Supplementary data
Construction of E. coliacs-, pta-, ackA-
Gene replacement with thyAOligos were used to amplify thyA with 50 bp overlaps to the ends of each of the genes. Products were used in transformation to replace acs, ackA and pta with thyA. / acs/ackA/pta thyA
l------l------l
acs
5’acs-thyAF1
GCCAACATCGCAGACCGTTGCCTGATAAACCCTCAGCAGTACGAGGCGATGCTGCTGCTGGAAGGTGTGGT
3’acs-thyAR3
TATCGCCCAGGTTGCTGGTATCGCCCGCCGCAATTTTGCGCAGAATACGGGGGTAAAAAAACCGACGCACAC
ackA
5’ackA-thyAF1
TGCGGTAGTTCTTCACTGAAATTTGCCATCATCGATGCAGTAAATGGTGAGCTGCTGCTGGAAGGTGTGGT
3’ackA-thyAR3
TTGTGTTGATGAAACCAGATCCGAAACGTGCAGCCAGGTTGCGTTCATGAGGGTAAAAAAACCGACGCACAC
pta
5’pta-thyAF1
ACCAGCGTCGGTCTGACCAGCGTCAGCCTTGGCGTGATCCGTGCAATGGAGCTGCTGCTGGAAGGTGTGGT
3’pta-thyAR3
GGACAGGTCGTTAACCGGCTTGCGCATACCCTGCAGCATCGGCCCGATGGGGGTAAAAAAACCGACGCACAC
thyA removal
Each pair of oligos was annealed (20 bp) and filled in to produce a 100 bp piece that was used in transformation to seamlessly remove the thyA gene and therefore generate the final deletion of each gene. / acs
5’acs-del
GCCAACATCGCAGACCGTTGCCTGATAAACCCTCAGCAGTACGAGGCGATCCGTATTCTG
3’acs-del
TATCGCCCAGGTTGCTGGTATCGCCCGCCGCAATTTTGCGCAGAATACGGATCGCCTCGT
ackA
5’ackA-del
TGCGGTAGTTCTTCACTGAAATTTGCCATCATCGATGCAGTAAATGGTGATCATGAACGC
3’ackA-del
TGTTGATGAAACCAGATTTGCCGAAACGTGCAGCCAGGTTGCGTTCATGATCACCATTTA
pta
5’pta-del
ACCAGCGTCGGTCTGACCAGCGTCAGCCTTGGCGTGATCCGTGCAATGGACCATCGGGCC
3’pta-del
GGACAGGTCGTTAACCGGCTTGCGCATACCCTGCAGCATCGGCCCGATGGTCCATTGCAC
Verification of gene deletions
Primer sets that were used in PCR to verify the gene deletions. / acs
acs-F:CACACCATTCCTGCCAACAT
acs-R:GCCGCAATTTTGCGCAGAAT
ackA
ackA-F:GGTTCTGAACTGCGGTAGTT
ackA-R:AGGACGGGTACCTTCTTTGT
pta
pta-F:CTTGGCGTGATCCGTGCAAT
pta-R:CGTTAACCGGCTTGCGCATA
Organism / ackA / pta
Methanosarcina acetivorans (mac) / AAM06961 / AAM06962
Clostridium phytofermentans (cph) / YP_001558443 / YP_001558442
Ruminococcus gnavus(rgn) / ZP_02042091 / ZP_02042092
Pelobacter carbinolicus (pca) / YP_355987 / YP_357950
Data 1alignments
Acetate Kinase multiple alignment
cph-ackA ---MKVLVINCGSSSLKYQLIDSVTEQALAVGLCERIGIDG-RLTHKSADGEKVVLEDAL 56
rgn-ackA ---MNVLVINCGSSSLKFQLINAKSEEVLAKGICERIGIDG-RLTYQPEGGEKEKSEKAM 56
mac-ackA ---MKVLVINAGSSSLKYQLIDMTNESPLAIGLCERIGIDNSIITQKRSDGKKLEKQTDL 57
pca-ackA ---MLILALNCGSSSVKYNLFDWAGKQTLAKGVVERVMIGESFITQSVPGREDYRLEKDC 57
k12-ackA MSSKLVLVLNCGSSSLKFAIIDAVNGEEYLSGLAECFHLPEARIKWKMDGNKQEAALGAG 60
:*.:*.****:*: ::: . *: *. : :.. . :.
cph-ackA PNHEVAIKNVIAALMNENYGVIKSLDEINAVGHRVVHGGEKFAHSVVINDEVLNAIEECN 116
rgn-ackA PTHTEAIQFVIEALTNPETGVVNSLDEIGAVGHRMVHGGEKFASSVVITDEVKKAVEECN 116
mac-ackA PNHKVALEEVVKALTDSELGVIKSMDEINAVGHRVVHGGEKFTSSALIDEGVEQAIKDCF 117
pca-ackA PDHTTAIQLIIRTLTEAPSAVLDNIDAISAVGHRVVHGGEKFTRSVRIDEEVLAAIRSVQ 117
k12-ackA AAHSEALNFIVNTILAQKP---ELSAQLTAIGHRIVHGGEKYTSSVVIDESVIQGIKDAA 117
.* *:: :: :: . : *:***:******:: *.* : * .:..
