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 plasmids
Cloning was performed using standard procedures in E. coli TOP10 cells (Invitrogen). Primers are listed
below. 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 transformed
into DH10B strain for methyl halide production assays. For yeast expression, the B. maritima MHT
coding region was cloned into vector pCM190.
Cloning was performed using standard procedures in E. coli TOP10 cells (Invitrogen). The B. maritima
MCT coding region was synthesized by DNA 2.0 (Menlo Park, CA) and amplified using specified
primers with PfuUltra II (Stratagene) according to manufacturers instructions. PCR products were
purified using a Zymo Gel Extraction kit according to manufacturers instructions. Purified expression
vector (pCM1902) and coding region insert were digested with restriction enzymes NotI and PstI
overnight at 37 degrees and gel purified on a 1 % agarose gel and extracted using a Promega Wizard SV
Gel kit according to manufacturers instructions. Vector and insert were quantitated and ligated (10 fmol
vector to 30 fmol insert) with T4 ligase (Invitrogen) for 15 minutes at room temperature and transformed
into chemically competent E. coli TOP10 cells (Invitrogen). Transformants were screened and plasmids
were sequenced using specified primers to confirm cloning.
Constructs were transformed into the S. cerevisiae W303a background using standard lithium acetate
technique and plated on selective media3. Briefly, competent W303a cells were prepared by sequential
washes with water and 100mM lithium acetate in Tris-EDTA buffer. 1 g of plasmid was incubated for
30 minutes at 30 degrees with 50 L of competent cells along with 300 L of PEG 4000 and 5 g of
boiled salmon sperm DNA as a carrier. Cells were then heat-shocked at 42 degrees for 20 minutes. Cells
were 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 streaking
on uracil dropout plates.
Media and growth conditions
Bacteria carrying MHT expression vectors were inoculated from freshly streaked plates and grown
overnight. Cells were diluted 100-fold into media containing 1 mM IPTG and 100 mM appropriate
sodium 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 complete
uracil dropout media and grown overnight at 30 degrees. Cultures were next inoculated into 100mL fresh
synthetic complete uracil dropout media and grown for 24 hours. Cells were spun down and concentrated
to high cell density (OD 50) in fresh YP media with 2% glucose and 100 mM sodium iodide salt. 10 mL
of 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 spectrometry
The GC-MS system consisted of a model 6850 Series II Network GC system (Agilent) and model 5973
Network mass selective system (Agilent). Oven temperature was programmed from 50 degrees (1 min) to
70 degrees (10 degrees / min). 100 L of culture headspace was withdrawn through the rubber stopper
with a syringe and manually injected into the GC-MS. Samples were confirmed as methyl iodide by
comparison with commercially obtained methyl iodide (Sigma), which had a retention time of 1.50
minutes 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 prepared
at 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 culture
tubes and sealed with rubber stoppers. Standards were incubated at 30 degrees for 1 hour and methyl
iodide in the headspace was measured as above. A standard curve was fit to the data to quantitate methyl
iodide concentration with headspace counts.

S1

1.5

standard

mw 142 counts, x 104

mw 142 counts, x 106

1.5

1.0

0.5

0.0
1.0

1.5

experiment
1.0

0.5

0.0
1.0

2.0

time (min)

1.5

2.0

time (min)

Supplemental Figure 1. GC-MS chromatograms of methyl iodide production

Chromatograms at molecular weight 142 for methyl iodide standard in YPD (left) and methyl iodide
producing S. cerevisiae (right). For the standard, commercially available methyl iodide (Sigma) was
aliquoted into 10mL of YPD media in a 14mL culture tube and sealed with a rubber stopper. For the
experimental sample, 9mL of a MHT-expressing culture (prepared as above) was added to 1mL of 1M
NaI in a 14mL culture tube and sealed with a rubber stopper. Both samples were incubated at 30 degrees
with 250 rpm shaking for 60 minutes. 100L of headspace was withdrawn with a syringe and manually
injected into the GC-MS as described above.

Methyl iodide toxicity assay

Individual colonies were inoculated in YP media with 2% glucose and grown overnight. Cultures were
diluted to an OD600 of 0.05 and methyl iodide was added to the specified amount. Cultures were grown at
30 degrees with 250 rpm shaking for 24 hours. OD600 was measured by spectometry with YP media used
as a blank. Each data point was performed in triplicate. The RAD50 mutant4 was obtained from the
Saccharomyces 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, assaying
methyl iodide at 1 hour, and venting the culture to simulate product extraction. Cultures were then resealed 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 by
summing the production each hour. An example of the raw data from methyl iodide measurements is
shown in Supplemental Figure 2, below.

