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SUPPLEMENTARY INFORMATION

ForMicrobial conversion of biomass to methyl halides, Bayer et al.

1. Construction of MHT-expressing strains, growth and assay conditions p.2

2. Cumulative methyl iodide production p.3

3. Targeting the B. maritima MHT to the yeast vacuole p.4

4. Methyl halide production from cellulosic feedstocks p.4

4. Metabolic pathway to methyl iodide p.5

5. Combustion of culture headspace p.6

6. Strains, plasmids, and primers used p.7

7. MHT sequences p.8

8. References p.15

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1. Construction of MHT-expressing strains, growth and assay conditions

Strains and plasmidsCloning was performed using standard procedures in E. coli TOP10 cells (Invitrogen). Primers are listedbelow. The MHT coding regions were synthesized by DNA 2.0 (Menlo Park, CA) in the pTRC99a1

inducible expression vector carrying a gene for chloramphenicol resistance. Constructs were transformedinto DH10B strain for methyl halide production assays. For yeast expression, the B. maritima MHTcoding region was cloned into vector pCM190.

Cloning was performed using standard procedures in E. coli TOP10 cells (Invitrogen). The B. maritimaMCT coding region was synthesized by DNA 2.0 (Menlo Park, CA) and amplified using specifiedprimers with PfuUltra II (Stratagene) according to manufacturers instructions. PCR products werepurified using a Zymo Gel Extraction kit according to manufacturers instructions. Purified expressionvector (pCM1902) and coding region insert were digested with restriction enzymes NotI and PstIovernight at 37 degrees and gel purified on a 1 % agarose gel and extracted using a Promega Wizard SVGel kit according to manufacturers instructions. Vector and insert were quantitated and ligated (10 fmolvector to 30 fmol insert) with T4 ligase (Invitrogen) for 15 minutes at room temperature and transformedinto chemically competent E. coli TOP10 cells (Invitrogen). Transformants were screened and plasmidswere sequenced using specified primers to confirm cloning.

Constructs were transformed into the S. cerevisiae W303a background using standard lithium acetatetechnique and plated on selective media3. Briefly, competent W303a cells were prepared by sequentialwashes with water and 100mM lithium acetate in Tris-EDTA buffer. 1 g of plasmid was incubated for30 minutes at 30 degrees with 50 L of competent cells along with 300 L of PEG 4000 and 5 g ofboiled salmon sperm DNA as a carrier. Cells were then heat-shocked at 42 degrees for 20 minutes. Cellswere spun down and resuspended in 100 L water and plated on synthetic complete uracil dropout plates.Plates were incubated at 30 degrees for 48 hours and positive transformants were confirmed by streakingon uracil dropout plates.

Media and growth conditionsBacteria carrying MHT expression vectors were inoculated from freshly streaked plates and grownovernight. Cells were diluted 100-fold into media containing 1 mM IPTG and 100 mM appropriatesodium halide salt. Culture tubes were sealed with a rubber stopper and grown at 37 degrees for 3 hours.Yeast carrying MHT expression vectors were streaked on uracil dropout plates from freezer stocks (15%glycerol) and grown for 48 hours. Individual colonies were inoculated into 2 mL of synthetic completeuracil dropout media and grown overnight at 30 degrees. Cultures were next inoculated into 100mL freshsynthetic complete uracil dropout media and grown for 24 hours. Cells were spun down and concentratedto high cell density (OD 50) in fresh YP media with 2% glucose and 100 mM sodium iodide salt. 10 mLof this concentrated culture was aliquoted into 14 mL culture tubes and sealed with a rubber stopper.Cultures were grown at 30 degrees with 250 rpm shaking.

Gas chromatography-mass spectrometryThe GC-MS system consisted of a model 6850 Series II Network GC system (Agilent) and model 5973Network mass selective system (Agilent). Oven temperature was programmed from 50 degrees (1 min) to70 degrees (10 degrees / min). 100 L of culture headspace was withdrawn through the rubber stopperwith a syringe and manually injected into the GC-MS. Samples were confirmed as methyl iodide bycomparison with commercially obtained methyl iodide (Sigma), which had a retention time of 1.50minutes and molecular weight of 142 (Supplemental Figure 1, below). Methyl iodide production was

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compared to a standard curve of commercially available methyl iodide in YPD. Standards were preparedat 0.1 g/L, 0.5 g/L 1.0 g/L, and 10 g/L in 10mL YP media plus 2% glucose, aliquoted into 14 mL culturetubes and sealed with rubber stoppers. Standards were incubated at 30 degrees for 1 hour and methyliodide in the headspace was measured as above. A standard curve was fit to the data to quantitate methyliodide concentration with headspace counts.

Supplemental Figure 1. GC-MS chromatograms of methyl iodide productionChromatograms at molecular weight 142 for methyl iodide standard in YPD (left) and methyl iodideproducing S. cerevisiae (right). For the standard, commercially available methyl iodide (Sigma) wasaliquoted into 10mL of YPD media in a 14mL culture tube and sealed with a rubber stopper. For theexperimental sample, 9mL of a MHT-expressing culture (prepared as above) was added to 1mL of 1MNaI in a 14mL culture tube and sealed with a rubber stopper. Both samples were incubated at 30 degreeswith 250 rpm shaking for 60 minutes. 100L of headspace was withdrawn with a syringe and manuallyinjected into the GC-MS as described above.

Methyl iodide toxicity assayIndividual colonies were inoculated in YP media with 2% glucose and grown overnight. Cultures werediluted to an OD600 of 0.05 and methyl iodide was added to the specified amount. Cultures were grown at30 degrees with 250 rpm shaking for 24 hours. OD600 was measured by spectometry with YP media usedas a blank. Each data point was performed in triplicate. The RAD50 mutant4 was obtained from theSaccharomyces Genome Deletion Project (Invitrogen).

2. Cumulative methyl iodide production

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Long-term (>2 hour) methyl iodide production was measured by inducing cultures as above, assayingmethyl iodide at 1 hour, and venting the culture to simulate product extraction. Cultures were then re-sealed and methyl iodide was measured again to determine how much methyl iodide had been vented.Cultures were again grown for 1 hour, measured, and vented. Data is displayed in the main text bysumming the production each hour. An example of the raw data from methyl iodide measurements isshown in Supplemental Figure 2, below.

Supplemental Figure 2. Cumulative methyl iodide production in shake flask.Methyl iodide headspace counts from MHT-expressing culture, vented each hour to simulate productextraction. Cells were grown and induced as above, sealed with a rubber stopper, grown for 1 hour, andmeasured via GC-MS (white data points). Cultures were then vented in a fume hood for 5 minutes,resealed, and assayed to determine how much methyl iodide had been removed (gray data points). Thisprocedure was repeated every hour for 8 hours. Figure 2b from the main text is shown for comparison.

3. Targeting the B. maritima MHT to the yeast vacuole

We fused a 16 amino acid vacuolar targeting tag (KAISLQRPLGLDKDVL) from yeast carboxypeptidaseY5 to the N-terminus of the B.maritima MCT and expressed the enzyme from the same vector as above(pCM190). Assays of methyl iodide production indicated that targeting the MCT to the vacuole resultedin a 50% increase in production rate. We next expressed the cytosolic and vacuolar targeted enzymes in aVPS33 background, which is unable to form functional vacuoles6. The difference in production rate wasabolished in the VPS33 strain, indicating that MCT targeting to fully formed vacuoles is necessary forenhancing the rate of methyl iodide formation.