cph-ackA DLAPLHNPANLIGINACKSIMPNVP--MVAVFDTAFHQTMPKEAYLYGIPFEYYDKYKVR 174
rgn-ackA DLAPLHNPANLIGVAACENLMPNTP--MVAVFDTAFHQTMPPKAYMYGLPYEYYEKYKVR 174
mac-ackA ELAPLHNPPNMMGITACQEIMPGVP--MVAVFDTAFHQTIPAYAYMYALPYTLYEKYGIR 175
pca-ackA HLAPLHNPPNIAGIEAVQNILPQVV--NIAIFDTAFHQTMPRHAYIYALPYAWYEQYGVR 175
k12-ackA SFAPLHNPAHLIGIEEALKSFPQLKDKNVAVFDTAFHQTMPEESYLYALPYNLYKEHGIR 177
:******.:: *: . :* :*:********:* :*:*.:*: *.:: :*
cph-ackA RYGFHGTSHSYVSKRATTLAGLDVNNSKVIVCHLGNGASISAVKNGESVDTSMGLTPLEG 234
rgn-ackA RYGFHGTSHSFVSKRAAELLGKAYDETKTIVCHLGNGSSVTAVLNGKSVDTSMGLTPLEG 234
mac-ackA KYGFHGTSHFYVARRAAAMLGKPEEEVKVITCHLGNGSSITAVNGGKSVETTMGFTPLEG 235
pca-ackA RYGFHGTSHLYVSKRAAALLGKHARDCNLITMHIGNGVSHCAIRNGISIDTSMGLTPLEG 235
k12-ackA RYGAHGTSHFYVTQEAAKMLNKPVEELNIITCHLGNGGSVSAIRNGKCVDTSMGLTPLEG 237
:******* :*::.*: : . : : *.*:**** *: .*.::*:**:*****
cph-ackA LIMGTRSGDLDPAIIDFVAKKENLSLDEVMNILNKKSGVLGMSG-VSSDFRDIEAAANEG 293
rgn-ackA LVMGTRCGDIDPAIMEFLAHKLDLDVDGVMKILNKDSGVYGLSGGLSSDFRDLTDASLAG 294
mac-ackA VAMGTRCGSIDPAVVPFVMEKEGLTTREIDTLMNKKSGVLGVSG-LSNDFRDLDEAASKG 294
pca-ackA AVMGTRCGDIDPAIPHFMMKMADLSPDEMDRLLNKKSGVLGITG-RYTDRRDIIQAAESG 294
k12-ackA LVMGTRSGDIDPAIIFHLHDTLGMSVDAINKLLTKESGLLGLTE-VTSDCRYVEDNYAT- 295
****.*.:***: .: . .: : ::.*.**: *:: .** :
cph-ackA NEHAKEALAVFAYRVAKYVGSYIVAMNG-VDAVVFTAGLGENDKNIRAAVSSHLEFLGVS 352
rgn-ackA DKKAQIAMEVFSYRVAKYIGAYVAVMNG-VDDIVFTAGIGENDDIVREQVCSYLGYLGIE 353
mac-ackA NQRAELALEIFAYKIKKVIGEYSAVLNG-ADAVVFTAGIGENSASIRKRILSGLDGLGIE 353
pca-ackA DERCQLSLEIECYRLRKYIGAYLAAVGP-LDAIVFTAGVGEHSWLIRQMALQHLEHLGIH 353
k12-ackA KEDAKRAMDVYCHRLAKYIGAYTALMDGRLDAVVFTGGIGENAAMVRELSLGKLGVLGFE 355
.: .: :: : .::: * :**. :. * :***.*:**: :* * **.