5.0

4.0

CH3I counts, x 10

S2
CH3I counts, x 107

S2

3.0

2.0

1.0

0.0
0.0

2.0

4.0

6.0

4.0

3.0

2.0

1.0

0.0
0.0

8.0

2.0

time (hr)

4.0

6.0

8.0

time (hr)

Supplemental Figure 2. Cumulative methyl iodide production in shake flask.

Methyl iodide headspace counts from MHT-expressing culture, vented each hour to simulate product
extraction. Cells were grown and induced as above, sealed with a rubber stopper, grown for 1 hour, and
measured 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). This
procedure 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 carboxypeptidase
Y5 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 resulted
in a 50% increase in production rate. We next expressed the cytosolic and vacuolar targeted enzymes in a
VPS33 background, which is unable to form functional vacuoles6. The difference in production rate was
abolished in the VPS33 strain, indicating that MCT targeting to fully formed vacuoles is necessary for
enhancing the rate of methyl iodide formation.

4. Methyl halide production from cellulosic feedstocks

Growth and assay conditions
Actinotalea fermentans was obtained from ATCC (43279). A. fermentans and S. cerevisiae cells were
inoculated 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 a
commercially available blender with a 1 HP, 1000W motor. Bagasse was aliquoted into the appropriate
dry weight, then washed 3 times with hot water to remove soil and residual sugar. Cultures were
incubated at 30 degrees with 250 rpm agitation for 36 hours. 9mL aliquots of cultures were placed in
14mL tubes with 1mL of 1M sodium chloride and sealed with a rubber stopper. Headspace samples were
assayed for GC-MS production as above. A. fermentans and S. cerevisiae were quantitated as described
below.
Yeast and bacteria quantitation
S. cerevisiae and A. fermentans were quantitated from cultures grown on cellulosic stocks by plating on
selective 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 were
incubated at 30 degrees for either 48 hours (for YPD) or 16 hours (for BH). Colonies were counted by
hand and counts from at least 4 plates were averaged. In the switchgrass and corn stover grown cultures
some unidentified background cultures were apparent but showed distinguishable morphology from A.
fermentans.

S5
S3

Supplemental Figure 3.
5. Preparation of cellulosic feedstocks
Cellulosic stocks were obtained from the sources cited in the text. We prepared the feedstocks by
blending for ~1 minute. a, poplar before blending (left) and after blending. B, corn stover before
blending (left) and after blending (right). c, bagasse and d, switchgrass. We found that blending
did not further decrease the size of the particles in bagasse and switchgrass, feedstocks are shown
as they were supplied. e, example of culture flask with 20 g/L switchgrass.

5. Metabolic pathway to methyl iodide

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

S4

Glycolysis
-ATP

-ATP

glucose

SAM Biosynthesis

F6P

G6P

Folate Metabolism

F1,6B

H2O

MET
-ATP

5-methyl H4folate

SAM

Glyceraldehyde

CO2

1,3bisphospho
glycerate

3PG

Serine Biosynthesis

Serine

N5,N10methylene-THF

Glycine Cleavage

+ATP

O-phosphoserine

3-phosphohydroxy
pyruvate

Glycine

-ATP

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 are
tracked throughout the pathway and are indicated with a red circle.

6. Combustion of culture headspace

As a further demonstration of methyl iodide productivity by the engineered yeast strains, we asked
whether the headspace of the culture could be ignited. Methyl halides are volatile and reactive, with
combustion yielding carbon monoxide, carbon dioxide, and hydrogen halide. To test this, we grew a
culture of B.maritima MHT-expressing yeast overnight in 100 mM sodium chloride to accumulate methyl
iodide. We then decanted the culture into a side-arm flask (Supplemental Movie 1 ). The mouth of the
flask was sealed with a rubber stopper with tubing running into the flask. The tubing was used to force
pressurized air (from a house line) into the flask. A Bunsen burner was used to heat the outlet air from
the flask. Combustion was performed in a fume hood and appropriate personal protective equipment was
used. 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 of
combustion, suggesting we exhausted the methyl iodide in the flask. These results suggest the relative
ease of extracting methyl halides from the yeast culture.

S6

S5

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 with
pressurized air. The off-gas was combusted with a flame from a Bunsen burner. Figure is taken from a
frame 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
pTRC99a1
pCM1902
pFA6a-GFP(S65T)-KAN15
Primers
Batis fwd
GG GCGGCCGC GGA ATT GTG AGC GGA TAA CAA TTG AAT TCA TTA AAG

S7

Batis rev
CC CTGCAG CAGCCAAGCT TGCATGCCT
pCM190 sequencing fwd
CACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG
Vac.Batis fwd
GG GCGGCCGC ATGAAGGCCATCTCTTTACAGAGGCCATTGGGTTTAGACAAGGACGTCTTA
TCT ACT GTT GCG AAT ATT GCT CC
VPS33.del fwd
ATGAATAGATTTTGGAATACTAAGAAATTTGGCAGATCCGCTAGGGATAA
VPS33.del rev
TTAAGATATAGAGTTCATGATCCTTGTGCCGAATTCGAGCTCGTTTAAAC