4. Methyl halide production from cellulosic feedstocks

Growth and assay conditionsActinotalea fermentans was obtained from ATCC (43279). A. fermentans and S. cerevisiae cells wereinoculated in either YP media + 2% glucose (for S. cerevisiae) or BH media + 2% glucose (for A.fermentans) and grown overnight. Cultures were diluted to OD600 = 0.05 in 50 mL of YP media with 20

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g/L of cellulosic stock as the sole carbon source. Corn stover and poplar were pulverized using acommercially available blender with a 1 HP, 1000W motor. Bagasse was aliquoted into the appropriatedry weight, then washed 3 times with hot water to remove soil and residual sugar. Cultures wereincubated at 30 degrees with 250 rpm agitation for 36 hours. 9mL aliquots of cultures were placed in14mL tubes with 1mL of 1M sodium chloride and sealed with a rubber stopper. Headspace samples wereassayed for GC-MS production as above. A. fermentans and S. cerevisiae were quantitated as describedbelow.

Yeast and bacteria quantitationS. cerevisiae and A. fermentans were quantitated from cultures grown on cellulosic stocks by plating onselective media.. Cultures were diluted in sterile water and 100 uL was plated on either YPD agar +ampicillin (to quantitate S. cerevisiae) or brain-heart agar (to quantitate A. fermentans). Plates wereincubated at 30 degrees for either 48 hours (for YPD) or 16 hours (for BH). Colonies were counted byhand and counts from at least 4 plates were averaged. In the switchgrass and corn stover grown culturessome unidentified background cultures were apparent but showed distinguishable morphology from A.fermentans.

5. Metabolic pathway to methyl iodide

Supplemental Figure 5. Preparation of cellulosic feedstocksCellulosic stocks were obtained from the sources cited in the text. We prepared the feedstocks byblending for ~1 minute. a, poplar before blending (left) and after blending. B, corn stover beforeblending (left) and after blending (right). c, bagasse and d, switchgrass. We found that blendingdid not further decrease the size of the particles in bagasse and switchgrass, feedstocks are shownas they were supplied. e, example of culture flask with 20 g/L switchgrass.

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The pathway from glucose to methyl iodide is shown below (Supplemental Figure 4). 3-phosphoglycerate from glycolysis is used in serine biosynthesis (non-fermentable carbon sources such asacetate would feed into serine biosynthesis from TCA cycle intermediates). Serine contributes a methylgroup to the folate pathway, which replenishes SAM. The glycine cleavage pathway allows an additionalcarbon to be funneled back into folate metabolism7-10. MHTs use SAM to methylate halide ions11.

Supplemental Figure 4. Pathway for theoretical yield of methyl iodide from glucose.The pathway that transforms the carbon in glucose to the fungible carbon in methyl iodide. Carbons aretracked throughout the pathway and are indicated with a red circle.

6. Combustion of culture headspaceAs a further demonstration of methyl iodide productivity by the engineered yeast strains, we askedwhether the headspace of the culture could be ignited. Methyl halides are volatile and reactive, withcombustion yielding carbon monoxide, carbon dioxide, and hydrogen halide. To test this, we grew aculture of B.maritima MHT-expressing yeast overnight in 100 mM sodium chloride to accumulate methyliodide. We then decanted the culture into a side-arm flask (Supplemental Movie 1 ). The mouth of theflask was sealed with a rubber stopper with tubing running into the flask. The tubing was used to forcepressurized air (from a house line) into the flask. A Bunsen burner was used to heat the outlet air fromthe flask. Combustion was performed in a fume hood and appropriate personal protective equipment wasused. We observed combustion of the headspace and the evolution of violet smoke indicating iodine(Supplemental Movie 1 and Figure 5, below). The flame appeared to diminish after 20 seconds ofcombustion, suggesting we exhausted the methyl iodide in the flask. These results suggest the relativeease of extracting methyl halides from the yeast culture.

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Supplemental Figure 5. Combustion of methyl iodide-producing yeast culture headspace.Methyl iodide producing culture was placed in a side-arm flask and the headspace was forced out withpressurized air. The off-gas was combusted with a flame from a Bunsen burner. Figure is taken from aframe in Supplemental Movie 1.

7. Strains, plasmids, and primers used

Strains

E. coli (Invitrogen TOP10)[F- mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 ara139 (ara-leu)7697 galU galK rpsL (StrR)endA1 nupG]

S. cerevisiae W303a(MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15)

A. fermentans (ATCC 43279)12-14

Plasmids

pTRC99a1pCM1902pFA6a-GFP(S65T)-KAN15

Primers

Batis fwdGG GCGGCCGC GGA ATT GTG AGC GGA TAA CAA TTG AAT TCA TTA AAG

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Batis revCC CTGCAG CAGCCAAGCT TGCATGCCT

pCM190 sequencing fwdCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG

Vac.Batis fwdGG GCGGCCGC ATGAAGGCCATCTCTTTACAGAGGCCATTGGGTTTAGACAAGGACGTCTTATCT ACT GTT GCG AAT ATT GCT CC

VPS33.del fwdATGAATAGATTTTGGAATACTAAGAAATTTGGCAGATCCGCTAGGGATAA

VPS33.del revTTAAGATATAGAGTTCATGATCCTTGTGCCGAATTCGAGCTCGTTTAAAC

8. MHT sequences

MHT sequences are rank ordered according to methyl iodide activity. The organism name (as used in themain text) and accession number are given.

> Batis_maritima_ AAD26120MSTVANIAPVFTGDCKTIPTPEECATFLYKVVNSGGWEKCWVEEVIPWDLGVPTPLVLHLVKNNALPNGKGLVPGCGGGYDVVAMANPERFMVGLDISENALKKARETFSTMPNSSCFSFVKEDVFTWRPEQPFDFIFDYVFFCAIDPKMRPAWGKAMYELLKPDGELITLMYPITNHEGGPPFSVSESEYEKVLVPLGFKQLSLEDYSDLAVEPRKGKEKLARWKKMNN> Oryza_sativa_2_ EAY92545MASAIVDVAGGGRQQALDGSNPAVARLRQLIGGGQESSDGWSRCWEEGVTPWDLGQPTPAVVELVHSGTLPAGDATTVLVPGCGAGYDVVALSGPGRFVVGLDICDTAIQKAKQLSAAAAAAADGGDGSSSFFAFVADDFFTWEPPEPFHLIFDYTFFCALHPSMRPAWAKRMADLLRPDGELITLMYLAEGQEAGPPFNTTVLDYKEVLNPLGLVITSIEDNEVAVEPRKGMEKIARWKRMTKSD> Vitis_vinifera_2_CAO46361MANDSTSIESNSELQKISQVIGSGFNGSWEEKWQQGLTPWDLGKATPIIEHLHQAGALPNGRTLIPGCGRGYDVVAIACPERFVVGLDISDSAIKKAKESSSSSWNASHFIFLKADFFTWNPTELFDLIIDYTFFCAIEPDMRPAWASRMQQLLKPDGELLTLMFPISDHTGGPPYKVSIADYEKVLHPMRFKAVSIVDNEMAIGSRKGREKLGRWKRTDEPLL> Burkholderia_xenovorans_YP_557005MSDPTQPAVPDFETRDPNSPAFWDERFERRFTPWDQAGVPAAFQSFAARHSGAAVLIPGCGSAYEAVWLAGQGNPVRAIDFSPAAVAAAHEQLGAQHAQLVEQADFFTYEPPFTPAWIYERAFLCALPLARRADYAHRMADLLPGGALLAGFFFLGATPKGPPFGIERAELDALLTPYFDLIEDEAVHDSIAVFAGRERWLTWRRRA> Brassica_rapa_chinensis_ABL86248MAEVQQNSAHINGENIIPPEDVAKFLPKTVEEGGWEKCWEDGVTPWDQGRATPLVVHLVESSSLPLGRALVPGCGGGHDVVAMASPERYVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVVFCAIEPETRPAWAKAMYELLKPDGELITLMYPITDHDGGPPYKVAFSTYEDVLVPVGFKAVSIEENPYSIATRKGKEKLARWKKIN> Burkholderia_pseudomallei_ YP_332262MKDRLMSQGDGVTNEANQPEAAGQATGDAQPASPAGPAHIANPANPANPANPPALPSLSPPAAAPSSASSAAHFSSRDPGDASFWDERFEQGVTPWDSARVPDAFAAFAARHARVPVLIPGCGSAYEARWLARAGWPVRAIDFSAQAVAAARRELGEDAGLVEQADFFTYAPPFVPQWIYERAFLCAIPRSRRADYARRMAELLPPGGFLAGFFFIGATPKGPPFGIERAELDALLCPHFALVEDEPVADSLPVFAGRERWLAWRRS> Burkholderia_thailandensis_YP_441114MTSEANKGDAAVQAAGDAQPASPASPPSADVQPARAALAPSSVPPAPSAANFASRDPGDASFWDERFERG