cph-ackA LDAEKNS--QRGKELIISNPDSKVKIMVIPTNEELAICREVVELV------395
rgn-ackA FNTELNKG-LRGKETELSMPGSKVKVFVIPTNEELAIARETLELVRK------399
mac-ackA IDEEKNK--IRGQEIDISTPDAKVRVLVIPTNEELTIARDTKEICETEVKLRRSVSI--- 408
pca-ackA FDKQRNRDLASGLEAAISSPDSPIKVFVIPTNEELVFIEDVAAILDGSYADHMDFAYSFA 413
k12-ackA VDHERNLAARFGKSGFINKEGTRP-AVVIPTNEELVIAQDASRLTA------400
.: : * *. :. .: .********.: .:. :
cph-ackA ------
rgn-ackA ------
mac-ackA ------
pca-ackA HPDYKPR 420
k12-ackA ------
Phosphotransacetylase multiple alignment
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta MSRIIMLIPTGTSVGLTSVSLGVIRAMERKGVRLSVFKPIAQPRTGGDAPDQTTTIVRAN 60
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta SSTTTAAEPLKMSYVEGLLSSNQKDVLMEEIVANYHANTKDAEVVLVEGLVPTRKHQFAQ 120
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta SLNYEIAKTLNAEIVFVMSQGTDTPEQLKERIELTRNSFGGAKNTNITGVIVNKLNAPVD 180
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta EQGRTRPDLSEIFDDSSKAKVNNVDPAKLQESSPLPVLGAVPWSFDLIATRAIDMARHLN 240
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta ATIINEGDINTRRVKSVTFCARSIPHMLEHFRAGSLLVTSADRPDVLVAACLAAMNGVEI 300
cph-pta ------
rgn-pta ------
mac-pta ------
pca-pta ------
k12-pta GALLLTGGYEMDARISKLCERAFATGLPVFMVNTNTWQTSLSLQSFNLEVPVDDHERIEK 360
cph-pta ------MGFIDDIKARAKQSIKTIVLPESMDRRTIEA 31
rgn-pta ------MAFIDEIKARAKADKKTIVLPETEDVRTYQA 31
mac-pta ------MVTFLEKISERAKKLNKTIALPETEDIRTLQA 32
pca-pta ------MHLADQIKAKARENRQVVVLPEGYDDRMIQA 31
k12-pta VQEYVANYINADWIESLTATSERSRRLSPPAFRYQLTELARKAGKRIVLPEGDEPRTVKA 420
: .:. *: : :.*** : * :*
cph-pta AAKTLEEGNANVIIIGSEEEVKKNSEGL--DISG-ATIVDPKTSDKLPAYINKLVELRQA 88
rgn-pta AEAVLREGTADLILVGSKEEIEKNGAGF--DLTG-AVIVDPATSDKTEGYIAKLVELRQK 88
mac-pta AAKILERGIANVVLVGDEADIKALAGDL--DLSK-AKIVNPKTYEKKDEYINTFYELRKH 89
pca-pta AGPIVKDNLADVVLLGEPAALQAKATELGVSLEG-VTLLDPKSAPQLDTYADELVEIRKK 90
k12-pta AAICAERGIATCVLLGNPAEINRVAASQGVELGAGIEIVDPEVVR--ESYVGRLVELRKN 478
* ..* :::*. :: . .: :::* * : *:*:
cph-pta KGMTPEKAKELLTT-DYITYGVMMVKMGDADGLVSGACHSTADTLRPCLQILKTAPNTKL 147
rgn-pta KGMTEEQAREILLT-NYLYYGVMMVKMGDADGMVSGACHSTADTLRPCLQILKTKPGTKL 147
mac-pta KGITPETAAEVMS--DYVYFAVMMAKLGEVDGVVSGAAHSSSDTLRPAVQIVKTAPGAAL 147
pca-pta KGLSKDDALKLLTADDNLFFATMMVRQGDAGGCVAGAYNTTGNVLRAAFQVIGPAPGMKT 150
k12-pta KGMTETVAREQLE--DNVVLGTLMLEQDEVDGLVSGAVHTTANTIRPPLQLIKTAPGSSL 536
**:: * : : : : ..:*..:..**:** :::.:.:*..*:: .*.
cph-pta VSAFFVMVVPNCDMGANGTFLFSDAGLNQNPNAEELAAIAGSTAKSFEQLVGSEPIVAML 207
rgn-pta VSAFFVMVVPDCDMGADGTFIFGDSGLEQNPDPEKLANIALSSAESFRTLTGKEPVVAML 207
mac-pta ASAFFIIAVPDCEYGSDGTFLFADSGMVEIPSVEDVANIAVISAKTFELLVQDTPYVAML 207
pca-pta VSSCFIMVTKTPEFGENGALIFADCGVNPNPDAAALAEIAVSSAQSCKSFLGAEARVAML 210
k12-pta VSSVFFMLLP------EQVYVYGDCAINPDPTAEQLAEIAIQSADSAAAFG-IEPRVAML 589
.*: *.: : . ::.*..: * :*** :*.: : .****
cph-pta SHSTKGSAKHADVDKVVEATKIANELYPEYKIDGEFQLDAAIVPSVGASKAPGSDIAGKA 267
rgn-pta SHSTKGSAKHADVDKVVEATRLAKEMAPELKLDGELQLDAAIVPAVGESKAPGSPVAGHA 267
mac-pta SYSTKGSAHSKLTEATVAATKLAQELAPDVAIDGELQVDAAVVPKVAASKAPGSPVAGKA 267
pca-pta SFSTKGSAKHEDPDKVIAALAIAKEKAPELQIDGELQADAALVPAVGNSKAPGSNVAGKA 270
k12-pta SYSTGTSGAGSDVEKVREATRLAQEKRPDLMIDGPLQYDAAVMADVAKSKAPNSPVAGRA 649
*.** *. : . * :*:* *: :** :****::.*.****.* :**:*
cph-pta NVLIFPDLDAGNIGYKLTQRLAKAEAYGPLTQGIAAPVNDLSRGCSSDDIVGVVAITAVQ 327
rgn-pta NVLIFPDLDAGNIGYKLVQRLAKAEAYGPLTQGIAAPVNDLSRGCSAEDIEGVVAITAVQ 327
mac-pta NVFIFPDLNAGNIAYKIAQRLAKAEAYGPITQGLAKPINDLSRGCSDEDIVGAVAITCVQ 327
pca-pta NTLVFPDLDAGNIAYKLVQRLAKAEAFGPIIQGLAKPVNDLSRGCSVDDIISVTAITAVQ 330
k12-pta TVFIFPDLNTGNTTYKAVQRSADLISIGPMLQGMRKPVNDLSRGALVDDIVYTIALTAIQ 709
..::****::** **.***. : **: **: *:******. :** .*:*.:*
cph-pta AQSK-- 331
rgn-pta AQAQ-- 331
mac-pta AAAQDK 333
pca-pta AQG--- 333
k12-pta SAQQQ- 714
:
Amino acid / From codon / To codonLeucine / CTG / CTT
Isoleucine / ATA / ATT
Threonine / ACA
ACT / ACC
ACG
Argenine / AGA
AGG
CGA
CGG / CGT
CGC
CGT
CGC
Glycine / GGA
GGG / GGT
GGC
Proline / CCT
CCC
CCA / CCG
CCG
CCG
Promoter /
lacI / GACACCATCGAATG GCGCAA AACCTTTC GCGGTATGG CATGAT AGCGCCCGGAAGAGAGT
ssb / TAGTAAAAGCGCTA TTGGTA ATGGTACAA TCGCGCGTT TACACT TATTCAGAACGATTTT
recA / TTTCTACAAAACAC TTGATA CTGTATGA GCATACAG TATAAT TGCTTCAACAGAACAT
Data 2
Gene cassettes with linker, rbs and ter sequences
Codon adjusted gene cassettes as ordered from IDT. 40 bp linkers are underlined: beginning of sequence – L1 (ackA), L2 (pta), end of sequence – R1(ackA), BR (pta). Green – ribosome binding site (rbs), Red – terminator.
ackA
Methanosarcina acetivorans