8. MHT sequences
MHT sequences are rank ordered according to methyl iodide activity. The organism name (as used in the
main text) and accession number are given.
> Batis_maritima_ AAD26120
MSTVANIAPVFTGDCKTIPTPEECATFLYKVVNSGGWEKCWVEEVIPWDLGVPTPLVLHLVKNNALPNG
KGLVPGCGGGYDVVAMANPERFMVGLDISENALKKARETFSTMPNSSCFSFVKEDVFTWRPEQPFDFIF
DYVFFCAIDPKMRPAWGKAMYELLKPDGELITLMYPITNHEGGPPFSVSESEYEKVLVPLGFKQLSLED
YSDLAVEPRKGKEKLARWKKMNN
> Oryza_sativa_2_ EAY92545
MASAIVDVAGGGRQQALDGSNPAVARLRQLIGGGQESSDGWSRCWEEGVTPWDLGQPTPAVVELVHSGTL
PAGDATTVLVPGCGAGYDVVALSGPGRFVVGLDICDTAIQKAKQLSAAAAAAADGGDGSSSFFAFVADDF
FTWEPPEPFHLIFDYTFFCALHPSMRPAWAKRMADLLRPDGELITLMYLAEGQEAGPPFNTTVLDYKEVL
NPLGLVITSIEDNEVAVEPRKGMEKIARWKRMTKSD
> Vitis_vinifera_2_CAO46361
MANDSTSIESNSELQKISQVIGSGFNGSWEEKWQQGLTPWDLGKATPIIEHLHQAGALPNGRTLIPGCGR
GYDVVAIACPERFVVGLDISDSAIKKAKESSSSSWNASHFIFLKADFFTWNPTELFDLIIDYTFFCAIEP
DMRPAWASRMQQLLKPDGELLTLMFPISDHTGGPPYKVSIADYEKVLHPMRFKAVSIVDNEMAIGSRKGR
EKLGRWKRTDEPLL
> Burkholderia_xenovorans_YP_557005
MSDPTQPAVPDFETRDPNSPAFWDERFERRFTPWDQAGVPAAFQSFAARHSGAAVLIPGCGSAYEAVWLA
GQGNPVRAIDFSPAAVAAAHEQLGAQHAQLVEQADFFTYEPPFTPAWIYERAFLCALPLARRADYAHRMA
DLLPGGALLAGFFFLGATPKGPPFGIERAELDALLTPYFDLIEDEAVHDSIAVFAGRERWLTWRRRA
> Brassica_rapa_chinensis_ABL86248
MAEVQQNSAHINGENIIPPEDVAKFLPKTVEEGGWEKCWEDGVTPWDQGRATPLVVHLVESSSLPLGRAL
VPGCGGGHDVVAMASPERYVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVV
FCAIEPETRPAWAKAMYELLKPDGELITLMYPITDHDGGPPYKVAFSTYEDVLVPVGFKAVSIEENPYSI
ATRKGKEKLARWKKIN
> Burkholderia_pseudomallei_ YP_332262
MKDRLMSQGDGVTNEANQPEAAGQATGDAQPASPAGPAHIANPANPANPANPPALPSLSPPAAAPSSASS
AAHFSSRDPGDASFWDERFEQGVTPWDSARVPDAFAAFAARHARVPVLIPGCGSAYEARWLARAGWPVRA
IDFSAQAVAAARRELGEDAGLVEQADFFTYAPPFVPQWIYERAFLCAIPRSRRADYARRMAELLPPGGFL
AGFFFIGATPKGPPFGIERAELDALLCPHFALVEDEPVADSLPVFAGRERWLAWRRS
> Burkholderia_thailandensis_YP_441114
MTSEANKGDAAVQAAGDAQPASPASPPSADVQPARAALAPSSVPPAPSAANFASRDPGDASFWDERFERG