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VTPWDSARVPDAFAAFAARHPRCPVLIPGCGSAYEARWLARAGWPVRAIDFSAQAVAAARRESGADAALVEQADFFAYVPPFVPQWIYERAFLCAIPTSRRADYARRVAELLPAGGFLAGFFFIGATPKGPPFGIERAELDALLSPNFELVEDEPVADSLPVFAGRERWLAWRRS> marine_gamma_proteobacterium_HTCC2080_ZP_01626954MEKFGASAMEPVLDWEARYQESSVPWERTGLNPAFVAWQSWLRDHQGGTVVVPGCGRSPELQAFADMGFNVIGVDLSPSAAQFQETVLAAKGLDGKLVVSNLFDWSPDTPVDFVYEQTCLCALKPDHWRAYENLLTRWLRPGGTLLALFMQTGESGGPPFHCGKAAMEQLFSEQRWIWDETSVRSEHPLGVHELGFRLTLR> Ralstonia_picketti_YP_001897927MAQPPVFQSRDAADPAFWDERFTREHTPWDAAGVPAAFRQFCEAQPAPLSTLIPGCGNAYEAGWLAERGWPVTAIDFAPSAVASARAVLGPHADVVQLADFFRFSPPRPVHWIYERAFLCAMPRRLWPDYAAQVAKLLPPRGLLAGFFAVVEGREAMPKGPPFETTQPELDALLSPAFERISDMPIAETDSIPVFAGRERWQVWRRRAD> Burkholderia_phytofirmans_YP_001894302MSDPTQPSAPEFESRDPNSPEFWDERFERGFMPWDQAGVPSAFESFAARHAGAAVLIPGCGSAYEAVWLAGHGYPVRAIDFSPAAVAAAHEQLGAQHADLVEQADFFTYELPFTPAWIYERAFLCALPLARRADYARRMADLLPGGALLAGFFFIGATPKGPPFGIERAELDGLLKPYFELIEDEPVHDSIAVFAGRERWLTWRRRV> Burkholderia_vietnamensis_YP_001120660MSNPTQPPPPSAADFATRDPANASFWDERFARGVTPWEFGGVPDGFRAFAQRRAPCTVLIPGCGSAQEAGWLAQAGWPVRAIDFAEQAVVAAKATLGAHADVVEQADFFAYQPPFVVQWVYERAFLCALPPSLRAGYAARMAELLPAGGLLAGYFFVMKKPKGPPFGIERAELDALLAPSFELIEDLPVTDSLAVFDGHERWLTWRRR> Aspergillus_clavatus_XP_001272206MSTPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDRLPWDRGFPNPALEDTLIQKRAIIGGPLGQDAQGKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVDVCQEEQAKNGDQYPVRDAEIGQGKITFVQGDFFEDTWLEKLNLTRNCFDVIYDYTFFCALNPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGPPWGSASEAYRAHLSHPGEEIPYDASRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIWQRPPQSSL> Phaeosphaeria_nodorum_XP_001792029MANPNQDRLRSHFAALDPSTHASGWDSLWAEGTFIPWDRGYANPALIDLLANPSSPPTSSDANPTPGAPKPNTIDGQGVQLPAPLEGGVRRKALVPGCGKGYDVALLASWGYDTWGLEVSRHAADAAKEYLKDAGEGALEGEYKIKDAKIGKGREECVVADFFDDAWLKDVGAGEFDVIYDNTFLCALPPLLRPKWAARMAQLLARDGVLICLEFPTHKPASSGGPPWSLPPTVHQELLKRPGEDISYDEGGVVVATDRAESENALVRVAHWTPKRTHNIAVINGVVRDCVSVWRHKKQS> Dechloromonas_aromatica_YP_286874MSETIKPPEQRPEHPDFWCKRFGEGVTPWDAGKVPMAFVDFVGAQTTPLNSLIPGCGSAWEAAHLAELGWPVTALDFSPLAIEKAREVLGDSPVKLVCADFFTFAPRQPLDLIYERAFLCALPRKLWADWGKQVAELLPSGARLAGFFFLCDQPKGPPFGILPAQLDELLRPNFELIEDQPVGDSVPVFAGRERWQVWRRR> Kortia_algicida_ZP_02160755MNSDATKEYWSQRYKDNSTGWDIGSPSTPLKTYIDQLKDRNLKILIPGAGNAYEAEYLLQQGFTNIYILDISEIPLQEFKQRNPEFPSDRLLCDDFFTHKNTYDLIIEQTFFCSFPPLPETRAQYAKHMADLLNPNGKLVGLWFDFPLTDDLEKRPFGGSKEEYLEYFKPYFDVKTFEKAYNSIAPRAGNELFGIFIKS> Methylibium_petroleiphilum_YP_001022598MSGPDLNFWQQRFDTGQLPWDRGAPSPQLAAWLGDGSLAPGRIAVPGCGSGHEVVALARGGFSVTAIDYAPGAVRLTQGRLAAAGLAAEVVQADVLTWQPTAPLDAVYEQTCLCALHPDHWVAYAARLHAWLRPGGTLALLAMQALREGAGQGLIEGPPYHVDVNALRALLPGDRWDWPRPPYARVPHPSSTWAELAIVLTRR> Burkholderia_dolosa_ZP_00987534MTGRSFAMSDPKQPGTPTAADFATRDPGDASFWDERFARGVTPWEFGGVPDGFRAFAQRLERCAVLIPGCGSAQEAGWLADAGWPVRAIDFAAQAVATAKAQLGAHADVVELADFFTYRPPFDVRWIYERAFLCALPPARRADYAAQMAALLPAGGLLAGYFFVTAKPKGPPFGIERAELDALLAPQFDLIDDWPVTDSLPVFEGHERWLTWRRR> Ralstonia_solanacearum_NP_518583MAQPPVFTTRDAAAPAFWDERFSRDHMPWDAHGVPPAFRQFCEAQPAPLSTLIPGCGSAYEAGWLAERGWPVAAIDFAPSAVASAQAVLGPHAGVVELADFFRFTPRQPVQWIYERAFLCAMPRRLWADYATQVARLLPPGGLLAGFFVVVDGRAAAPSGPPFEITAQEQEALLSPAFERIADALVPENESIPVFAGRERWQVWRRRAD> Ustilago_maydis_XP_762636MTSSLSKDDQIQNLRRLFADSGVPNDPKAWDQAWIDSTTPWDANRPQPALVELLEGAHDADAKVPDVDGNLIPVSQAIPKGDGTAVVPGCGRGYDARVFAERGLTSYGVDISSNAVAAANKWLGDQDLPTELDDKVNFAEADFFTLGTSKSLVLELSKPGQATLAYDYTFLCAIPPSLRTTWAETYTRLLAKHGVLIALVFPIHGDRPGGPPFSISPQLVRELLGSQKNADGSAAWTELVELKPKGPETRPDVERMMVWRRS> Hahella_chejuensis_YP_432621