CTGTAGTGATTGTGATTCTAATCAGTATGCATGTAGTCAAAGGAGGACAGCTATGAAAGTACTGGTTATTAACGCAGGCAGTTCTTCTCTCAAATATCAATTAATTGATATGACCAACGAGTCCCCGCTTGCAATTGGCCTCTGTGAGCGCATTGGCATCGACAACTCGATCATTACCCAGAAGCGCTCTGACGGCAAGAAACTGGAAAAGCAGACCGACCTTCCGAACCACAAGGTTGCCCTTGAGGAAGTCGTCAAGGCTCTTACGGACTCCGAGCTCGGTGTCATCAAAAGCATGGACGAAATCAATGCAGTCGGCCACCGTGTTGTGCACGGTGGTGAAAAGTTCACGTCCTCGGCTTTGATCGATGAAGGCGTAGAACAGGCAATCAAGGACTGCTTTGAACTGGCTCCGCTCCACAATCCGCCGAACATGATGGGTATTACGGCCTGTCAGGAGATCATGCCGGGCGTCCCGATGGTTGCTGTTTTTGACACCGCATTCCACCAGACCATCCCGGCCTATGCCTACATGTATGCTCTGCCGTACACCCTGTACGAAAAGTACGGCATCCGTAAATACGGTTTCCACGGCACGTCTCACTTTTACGTTGCCCGTCGCGCTGCCGCTATGCTCGGTAAACCGGAAGAAGAAGTCAAAGTCATCACCTGCCACCTTGGTAACGGCTCAAGCATTACGGCTGTTAACGGCGGTAAATCCGTTGAGACCACCATGGGCTTTACCCCGCTCGAAGGTGTTGCAATGGGTACCCGCTGCGGTTCGATTGACCCGGCAGTAGTCCCGTTCGTTATGGAAAAGGAAGGCCTTACGACCCGTGAAATCGATACCCTCATGAACAAGAAGTCAGGCGTGCTTGGTGTCTCCGGCCTCAGCAACGACTTCCGCGACCTCGATGAAGCAGCTTCCAAAGGCAACCAGCGTGCCGAACTCGCCCTTGAAATCTTTGCATACAAGATTAAGAAGGTCATTGGTGAGTATTCAGCCGTACTCAATGGTGCGGATGCGGTAGTCTTTACCGCAGGCATTGGTGAAAACAGTGCAAGTATCCGCAAGCGTATCCTCTCCGGCCTTGACGGTCTCGGCATTGAGATCGACGAAGAAAAGAACAAGATCCGTGGTCAGGAAATCGATATCAGCACGCCGGATGCAAAAGTACGTGTCCTTGTTATCCCGACCAACGAAGAACTTACCATTGCACGCGACACCAAGGAAATCTGTGAGACCGAAGTAAAGCTGCGCCGTTCAGTATCAATCTGACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTATGAGTCATAGCAATGTTTAGCAGATTTGCATTCATCAATT
Clostridium phytofermentans
CTGTAGTGATTGTGATTCTAATCAGTATGCATGTAGTCAAAGGAGGACAGCTATGAAAGTTTTAGTTATTAATTGCGGTAGTTCTTCCCTTAAATATCAGTTAATCGACTCTGTGACCGAGCAAGCATTAGCAGTAGGTCTTTGTGAACGTATCGGTATTGATGGCCGTCTTACGCACAAGTCAGCTGACGGTGAGAAGGTAGTTCTTGAGGATGCACTTCCGAACCATGAGGTTGCTATTAAAAATGTAATCGCTGCTCTTATGAATGAAAATTATGGTGTGATTAAGTCCTTAGATGAAATCAACGCTGTTGGTCATCGTGTAGTACATGGTGGTGAGAAATTTGCTCATTCCGTAGTAATCAATGATGAAGTCTTAAATGCAATTGAAGAGTGTAATGATCTTGCACCGTTACACAACCCGGCAAACCTTATTGGTATCAACGCTTGTAAATCAATTATGCCGAATGTACCGATGGTAGCTGTTTTTGATACGGCATTCCATCAGACCATGCCGAAAGAAGCTTACCTTTATGGTATTCCGTTTGAGTACTATGATAAATATAAGGTACGTCGTTATGGTTTCCACGGTACCAGTCACAGCTATGTTTCTAAACGTGCAACCACGCTTGCTGGCTTAGATGTAAATAACTCAAAAGTTATCGTTTGTCACCTTGGTAATGGCGCATCCATTTCCGCAGTTAAAAACGGTGAGTCTGTAGATACCAGTATGGGTCTTACCCCGCTTGAAGGTTTAATCATGGGTACCCGTAGTGGTGATCTTGATCCGGCAATCATTGATTTCGTTGCTAAGAAAGAAAACTTATCCTTAGATGAAGTAATGAATATCTTAAATAAGAAATCTGGTGTATTAGGTATGTCCGGTGTATCTTCTGACTTTCGTGATATCGAAGCAGCAGCAAACGAAGGCAATGAGCATGCAAAAGAAGCTTTAGCAGTTTTTGCATACCGTGTTGCTAAATATGTAGGTTCTTATATCGTAGCTATGAATGGTGTAGATGCTGTTGTATTTACCGCAGGTCTTGGTGAGAATGATAAGAACATCCGTGCAGCAGTAAGTTCACACCTTGAGTTCCTTGGTGTATCTTTAGATGCTGAGAAGAATTCTCAACGTGGTAAAGAATTAATCATCTCTAACCCGGATTCTAAGGTTAAGATTATGGTTATCCCGACGAACGAAGAGCTTGCAATCTGTCGTGAAGTTGTTGAATTAGTGTAGCGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTATGAGTCATAGCAATGTTTAGCAGATTTGCATTCATCAATT
Pelobacter carbinolicus
