E V T D W A R Q T P E Q V Q A W R E K L A N S - - - K G K I I NN G A G L A A A L A N G S G K E R V Y I V E P T G T F E S D P N V T D K K F P G N P T R S Y R T D A P L K I V G E L R D W D R Q T P E D I Q K W K E K L E N N - - - K G E I I N

rif2_ARTrif4_ARTrif6_ARTrif1_ART