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MDANFWHERWAENSIAFHQCEANPLLVAHFNRLDLAKGSRVFVPLCGKTLDISWLLSQGHRVVGCELSEMAIEQFFKELGVTPAISEIVAGKRYSAENLDIIVGDFFDLTVETLGHVDATYDRAALVALPKPMRDSYAKHLMALTNNAPQLMLCYQYDQTQMEGPPFSISAEEVQHHYADSYALTALATVGVEGGLRELNEVSETVWLLESR> Brassica_oleracea_2_AAK69761MAEVQQNSGNSNGENIIPPEDVAKFLPKTVDEGGWEKCWEDGVTPWDQGRATPLVVHLVESSSLPLGRGLVPGCGGGHDVVAMASPERYVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVVFCAIEPETRPAWAKAMYELLKPDGELITLMYPITDHDGGPPYKVAVSTYEDVLVPVGFKAVSIEENPYSIATRKGKEKLARWKKIN> Burkholderia_ambifaria_YP_774669MSEPKQPSTPGAADFATRDPGDASFWDERFARGVTPWEFGGVPEGFRAFAQRLGPCAVLIPGCGSAQEAGWLAQAGWPVRAIDFAAQAVAAAKAQLGAHADVVEQADFFMYRPPFDVQWVYERAFLCALPPSLRAGYAARMAELLPAGALLAGYFFVTKKPKGPPFGIERAELDALLAPHFELIDDLPVTDSLAVFEGHERWLTWRRR> Rhodoferax_ferrireducens_YP_522685MAGPTTEFWQERFEKKETGWDRGSPSPQLLAWLASGALRPCRIAVPGCGSGWEVAELAQRGFDVVGLDYTAAATTRTRALCDARGLKAEVLQADVLSYQPEKKFAAIYEQTCLCAIHPDHWIDYARQLHQWLEPQGSLWVLFMQMIRPAATEEGLIQGPPYHCDINAMRALFPQKDWVWPKPPYARVSHPNLSHELALQLVRR> Coccidioides_immitis_XP_001248254MANEILRSAPNLSDRFKNLDGRNQGEVWDDLWKESRTPWDRGSHNPALEDALVEKRGFFGAPVFEDEPLRRKKALVPGCGRGVDVFLLASFGYDAYGLEYSKTAVDVCLKEMEKYGEGGKVPPRDEKVGSGKVMFLEGDFFKDDWVKEAGVEDGAFDLIYDYTFFCALNPALRPQWALRHRQLLAPSPRGNLICLEFPTTKDPAALGPPFASTPAMYMEHLSHPGEDIPYDDKGHVKSNPLQQPSDKGLERVAHWQPKRTHTVGMDDKGNVLDWVSIWRRRD> Synechococcus_elongatus_YP_172090MTNAVNQAQFWEQRYQEGSDRWDLGQAAPVWRSLLAGTNAPAPGRIAVLGCGRGHDARLFAEQGFEVVGFDFAPSAIAAAQALAQGTTAQFLQRDIFALPQEFAGQFDTVLEHTCFCAIDPDRRAEYVEVVRQILKPKGCLLGLFWCHDRPSGPPYGCSLTELRDRFAQGWQEEQLESVTESVEGRRGEEYLGRWRRLD> Ralstonia_eutropha_YP_724967MSDPAKPVPTFATRNAADPAFWDERFEQGFTPWDQGGVPEEFRQFIEGRAPCPTLVPGCGNGWEAAWLFERGWPVTAIDFSPQAVASARQTLGPAGVVVQQGDFFAFTPQPPCELIYERAFLCALPPAMRADYAARVAQLLPPGGLLAGYFYLGENRGGPPFAMPAEALDALLAPAFERLEDRPTAAPLPVFQGQERWQVWRRRSG> Oryza_sativa_japonica_2_NP_001056843MSSSAARVGGGGGRDPSNNPAVGRLRELVQRGDAADGWEKSWEAAVTPWDLGKPTPIIEHLVKSGTLPKGRALVPGCGTGYDVVALASPERFVVGLDISSTAVEKAKQWSSSLPNADCFTFLADDFFKWKPSEQFDLIFDYTFFCALDPSLRLAWAETVSGLLKPHGELITLIYLISDQEGGPPFNNTVTDYQKVLEPLGFKAILMEDNELAIKPRKGQEKLGRWKRFVPGSSL> Burkholderia_multivorans_YP_001578763MSDPKHAAAPAAASFETRDPGDASFWDERFARGMTPWEFGGVPAGFRAFASARPPCAVLIPGCGSAREAGWLAQAGWPVRAIDFSAQAVAAAKAQLGAHADVVEQADFFAYRPPFDVQWIYERAFLCALPPARRADYAATMAALLPAQGLLAGYFFVADKQKGPPFGITRGELDALLGAHFELIDDAPVSDSLPVFEGHERWLAWRRR> Cellulophaga_sp._ZP_01049440MELTSTYWNNRYAEGSTGWDLKEVSPPIKAYLDQLENKELKILIPGGGYSYEAQYCWEQGFKNVYVVDFSQLALENLKQRVPDFPSLQLIQEDFFTYDGQFDVIIEQTFFCALQPDLRPAYVAHMHTLLKAKGKLVGLLFNFPLTEKGPPYGGSTTEYESLFSEHFDIQKMETAYNSVAARAGKELFIKMVKK> Neosartorya_fischeri_XP_001266691MSNDPRLLSSIPEFIARYKENYVEGWAELWNKSEGKPLPFDRGFPNPALEDTLIEKRDIIGGPIGRDAQGNTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSDTAVQVCKEEQAKNGDKYPVRDAEIGQGKITFVQGDFFKDTWLEKLQLPRNSFDLIYDYTFFCALDPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDTSLQGPPWASTSEAYMAHLNHPGEEIPYDANRQCSIDPSKAPSPQGLERVAYWQPARTHEVGIVEGEVQDRVSIWRRPN> Tenacibaculum_sp._ZP_01053731MIFDEQFWDNKYITNKTGWDLGQVSPPLKAYFDQLTNKDLKILIPGGGNSHEAEYLLENGFTNVYVIDISKLALTNLKNRVPGFPSSNLIHQNFFELNQTFDLVIEQTFFCALNPNLREEYVSKMHSVLNDNGKLVGLLFDAKLNEDHPPFGGSKKEYTSLFRNLFTIEVLEECYNSIENRKGMELFCKFVK> Arabidopsis_thaliana_3_NP_850403MENAGKATSLQSSRDLFHRLMSENSSGGWEKSWEAGATPWDLGKPTPVIAHLVETGSLPNGRALVPGCGTGYDVVAMASPDRHVVGLDISKTAVERSTKKFSTLPNAKYFSFLSEDFFTWEPAEKFDLIFDYTFFCAFEPGVRPLWAQRMEKLLKPGGELITLMFPIDERSGGPPYEVSVSEYEKVLIPLGFEAISIVDNELAVGPRKGM