CTGTAGTGATTGTGATTCTAATCAGTATGCATGTAGTCAAAGGAGGACAGCTATGTTGATTCTGGCACTGAACTGCGGCAGTTCGTCGGTCAAATACAATCTTTTCGACTGGGCTGGCAAACAGACCCTCGCCAAAGGCGTGGTCGAGCGCGTCATGATCGGCGAATCGTTCATCACCCAGAGTGTACCGGGTCGCGAAGATTATCGTCTGGAAAAAGATTGTCCGGACCACACCACCGCCATCCAACTCATCATCCGTACCCTGACCGAAGCCCCGAGCGCGGTTCTGGACAACATCGATGCCATTTCGGCCGTCGGCCACCGCGTGGTGCACGGCGGTGAAAAGTTCACCCGTTCGGTACGTATCGACGAGGAAGTCCTGGCGGCGATTCGCTCGGTGCAGCACCTGGCGCCGCTGCATAACCCGCCGAACATTGCCGGCATCGAGGCGGTGCAGAACATTTTGCCGCAGGTAGTCAACATCGCCATTTTCGATACGGCTTTCCACCAGACCATGCCGCGCCACGCTTATATCTATGCCCTGCCGTACGCATGGTACGAACAGTACGGCGTGCGCCGTTACGGTTTTCACGGCACCTCCCACCTCTATGTGTCGAAGCGCGCCGCGGCCCTGCTCGGCAAACATGCCCGCGACTGCAACCTGATCACCATGCATATCGGCAACGGCGTCTCCCACTGCGCCATCCGCAACGGTATTTCCATCGATACCAGCATGGGCCTTACGCCGCTGGAAGGCGCCGTGATGGGCACGCGCTGCGGCGACATCGATCCGGCCATCCCGCACTTCATGATGAAAATGGCAGATCTTTCGCCGGATGAGATGGACCGCCTTCTCAACAAAAAAAGCGGCGTACTTGGCATCACCGGCCGCTATACCGACCGCCGCGATATTATCCAGGCAGCAGAATCCGGCGATGAACGCTGCCAACTGAGCCTGGAAATCGAATGTTATCGCCTGCGCAAATACATTGGCGCCTACCTGGCAGCGGTCGGTCCGCTGGATGCCATCGTCTTTACCGCCGGCGTCGGTGAGCATTCGTGGCTCATCCGCCAAATGGCCCTGCAGCACCTTGAGCACCTGGGCATCCATTTCGACAAGCAACGCAACCGCGATCTGGCAAGCGGCCTGGAAGCGGCCATTTCCAGTCCGGACTCCCCGATCAAGGTGTTTGTCATTCCGACCAATGAAGAATTGGTGTTCATCGAAGATGTCGCCGCCATTCTTGACGGTAGCTATGCCGACCACATGGACTTTGCCTACTCTTTCGCCCACCCGGACTACAAGCCGCGTTGACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTATGAGTCATAGCAATGTTTAGCAGATTTGCATTCATCAATT
Ruminococcus gnavus
CTGTAGTGATTGTGATTCTAATCAGTATGCATGTAGTCAAAGGAGGACAGCTATGAACGTATTAGTAATCAACTGTGGTAGTTCTTCTTTGAAATTCCAGCTGATCAATGCAAAATCTGAGGAGGTTCTGGCAAAAGGTATTTGTGAACGTATCGGTATTGACGGTCGTCTGACGTATCAGCCGGAAGGTGGTGAAAAAGAAAAATCAGAAAAAGCAATGCCGACGCACACCGAGGCGATCCAGTTTGTGATCGAGGCTCTGACCAATCCGGAGACCGGTGTTGTAAACAGCCTGGATGAGATCGGTGCAGTGGGTCATCGTATGGTACACGGTGGTGAAAAATTTGCTTCTTCTGTAGTGATCACCGATGAAGTGAAAAAAGCGGTAGAAGAGTGCAATGATCTTGCACCGCTTCACAATCCGGCAAACCTGATCGGTGTTGCAGCATGTGAAAATCTGATGCCGAACACCCCGATGGTAGCAGTGTTTGATACCGCATTCCATCAGACCATGCCGCCGAAAGCATACATGTACGGTCTTCCGTATGAATACTATGAGAAATACAAAGTACGTCGTTACGGTTTCCACGGTACCAGCCACAGCTTTGTTTCTAAGCGTGCAGCTGAGTTGTTAGGTAAAGCATATGATGAGACCAAAACGATCGTATGCCACCTTGGTAACGGTTCTAGCGTAACCGCTGTTTTAAACGGTAAATCCGTAGATACCTCCATGGGTCTGACGCCGTTGGAAGGTCTTGTAATGGGTACCCGCTGCGGTGATATCGATCCGGCAATTATGGAATTTTTGGCACACAAGCTGGATCTGGATGTTGACGGCGTTATGAAGATCTTGAATAAGGATTCTGGTGTATACGGTCTTTCCGGCGGTCTTTCCAGCGATTTCCGTGATCTGACCGATGCCTCTCTGGCAGGCGATAAGAAAGCACAGATCGCAATGGAAGTATTCTCATACCGTGTTGCAAAATATATCGGTGCATACGTAGCTGTTATGAATGGTGTAGATGACATTGTATTTACCGCAGGTATCGGTGAAAATGATGATATCGTACGTGAACAGGTATGCAGCTACCTGGGTTACCTTGGTATCGAATTCAATACCGAATTAAACAAAGGTCTCCGTGGTAAAGAAACCGAGCTCTCAATGCCGGGTTCCAAAGTAAAAGTGTTTGTAATTCCGACCAATGAAGAGCTTGCGATTGCACGCGAGACCCTGGAACTGGTGCGCAAATAACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTATGAGTCATAGCAATGTTTAGCAGATTTGCATTCATCAATT
Pta
Methanosarcina acetivorans
TCAATGTACATCGATTCACGTATTAATGCATCAGAATTGAAGGAGGACAGCTTTGGTAACCTTTTTAGAGAAGATCAGTGAACGTGCAAAGAAACTCAACAAGACCATCGCTTTACCGGAAACGGAAGATATTCGCACCCTCCAGGCAGCTGCCAAGATCCTTGAACGTGGTATTGCAAACGTTGTCCTTGTCGGTGATGAAGCCGACATTAAGGCGCTCGCAGGTGATCTTGATCTCTCAAAAGCAAAAATTGTAAATCCGAAAACCTATGAGAAAAAAGACGAATACATTAACACGTTCTATGAGCTGCGTAAGCACAAAGGTATTACCCCGGAAACGGCAGCTGAAGTTATGAGCGATTACGTTTACTTCGCCGTTATGATGGCAAAACTCGGTGAAGTAGACGGTGTAGTATCAGGTGCTGCTCACTCTTCTTCAGACACCCTGCGTCCGGCTGTCCAGATTGTTAAGACGGCTCCGGGCGCAGCACTTGCATCCGCTTTCTTCATCATTGCCGTACCGGACTGTGAATACGGTTCAGATGGCACCTTCCTCTTTGCCGACTCGGGTATGGTTGAAATTCCGAGTGTAGAGGATGTTGCAAACATTGCAGTTATTTCTGCAAAGACCTTCGAACTGCTGGTTCAGGACACGCCGTATGTTGCAATGCTTTCCTATTCCACCAAAGGTAGTGCACACAGCAAACTGACCGAGGCAACCGTCGCTGCCACCAAGCTTGCACAGGAACTTGCTCCGGACGTTGCAATCGACGGTGAACTGCAGGTTGATGCAGCAGTTGTCCCGAAAGTTGCAGCTTCAAAGGCTCCGGGTAGCCCGGTCGCAGGTAAAGCCAATGTCTTTATCTTCCCGGATCTGAACGCTGGTAACATCGCATACAAGATCGCCCAGCGCCTTGCCAAGGCTGAAGCATATGGTCCGATCACCCAGGGTCTTGCAAAGCCGATTAACGACCTGTCCCGTGGTTGCAGTGACGAAGATATTGTCGGTGCTGTTGCGATTACCTGTGTCCAGGCCGCAGCCCAGGACAAATAACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTACGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAG
Clostridium phytofermentans
TCAATGTACATCGATTCACGTATTAATGCATCAGAATTGAAGGAGGACAGCTATGGGTTTTATTGATGACATCAAGGCACGTGCTAAACAAAGTATTAAGACGATTGTTTTACCGGAGAGTATGGACCGTCGTACCATTGAGGCAGCTGCTAAGACGTTAGAAGAGGGCAATGCTAACGTAATTATTATCGGTAGTGAGGAAGAAGTTAAGAAGAATTCAGAAGGTCTTGACATTTCGGGTGCTACCATCGTTGACCCGAAGACCTCGGACAAGCTTCCGGCTTACATTAACAAGCTTGTAGAACTTCGTCAGGCAAAAGGCATGACCCCGGAAAAAGCAAAAGAGCTTTTAACCACCGACTACATTACCTACGGTGTAATGATGGTTAAGATGGGCGATGCAGATGGTTTAGTATCTGGTGCTTGTCACTCTACCGCAGATACCTTACGTCCGTGTCTTCAGATTTTAAAAACGGCTCCGAATACGAAGTTAGTTTCTGCTTTCTTCGTAATGGTAGTACCGAATTGTGATATGGGCGCAAATGGTACGTTCCTTTTCTCTGATGCTGGTTTAAATCAGAATCCGAATGCTGAAGAGTTAGCAGCAATCGCTGGTTCCACCGCGAAGAGTTTTGAACAATTAGTTGGCTCTGAACCGATCGTAGCTATGCTTTCTCATTCAACCAAGGGTAGCGCAAAGCATGCAGATGTTGATAAGGTTGTAGAAGCAACGAAGATTGCAAATGAATTATACCCGGAATATAAGATCGACGGCGAGTTCCAGTTAGATGCAGCAATCGTTCCGAGTGTAGGTGCTTCAAAAGCTCCGGGTAGTGATATTGCTGGTAAAGCTAACGTATTAATCTTCCCGGACCTTGATGCTGGTAACATTGGTTATAAGTTAACCCAGCGTCTTGCAAAGGCAGAAGCTTATGGTCCGTTAACGCAGGGTATTGCAGCTCCGGTAAATGATTTATCACGTGGTTGTTCTTCTGATGATATCGTTGGTGTTGTTGCAATCACGGCTGTTCAGGCACAGAGTAAATAACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTACGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAG
Pelobacter carbinolicus