S11

EKLGRWKKSSTFHSTL> Giberella_zeae_XP_390285MATENPLEDRISSVPFAEQGPKWDSCWKDALTPWDRGTASIALHDLLAQRPDLVPPSQHQDHRGHPLRDATGAIQKKTALVPGCGRGHDVLLLSSWGYDVWGLDYSAAAKEEAIKNQKQAESEGLYMPVDGLDKGKIHWITGNFFAQDWSKGAGDDGKFDLIYDYTFLCALPPDARPKWAKRMTELLSHDGRLICLEFPSTKPMSANGPPWGVSPELYEALLAAPGEEIAYNDDGTVHEDPCSKPWADALHRLSLLKPTRTHKAGMSPEGAVMDFLSVWSR> Legionella_pneumophila_YP_128074MNKGQYFWNELWCEGRISFHKKEVNPDLIAYVSSLNIPAKGRVLVPLCGKSVDMLWLVRQGYHVVGIELVEKAILQFVQEHQITVRENTIGQAKQYFTDNLNLWVTDIFALNSALIEPVDAIYDRAALVALPKKLRPAYVDICLKWLKPGGSILLKTLQYNQEKVQGPPYSVSPEEIALSYQQCAKIKLLKSQKRIQEPNDHLFNFGISEVNDSVWCIRKG> Burkholderia_cenocepacia_AU_1054_YP_621990MSDPKQPAAPSAADFATRDPGSASFWDERFARGVTPWEFGGVPDGFRVFAQRREPCAVLIPGCGSAQEAGWLAQAGWPVRAIDFAAQAVAAAKAQLGAHADVVEQADFFQYRPPFDVQWVYERAFLCALPPGLRAGYAARMAELLPTGGLLAGYFFVVAKPKGPPFGIERAELDALLAPHFELLEDLPVTDSLAVFDGHERWLTWRRR> Aspergillus_fumigatus_XP_751474MSNDPRLVSSIPEFIARYKENYVEGWAELWDKSEGKPLPFDRGFPNPALEDTLIEKRDIIGDPIGRDAQGNTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVKVCKEEQAKNGDKYPVRDAEIGQGKITYVQGDFFKDTWWEKLQLPRNSFDLIYDYTFFCALDPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDTSLQGPPWASTSEAYMAHLNHPGEEIPYDANRQCSIDPSKAPSPQGLERVAYWQPARTHEVGIVEGEVQDRVSIWRRPN> Microscilla_marina_ZP_01688206MHTTLDKDFWSNRYQAQDTGWDAGSITTPIKAYVDQLEDKHLKILVPGAGNSHEAEYLHQQGFTNVTVIDIVQAPLDNLKSRSPDFPEAHLLQGDFFELVGQYDLIIEQTFFCALNPSLRESYVQKVKSLLKPEGKLVGVLFCNVFLDRTEPPFGATEQQHQEYFLPHFIAKHFASCYNSIAPRQGAEWFICLIND> Oceanicaulis_alexandrii_ZP_00951977MTQASSDTPRSEDRSGFDWESRFQSDDAPWERQGVHPAAQDWVRNGEIKPGQAILTPGCGRSQEPAFLASRGFDVTATDIAPTAIAWQKTRFQTLGVMAEAIETDALAWRPETGFDALYEQTFLCAIHPKRRQDYEAMAHASLKSGGKLLALFMQKAEMGGPPYGCGLDAMRELFADTRWVWPDGEARPYPHPGLNAKAELAMVLIRR> Burkholderia_cenocepacia_MC0-3_ZP_01560172MSDPKQPAAPSAAEFATRDPGSASFWDERFARGVTPWEFGGVPDGFRAFAQRHEPCAVLIPGCGSAQEAGWLAQAGWPVRAIDFAAQAVAAAKVQLGAHADVVEQADFFQYRPPFDVQWVYERAFLCALPPSLRADYAARMAELLPTGGLLAGYFFVVAKPKGPPFGIERAELDALLAPHFELLEDLPVTDSLAVFDGHERWLTWRRR> Leptospirillum_sp._EAY56191MPDKIFWNQRYLDKNTGWDLGQPAPPFVRLVEKGEFGPPGRVLIPGAGRSYEGIFLASRGYDVTCVDFAPQAVREAREAARQAGVKLTVVEEDFFRLDPRTIGVFDYLVEHTCFCAIDPPMRQAYVDQSHALLAPGGLLIGLFYAHGREGGPPWTTTEEEVRGLFGKKFDLLSLGLTDWSVDSRKGEELLGRLRRKNDRIE> Mariprofundus_ferrooxydans_ZP_01453074MTVWEERYQRGETGWDRGGVSPALTQLVDHLHLEARVLIPGCGRGHEVIELARLGFRVTAIDIAPSAIAHLSQQLEQEDLDAELVNGDLFAYAPDHCFDAVYEQTCLCAIEPEQRADYEQRLHGWLKPEGVLYALFMQTGIRGGPPFHCDLLMMRELFDASRWQWPEETGAVLVPHKNGRFELGHMLRRTGR> Polaromonas_naphthalenivorans_YP_973752MAGPTTDFWQARFDNKETGWDRGAPGPQLLAWLESGALQPCRIAVPGCGSGWEVAELARRGFEVVGIDYTPAAVERTRALLAAQGLAAEVVQADVLAYQPHKPFEAIYEQTCLCALHPDHWVAYARQLQQWLKPQGSIWALFMQMVRPEATDEGLIQGPPYHCDINAMRALFPAQHWAWPRPPYAKVPHPNVGHELGLRLMLRQGR> Synechococcus_sp._1_YP_001224608MTNVHLPQAWDARYQHGTDGWELGKAAPPLQAFLEHHPRAPQPEGTVLVPGCGRGHEAALLARLGFEVIGLDFSSEAIREARRLHGEHPRLRWLQADLFDADALSGAGLASGSLSGVLEHTCFCAIDPSQRAHYRSTVDRLLRAEGWLLGLFFCHPRPGGPPFGSDPEQLAASWAQIGFYPLIWEPARGSVAGRSEEWLGFWRKPEQRSA> Flavobacteriales_bacterium_1_ ZP_01732881MNYWEERYKKGETGWDAGTITTPLKEYIDQLTDKNLTILIPGAGNGHEFDYLIDNGFKNVFVVDIAITPLENIKKRKPKYSSHLINADFFSLTTTFDLILEQTFFCALPPEMRQRYVEKMTSLLNPNGKLAGLLFDFPLTSEGPPFGGSKSEYITLFSNTFSIKTLERAYNSIKPRENKELFFIFETK> Gramella_forsetii_YP_862431MNKDFWSLRYQKGNTGWDIGNISTPLKEYIDHLHKKELKILIPGAGNSYEAEYLFEKGFKNIWICDIAKEPIENFKKRLPEFPESQILNRDFFELKDQFDLILEQTFFCALPVNFRENYAKKVFELLKVNGKISGVLFDFPLTPDGPPFGGSKEEYLAYFSPYFKINTFERCYNSINPRQGKELFFNFSKK