TCAATGTACATCGATTCACGTATTAATGCATCAGAATTGAAGGAGGACAGCTATGCATTTAGCCGATCAAATCAAAGCCAAAGCCCGCGAAAACCGCCAAGTTGTCGTATTGCCGGAAGGCTACGACGACCGTATGATCCAGGCTGCCGGCCCGATCGTTAAAGACAACCTCGCGGACGTTGTGCTGCTTGGCGAGCCGGCTGCCTTGCAGGCCAAAGCCACGGAACTCGGTGTATCCCTCGAAGGCGTCACCCTGCTCGACCCGAAAAGTGCTCCGCAGCTCGACACCTATGCCGACGAACTGGTCGAAATCCGCAAGAAAAAAGGCCTGTCCAAAGATGACGCATTGAAACTTCTGACCGCCGATGACAACCTGTTTTTCGCCACCATGATGGTACGTCAAGGTGATGCCGGCGGTTGTGTTGCCGGCGCCTACAACACCACGGGCAACGTACTGCGCGCTGCGTTCCAGGTCATTGGCCCGGCACCGGGCATGAAAACCGTATCGTCCTGCTTTATCATGGTAACCAAGACCCCGGAATTCGGCGAAAACGGTGCTCTGATTTTTGCTGACTGCGGCGTCAACCCGAATCCGGACGCAGCTGCCCTTGCGGAAATCGCCGTTTCATCGGCTCAAAGCTGCAAAAGCTTCCTCGGTGCCGAGGCCCGCGTAGCGATGCTGTCCTTTTCCACCAAAGGCAGCGCAAAGCATGAAGACCCGGACAAGGTCATTGCGGCCCTTGCAATCGCTAAGGAAAAAGCCCCGGAGCTGCAGATCGATGGCGAGTTGCAGGCCGACGCCGCCCTGGTACCGGCAGTAGGCAACAGCAAAGCTCCGGGCTCAAACGTCGCCGGCAAAGCCAACACGCTGGTTTTCCCGGATCTCGATGCCGGCAACATTGCTTACAAACTTGTTCAGCGTCTGGCCAAAGCCGAAGCTTTCGGTCCGATCATTCAGGGTCTTGCCAAACCGGTCAACGACCTGTCCCGCGGCTGCTCGGTGGATGACATCATCAGCGTTACCGCTATCACGGCGGTTCAAGCCCAAGGCTAACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTACGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAG
Ruminococcus gnavus
TCAATGTACATCGATTCACGTATTAATGCATCAGAATTGAAGGAGGACAGCTATGGCGTTTATTGATGAAATTAAAGCACGTGCAAAAGCTGACAAAAAGACCATCGTTCTTCCGGAGACCGAAGACGTACGTACGTACCAGGCTGCAGAGGCAGTTTTACGTGAGGGCACGGCCGACCTGATCCTTGTAGGTAGCAAGGAAGAGATCGAAAAGAACGGTGCCGGTTTTGATCTCACCGGTGCTGTGATCGTAGATCCGGCTACCAGCGACAAGACCGAGGGCTATATTGCAAAACTGGTAGAACTCCGTCAGAAAAAAGGTATGACCGAAGAGCAGGCACGTGAGATTCTTCTGACCAACTATCTGTACTACGGTGTTATGATGGTGAAGATGGGTGATGCTGACGGTATGGTATCCGGTGCATGTCATTCTACCGCAGATACCCTTCGTCCGTGTCTGCAGATCCTGAAGACCAAACCGGGTACCAAATTAGTATCCGCATTCTTTGTAATGGTAGTGCCGGATTGCGATATGGGTGCAGACGGTACCTTTATTTTTGGTGATTCCGGTCTGGAGCAGAATCCGGATCCGGAAAAGCTTGCAAATATCGCACTTTCTTCCGCAGAATCTTTCCGTACGCTGACCGGTAAAGAGCCGGTTGTGGCAATGCTTTCACATTCTACCAAAGGTAGTGCAAAACATGCCGATGTTGATAAAGTTGTAGAGGCTACCCGTCTTGCAAAAGAGATGGCACCGGAACTGAAACTGGACGGTGAACTGCAGCTTGACGCAGCGATCGTTCCGGCAGTAGGTGAGTCCAAAGCACCGGGTAGCCCGGTTGCAGGTCATGCAAATGTTCTGATCTTCCCGGATCTGGATGCAGGTAATATCGGTTACAAGCTTGTACAGCGTCTTGCAAAAGCAGAAGCATATGGTCCGCTGACCCAGGGTATTGCAGCACCGGTCAACGATCTGTCCCGTGGTTGCAGCGCAGAGGATATCGAAGGTGTAGTAGCGATTACCGCAGTACAGGCTCAGGCACAGTAACGAAAGGCTCAGTCGAAAGACTGAGCCTTTCGTTTTACGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAG
Fig. 3Gene identification by PCR. 7/12 clones exhibited a restriction pattern similar to 4 different gene combinations. 5/12 clones exhibited a restriction pattern similar to 2 different gene combinations. Every PCR reaction contained 2 sets of primers (to identify sources for both ackA and pta). Double bands present the correct gene combinations.