S12

> Brassica_oleracea_1_AAK69760MAEEQQKAGHSNGENIIPPEEVAKFLPETVEEGGWEKCWEDGITPWDQGRATPLVVHLVDSSSLPLGRALVPGCGGGHDVVAMASPERFVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVVFCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHDGGPPYKVAVSTYEDVLVPVGFKAVSIEENPYSIATRKGKEKLGRWKKIN> Vitis_vinifera_3_CAO46360MGLCVPSGRISGGVCGLLSGRSLTWAKNLGVSTTQLRMSNNGSSIESNPKVQKLNQIIGSDSAGGWEKSWQQGHTPWDLGKPTPIIQHLHQTGTLPSGKTLVPGCGCGYDVVTIACPERFVVGLDISDSAIKKAKELSSSLWNANHFTFLKEDFFTWNPTELFDLIFDYTFFCAIEPDMRSVWAKRMRHLLKPDGELLTLMFPISDHAGGPPYKVSVADYEEVLHPMGFKAVSIVDNKMAIGPRKGREKLGRWKRTPSKSLL> Burkholderia_sp._YP_370293MSDPKQPKPNAPAAADFTTRDPGNASFWNERFERGVTPWEFGGVPEGFSVFAHRLELCAVLIPGCGSAQEAGWLAEAGWPVRAIDFAAQAVAAAKAQLGAHAGVVEQADFFAYRPPFDVQWVYERAFLCALPPAMRADYAARMAELLPADGLLAGYFFLMAKPKGPPFGIERAELDALLTPHFELIEDLPVTDSLAVFEGHERWLTWRRR> Flavobacteriales_bacterium_2_ZP_02183094MISMKKNKLDSDYWEDRYTKNSTSWDIGYPSTPIRTYIDQLKDKSLKILIPGAGNSFEAEYLWNLGFKNIYILDFAKQPLENFKKRLPDFPENQLLHIDFFKLDIHFDLILEQTFFCALNPSLREKYVEQMHQLLKPKGKLVGLFFNFPLTKSGPPFGGSLTEYQFLFDKKFKIKILETSINSIKEREGKELFFIFESP> Coprinopsis_cinerea_okayama_ XP_001831730MADPNLAPEIRAKMQEIFKPDDRHSWDLLWKENITPWDAGDAQPSLIELIEESGLDFARKGRALVPGCGTGYDAVYLASALGLQTIGMDISESAVEAANRYRDSSGVQGADRAIFQKADFFTYKVPDEERFDLIMDHTFFCAIHPSLRPEWGQRMSELIKPGGYLITICFPMIPKVETGPPYYLRPEHYDEVLKETFEKVYDKVPTKSSENHKDKERMLVWKKK> Flavobacterium_sp._ZP_01059895MKTDLNKLYWEDRYQNQQTGWDIGSVSTPLKEYIDQIDDKNIQILVPGAGYGHEVRYLAQQGFKNVDVIDLSVSALTQLKKALPDTTAYQLIEGDFFEHHTSYDLILEQTFFCALEPDKRPDYAAHAASLLKDSGKISGVLFNFPLTEKGPPFGGSSEEYKKLFSEYFNIKTLEACYNSIKPRLGNELFFIFEKSNQES> Vibrio_alginolyticus_ZP_01260043MKQAPMINTQFWDDLFIRGTMPWDAQSTPQELKDYLDNSLHVGQSVFIPGCGAAYELSTFIQYGHDVIAMDYSQEAVKMAQSALGNYKDKVVLGDVFNADFSHSFDVIYERAFLAALPRDMWSEYFSTVDKLLPSGGFLIGFFVIDDDYCSRFPPFCLRSGELASFLEPTFELVKSSVVANSVEVFKGREQWMVWQKR> Cryptococcus_neoformans_XP_566834MAQASGDDNAWEERWAQGRTAFDQSAAHPVFVKFLKSDIARELGVPKSGKALVPGCGRGYDVHLLASTGLDAIGLDLAPTGVEAARRWIGSQPSTSGKADILVQDFFTYDPLEKFDLIYDYTFLCALPPSLRQEWARQTTHLANIAADTNPILITLMYPLPPSAKSGGPPFALSEEIYQELLKEQGWKMVWSEDIEEPTRMVGAPGGEKLAVWKRI> Algoriphagus_sp._ZP_01720187MAELDEKYWSERYKSGLTGWDIGFPSTPIVQYLDQIVNKDVEILIPGAGNAYEAYYAFQSGFSNVHVLDISQEPLRNFKDKFPNFPSSNLHHGDFFEHHGSYNLILEQTFFCALNPSLRPKYVKKMSELLLKGGKLVGLLFNKEFNSPGPPFGGGIKEYQKLFHNSFEIDVMEECYNSIPARAGSEAFIRLINSKG> Vibrio_parahaemolyticus_NP_800656MKSKDSPIINEQFWDALFFNGTMPWDRSQTPNELKHYLKRIADKTHSVFIPGCGAAYEVSHFVDCGHDVIAMDYSAEAVNLAKSQLGQHQDKVMLGDVFNADFSREFDVIYERAFLAALPREIWGDYFAMIERLLPSNGLLVGYFVISDDYRSRFPPFCLRSGEIEQKLEANFHLIESTPVTDSVDVFKGKEQWMVWQKK> Psychrobacter_cryohalolentis_YP_581342MENVNQAQFWQQRYEQDSIGWDMGQVSPPLKAYIDQLPEAAKNQAVLVPGAGNAYEVGYLHEQGFTNVTLVDFAPAPIAAFAERYPNFPAKHLICADFFELSPEQYQFDWVLEQTFFCAINPSRRDEYVQQMASLVKPNGKLIGLLFDKDFGRDEPPFGGTKDEYQQRFATHFDIDIMEPSYNSHPARQGSELFIEMHVKD> Croceibacter_atlanticus_ZP_00949128MTSNFWEQRYANNNTGWDLNTVSPPLKHYIDTLSNKTLFILIPGCGNAYEAEYLHNQGFENVFIVDLAEHPLLEFSKRVPDFPKSHILHLDFFNLTQKFDLILEQTFFCALHPEQRLHYAHHTSKLLNSNGCLVGLFFNKEFDKTGPPFGGNKKEYKNLFKNLFKIKKLENCYNSIKPRQGSELFFIFEKK> Aspergillus_niger_3_XP_001388537MTTPTDNKFKDAQAYLAKHQGDSYLKGWDLLWDKGDYLPWDRGFPNPALEDTLVERAGTIGGPIGPDGKRRKVLVPGCGRGVDVLLFASFGYDAYGLECSAAAVEACKKEEEKVNNIQYRVRDEKVGKGKITFVQGDFFDDAWLKEIGVPRNGFDVIYDYTFFCALNPELRPKWALRHTELLAPFPAGNLICLESPRHRDPLAPGPPFASPSEAYMEHLSHPGEEISYNDKGLVDADPLREPSKAGLERVAYWQPERTHTVGKDKNGVIQDRVSIWRRRD> Ostreococcus_tauri_CAL52768

S13

MTTSSAPTRHTSMRVALAAPATVTRRLGTYKRVFDRRAMSTRAIDGAVTSNAGDFARQDGSTDWEGMWSRGITKGAAFDCSRTEPAFQNALDAKEIAIGSGRALVPGCGRGYALASLARAGFGDVVGLEISETAKEACEEQLKAESIPETARVEVVVADFFAYDPKEAFDAAYDCTFLCAIDPRRREEWARKHASLIKPGGTLVCLVFPVGDFEGGPPYALTPEIVRELLAPAGFEEIELRETPAEMYARGRLEYLFTWRRRS> Oryza_sativa_1_EAY99736MDRALPLALSVSLWWLLVGDLGGRWTLEDDGGGGGVSRFGSWYRMCGWWWVWADWIIELGASSWGNLFGLVLKRRKNEAVERDSSDGWEKSWEAAVTPWDLGKPTPIIEHLVKSGTLPKGRALGYDVVALASPERFVVGLGISSTAVEKAKQWSSSLPNADCFTFLADDFFKWKPSEQFDLIFDYTFFCALDPSLRLAWAETVSGLLKPHGELITLIYLVTEESIYSFVYFSIEDVMVLIISYCAERISYYRSVTKKEDHHSIIQSPILLRCPFRNHSYQKVLEPLGFKAILMEDNELAIKPRKAISAFRTSEQPSLAAQDVTE> Oryza_sativa_japonica_1_ABF99844MASAIVDVAGGGRQQALDGSNPAVARLRQLIGGGQESSDGWSRCWEEGVTPWDLGQRTPAVVELVHSGTLPAGDATTVLVPGCGAGYDVVALSGPGRFVVGLDICDTAIQKAKQLSAAAAAAADGGDGSSSFFAFVADDFFTWEPPEPFHLIFDYTFFCALHPSMRPAWAKRMADLLRPDGELITLMYLVINRRYQHV> Polaribacter_irgensii_ZP_01117536MNLSADAWDERYTNNDIAWDLGEVSSPLKAYFDQLENKEIKILIPGGGNSHEAAYLFENGFKNIWVVDLSETAIGNIQKRIPEFPPSQLIQGDFFNMDDVFDLIIEQTFFCAINPNLRADYTTKMHHLLKSKGKLVGVLFNVPLNTNKPPFGGDKSEYLEYFKPFFIIKKMEACYNSFGNRKGRELFVILRSK> Aspergillus_niger_1_XP_001389353MTDQSTLTAAQQSVHNTLAKYPGEKYVDGWAEIWNANPSPPWDKGAPNPALEDTLMQRRGTIGNALATDAEGNRYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSGAAVQACRQEEKESTTSAKYPVRDEEGDFFKDDWLEELGLGLNCFDLIYDYTFFCALSPSMRPDWALRHTQLLAPSPHGNLICLEYPRHKDPSLPGPPFGLSSEAYMEHLSHPGEQVSYDAQGRCRGDPLREPSDRGLERVAYWQPARTHEVGKDANGEVQDRVSIWRRR> Aspergillus_nidulans_XP_663698MSSPSQQPIKGRLISHFENRPTPSHPKAWSDLWDSGKSSLWDRGMPSPALIDLLESYQDTLLHPFEIDIEDEEDSSDAGKTRKRKRALVPGCGRGYDVITFALHGFDACGLEVSTTAVSEARAFAKKELCSPQSGNFGRRFDRERARHIGVGKAQFLQGDFFTDTWIENESTGLDQGRTENGKFDLVYDYTFLCALHPAQRTRWAERMADLLRPGGLLVCLEFPMYKDPALPGPPWGVNGIHWELLAGGDTGQGKFTRKAYVQPERTFEVGRGTDMISVYERK> Gloeobacter_violaceus_NP_923764MPSEESSGVDQPAFWEYRYRGGQDRWDLGQPAPTFVHLLSGSEAPPLGTVAVPGCGRGHDALLFAARGYKVCGFDFAADAIADATRLALRAGAAATFLQQDLFNLPRPFAGLFDLVVEHTCFCAIDPVRREEYVEIVHWLLKPGGELVAIFFAHPRPGGPPYRTDAGEIERLFSPRFKITALLPAPMSVPSRRGEELFGRFVRA> Anaeromyxobacter_dehalogenans_YP_466408MGTSYRLAYLIGFTPWEDQPLPPELSALVEGLRARPPGRALDLGCGRGAHAVYLASHGWKVTGVDLVPAALAKARQRATDAGVDVQFLDGDVTRLDTLGLSPGYDLLLDAGCFHGLSDPERAAYARGVTALRAPRAAMLLFAFKPGWRGPAPRGASAEDLTSAFGPSWRLVRSERARESRLPLPLRNADPRWHLLEAA> Lentisphaera_araneosa_ZP_01876851MRTKGNEKAESWDKIYREGNPGWDIKKPAPPFEDLFKQNPSWLKAGSLISFGCGGGHDANFFAQNDFNVTAVDFASEAVKLARSNYPQLNVIQKNILELSPEYDEQFDYVLEHTCFCAVPLDHRRAYMESAHAILKAGAYLFGLFYRFDPPDQDGPPYSLSLEDLEDAYSGLFTLEENAIPKRSHGRRTQRERFIVLKKI> Psychrobacter_arcticus_YP_265081MGNVNQAEFWQQRYEQDSIGWDMGQVSPPLKVYIDQLPEAAKEQAVLVPGAGNAYEVGYLYEQGFTNITLVDFAPAPIKDFAERYPDFPADKLICADFFDLLPKQHQFDWVLEQTFFCAINPARRDEYVQQMARLLKPKGQLVGLLFDKDFGRNEPPFGGTKEEYQQRFSTHFDTEIMEQSYNSHPARQGSELFIKMRVKD> Mycobacterium_vanbaalenii_YP_952715MDLTPRLSRFDEFYKNQTPPWVIGEPQQAIVELEQAGLIGGRVLDVGCGTGEHTILLARAGYDVLGIDGAPTAVEQARRNAEAQGVDARFELADALHLGPDPTYDTIVDSALFHIFDDADRATYVRSLHAATRPGSVVHLLALSDSGRGFGPEVSEHTIRAAFGAGWEVEALTETTYRGVVIDAHTEALNLPAGTVVDEPAWSARIRRL> Aspergillus_niger_2_XP_001390073MSEAPNPPVQGRLISHFADRRAEDQGSGWSALWDSNESVLWDRGSPSIALVDVVEQQQDVFFPYTRDGRRKKALVPGCGRGYDPVMLALHGFDVYGLDISATGVSEATKYATSEMQSPQDVKFIAGDFFSSEWESQALQDGDKFDLIYDYTFLCALHPDLRRKWAERMSQLLHPGGLLVCLEFPMYKDTSLPGPPWGLNGVHWDLLARGGDGITNITKEEEDEDSGIQLSGQFRRAQYFRPIRSYPSGKGTDMLSIYVRR> Methanosarcina_acetivorans_NP_617055MFWDEVYKGTPPWDIDHPQPAFQALIESGEIRPGRALDIGCGRGENAIMLAKNGCDVTGIDLAKDAISDAKAKAIERHVKVNFIVGNVLEMDQLFTEDEFDIVIDSGLFHVITDEERLLFTRHVHKVLKEGGKYFMLCFSDKEPGEYELPRRASKAEIESTFSPLFNIIYIKDVIFDSLLNPGRRQAYLLSATKS

S14

> Rhodococcus_sp._YP_709003MVDAPRFPYPGSPPVHGPDDLYVTPPPWDIGRAQPVFVALAEGGAIRGRVLDCGCGTGEHVLLAAGLGLDATGVDLAATALRIAEQKARDRGLTARFLHHDARRLAELGERFDTVLDCGLFHIFDPDDRAAYVDSLRDVLVPGGRYLMLGFSDQQPGDWGPHRLTRDEITTAFDDGWTIDSLESATLEVTLDPAGMRAWQLAATRTWPHPIERECSAPC> Flavobacterium_psychrophilum_YP_001296326MKKIDQKYWQNRYQTNDIAWDTGKITTPIKAYIDQIEDQSIKILIPGCGNGYEYEYLIKKGFYNSFVADYAQTPIDNLKKRIPNCNANQLLISDFFELEGSYDLIIEQTFFCALNPELRVKYAQKMLSLLSPKGKIIGLLFQFPLTEAGPPFGGSKEEYLKLFSTNFNIKTIETAYNSIKPREGNELFFIFTKK> Synechococcus_sp._2_ZP_01080043MQLDGASSAPTLTARDWDARYRQGTDRWELGMAAPPLQAFLEQHPLAPKPTGTVLVPGCGRGHEAALLARLGFDVVGLDFSVEAIREARRLQGEHENLRWLQADLFNGAALDRAGLGAHSLSGVVEHTCFCAIDPSQRDHYRSTVDRLLEPGGWLLGVFFCHDRPGGPPYGSDAEQLAASWSQIGFTGVIWEPAQGSVAQRSDEWLGLWRKPSQADNEAIPAGSR> Alkaliphilus_metalliredigens_YP_001319591MNDKLDQEVILNQEDLLNMLDSLLEKWDEEWWNEFYSDKGKPIPFFVNAPDENLVTYFDKYFDDIGRALDVGCGNGRNSRFIASRGYDVEGLDFSKKSIEWAKEESKKTGDIALYVNDSFFNINRELSSYDLIYDSGCLHHIKPHRRSQYLEKVHRLLKPGGYFGLVCFNLKGGANLSDHDVYKKSSMAGGLGYSDIKLKKILGTYFEIVEFREMRECADNALYGKDICWSILMRRLAK> Halorhodospira_halophila_YP_001002952MSGDPDPRRAPWEARWREGRTGWDRGGVSPTLEAWLSAGVIPGRRVLVPGAGRGYEVEALARRGYKVTAVDIAAEACQQLRDGLDAAGVEARVVQADLLAWQPDTPFDAVYEQTCLCALDPADWPAYEQRLYGWLRPGGVLLALFMQTGASGGPPFHCALPEMATLFDSERWQWPAEPPRQWPHPSGRWEEAVRLLRR> Mycobacterium_smegmatis_YP_886428MDTTPTRELFDEAYESRTAPWVIGEPQPAVVELERAGLIRSRVLDVGCGAGEHTILLTRLGYDVLGIDFSPQAIEMARENARGRGVDARFAVGDAMALGDLGDGAYDTILDSALFHIFDDADRQTYVASLHAGCRPGGTVHILALSDAGRGFGPEVSEEQIRKAFGDGWDLEALETTTYRGVVGPVHAEAIGLPVGTQVDEPAWLARARRL> Plesiocystis_pacifica_ZP_01912520MRVIVPGAGVGHDALAWAQAGHEVVALDFAPAAVARLRERAAEAGLTIEAHVADVTNPGPALNDGLGGRFDLVWEQTCLCAITPELRGAYLAQARSWLTPDGSMLALLWNTGNEGGPPYDMPPELVERLMTGLFVIDKFAPVTGSNPNRREHLYWLRPEPT> Arabidopsis_thaliana_1_ NP_181920MAEEQQNSSYSIGGNILPTPEEAATFQPQVVAEGGWDKCWEDGVTPWDQGRATPLILHLLDSSALPLGRTLVPGCGGGHDVVAMASPERFVVGLDISDKALNKANETYGSSPKAEYFSFVKEDVFTWRPNELFDLIFDYVFFCAIEPEMRPAWGKSMHELLKPDGELITLMYPMTDHEGGAPYKVALSSYEDVLVPVGFKAVSVEENPDSIPTRKGKEKLARWKKIN> Burkholderia_mallei_YP_102027MKDRLMSQGDGVTNEANQPEAAGQAAGDAQPASPAGPAHIANPANPANPPALPSFSPPAAASSSASSAAPFSSRDPGDASFWDERFEQGVTPWDSARVPDAFAARHARVPVLIPGCGSAYEARWLARAGWPVRAIDFSAQAVAAARRELGEDAGLVEQADFFTYAPPFVPQWIYERAFLCAIPRSRRADYARRMAELLPPGGFLAGFFFIGATPKGPPFGIERAELDALLCPHFALVEDEPVADSLPVFAGRERWLAWRRS> Saccharopolyspora_erythraea_YP_001106907MDDELAESQRAHWQDTYSAHPGMYGEEPSAPAVHAAGVFRAAGARDVLELGAGHGRDALHFAREGFTVQALDFSSSGLQQLRDAARAQQVEQRVTTAVHDVRHPLPSADASVDAVFAHMLLCMALSTEEIHALVGEIHRVLRPGGVLVYTVRHTGDAHHGTGVAHGDDIFEHDGFAVHFFPRGLVDSLADGWTLDEVHAFEEGDLPRRLWRVTQTLPR> Vibrio_sp_ZP_01477195MKQAPTINQQFWDNLFTQGTMPWDAKTTPQELKAYLENALHSGQSVFIPGCGAAYELSSFIQYGHDVIAMDYSEQAVKMAQSTLGKHKDKVVLGDVFNADSTHSFDVIYERAFLAALPRDQWPEYFAMVDKLLPRGGLLIGYFVIDDDYHSRFPPFCLRSGELEGYLEPVFKLVESSVVANSVEVFKGRERWMVWQKSCRI> Vitis_vinifera_1_CAN68137MASPDNTKPKARSSESVTGQRRGRRPSDRHWPCVGEESGSFYNTIADGERQYQHRIELRASKNKPSSWEEKWQQGLTPWDLGKATPIIEHLHQAGALPNGRTLIPGCGRGYDVVAIACPERFVVGLDISDSAIKKAKESSSSSWNASHFIFLKADFFTWNPTELFDLIIDYTFFCAIEPDMRPAWASRMQQLLKPDGELLTLMFPISDHTGGPPYKVSIADYEKVLHPMRFKAVSIVDNEMAIGSRKKKYPLKPDLSLFGFVDRPKRAYEARSEEFRISDWVCGWMGLCVPSGRISGGVCGLLSGRSLTWAKNLGVSTTQLRMSNNGSSIESNPKVQKLNQIIGSDSAGGWEKSWQQGHTPWDLGKPTPIIQHLHQTGTLPSGKTLVPGCGCGYDVVTIACPERFVVGLDISDSAIKKAK

S15

EISDHAGGPPYKVSVADYEEVLHPMGFKAVSIVDNKMAIGPRKGREKLGRWKRTPSKSLL> Arabidopsis_thaliana_2_NP_001078053MAEEQQNSDQSNGGNVIPTPEEVATFLHKTVEEGGWEKCWEEEITPWDQGRATPLIVHLVDTSSLPLGRALVPGCGGGHDVVAMASPERFVVGLDISESALAKANETYGSSPKAEYFSFVKEDVFTWRPTELFDLIFDYVFFCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHVGGPPYKVDVSTFEEVLVPIGFKAVSVEENPHAIPTRQREAGKVEEDQLIPKKEILLFGKSVICVIYKE

9. References

(1) Amann, E.; Ochs, B.; Abel, K. J. Gene 1988, 69, 301-15.(2) Gari, E.; Piedrafita, L.; Aldea, M.; Herrero, E. Yeast 1997, 13, 837-48.(3) Gietz, R. D.; Woods, R. A. Methods Enzymol 2002, 350, 87-96.(4) Symington, L. S. Microbiol Mol Biol Rev 2002, 66, 630-70, table of contents.(5) Valls, L. A.; Winther, J. R.; Stevens, T. H. J Cell Biol 1990, 111, 361-8.(6) Chan, S. Y.; Appling, D. R. J Biol Chem 2003, 278, 43051-9.(7) Gelling, C. L.; Piper, M. D.; Hong, S. P.; Kornfeld, G. D.; Dawes, I. W. J Biol Chem 2004, 279, 7072-81.(8) Hong, S. P.; Piper, M. D.; Sinclair, D. A.; Dawes, I. W. J Biol Chem 1999, 274, 10523-32.(9) Piper, M. D.; Hong, S. P.; Ball, G. E.; Dawes, I. W. J Biol Chem 2000, 275, 30987-95.(10) Piper, M. D.; Hong, S. P.; Eissing, T.; Sealey, P.; Dawes, I. W. FEMS Yeast Res 2002, 2, 59-71.(11) Wuosmaa, A. M.; Hager, L. P. Science 1990, 249, 160-2.(12) Bagnara, C.; Gaudin, C.; Belaich, J. P. Biochem Biophys Res Commun 1986, 140, 219-29.(13) Bagnara, C.; Gaudin, C.; Belaich, J. P. Applied Microbiology and Biotechnology 1987, 26, 170-176.(14) Bagnara, C.; Toci, R.; Gaudin, C.; Belaich, J. P. International Journal of Systematic Bacteriology 1985, 35, 502-507.(15) Wach, A.; Brachat, A.; Alberti-Segui, C.; Rebischung, C.; Philippsen, P. Yeast 1997, 13, 1065-75.

S16

Complete refs 10, 50, and 80:

(10) Rondon, M. R.; August, P. R.; Bettermann, A. D.; Brady, S. F.; Grossman, T. H.; Liles, M. R.; Loiacono, K. A.; Lynch, B. A.; MacNeil, I. A.; Minor, C.; Tiong, C. L.; Gilman, M.; Osburne, M. S.; Clardy, J.; Handelsman, J.; Goodman, R. M. Appl. Environ. Microbiol. 2000, 66, 2541-2547. (50) Venter, J. C.; Remington, K.; Heidelberg, J. F.; Halpern, A. L.; Rusch, D.; Eisen, J. A.; Wu, D.; Paulsen, I.; Nelson, K. E.; Nelson, W.; Fouts, D. E.; Levy, S.; Knap, A. H.; Lomas, M. W.; Nealson, K.; White, O.; Peterson, J.; Hoffman, J.; Parsons, R.; Baden-Tillson, H.; Pfannkoch, C.; Rogers, Y. H.; Smith, H. O. Science 2004, 304, 66-74. (80) Warnecke, F.; Luginbuhl, P.; Ivanova, N.; Ghassemian, M.; Richardson, T. H.; Stege, J. T.; Cayouette, M.; McHardy, A. C.; Djordjevic, G.; Aboushadi, N.; Sorek, R.; Tringe, S. G.; Podar, M.; Martin, H. G.; Kunin, V.; Dalevi, D.; Madejska, J.; Kirton, E.; Platt, D.; Szeto, E.; Salamov, A.; Barry, K.; Mikhailova, N.; Kyrpides, N. C.; Matson, E. G.; Ottesen, E. A.; Zhang, X.; Hernandez, M.; Murillo, C.; Acosta, L. G.; Rigoutsos, I.; Tamayo, G.; Green, B. D.; Chang, C.; Rubin, E. M.; Mathur, E. J.; Robertson, D. E.; Hugenholtz, P.; Leadbetter, J. R. Nature 2007, 450, 560-565.