S8

VTPWDSARVPDAFAAFAARHPRCPVLIPGCGSAYEARWLARAGWPVRAIDFSAQAVAAARRESGADAALV
EQADFFAYVPPFVPQWIYERAFLCAIPTSRRADYARRVAELLPAGGFLAGFFFIGATPKGPPFGIERAEL
DALLSPNFELVEDEPVADSLPVFAGRERWLAWRRS
> marine_gamma_proteobacterium_HTCC2080_ZP_01626954
MEKFGASAMEPVLDWEARYQESSVPWERTGLNPAFVAWQSWLRDHQGGTVVVPGCGRSPELQAFADMGFN
VIGVDLSPSAAQFQETVLAAKGLDGKLVVSNLFDWSPDTPVDFVYEQTCLCALKPDHWRAYENLLTRWLR
PGGTLLALFMQTGESGGPPFHCGKAAMEQLFSEQRWIWDETSVRSEHPLGVHELGFRLTLR
> Ralstonia_picketti_YP_001897927
MAQPPVFQSRDAADPAFWDERFTREHTPWDAAGVPAAFRQFCEAQPAPLSTLIPGCGNAYEAGWLAERGW
PVTAIDFAPSAVASARAVLGPHADVVQLADFFRFSPPRPVHWIYERAFLCAMPRRLWPDYAAQVAKLLPP
RGLLAGFFAVVEGREAMPKGPPFETTQPELDALLSPAFERISDMPIAETDSIPVFAGRERWQVWRRRAD
> Burkholderia_phytofirmans_YP_001894302
MSDPTQPSAPEFESRDPNSPEFWDERFERGFMPWDQAGVPSAFESFAARHAGAAVLIPGCGSAYEAVWLA
GHGYPVRAIDFSPAAVAAAHEQLGAQHADLVEQADFFTYELPFTPAWIYERAFLCALPLARRADYARRMA
DLLPGGALLAGFFFIGATPKGPPFGIERAELDGLLKPYFELIEDEPVHDSIAVFAGRERWLTWRRRV
> Burkholderia_vietnamensis_YP_001120660
MSNPTQPPPPSAADFATRDPANASFWDERFARGVTPWEFGGVPDGFRAFAQRRAPCTVLIPGCGSAQEAG
WLAQAGWPVRAIDFAEQAVVAAKATLGAHADVVEQADFFAYQPPFVVQWVYERAFLCALPPSLRAGYAAR
MAELLPAGGLLAGYFFVMKKPKGPPFGIERAELDALLAPSFELIEDLPVTDSLAVFDGHERWLTWRRR
> Aspergillus_clavatus_XP_001272206
MSTPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDRLPWDRGFPNPALEDTLIQKRAIIGGPLGQDAQ
GKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVDVCQEEQAKNGDQYPVRDAEIGQGKITFVQG
DFFEDTWLEKLNLTRNCFDVIYDYTFFCALNPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGP
PWGSASEAYRAHLSHPGEEIPYDASRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIW
QRPPQSSL
> Phaeosphaeria_nodorum_XP_001792029
MANPNQDRLRSHFAALDPSTHASGWDSLWAEGTFIPWDRGYANPALIDLLANPSSPPTSSDANPTPGAPK
PNTIDGQGVQLPAPLEGGVRRKALVPGCGKGYDVALLASWGYDTWGLEVSRHAADAAKEYLKDAGEGALE
GEYKIKDAKIGKGREECVVADFFDDAWLKDVGAGEFDVIYDNTFLCALPPLLRPKWAARMAQLLARDGVL
ICLEFPTHKPASSGGPPWSLPPTVHQELLKRPGEDISYDEGGVVVATDRAESENALVRVAHWTPKRTHNI
AVINGVVRDCVSVWRHKKQS
> Dechloromonas_aromatica_YP_286874
MSETIKPPEQRPEHPDFWCKRFGEGVTPWDAGKVPMAFVDFVGAQTTPLNSLIPGCGSAWEAAHLAELGW
PVTALDFSPLAIEKAREVLGDSPVKLVCADFFTFAPRQPLDLIYERAFLCALPRKLWADWGKQVAELLPS
GARLAGFFFLCDQPKGPPFGILPAQLDELLRPNFELIEDQPVGDSVPVFAGRERWQVWRRR
> Kortia_algicida_ZP_02160755
MNSDATKEYWSQRYKDNSTGWDIGSPSTPLKTYIDQLKDRNLKILIPGAGNAYEAEYLLQQGFTNIYILD
ISEIPLQEFKQRNPEFPSDRLLCDDFFTHKNTYDLIIEQTFFCSFPPLPETRAQYAKHMADLLNPNGKLV
GLWFDFPLTDDLEKRPFGGSKEEYLEYFKPYFDVKTFEKAYNSIAPRAGNELFGIFIKS
> Methylibium_petroleiphilum_YP_001022598
MSGPDLNFWQQRFDTGQLPWDRGAPSPQLAAWLGDGSLAPGRIAVPGCGSGHEVVALARGGFSVTAIDYA
PGAVRLTQGRLAAAGLAAEVVQADVLTWQPTAPLDAVYEQTCLCALHPDHWVAYAARLHAWLRPGGTLAL
LAMQALREGAGQGLIEGPPYHVDVNALRALLPGDRWDWPRPPYARVPHPSSTWAELAIVLTRR
> Burkholderia_dolosa_ZP_00987534
MTGRSFAMSDPKQPGTPTAADFATRDPGDASFWDERFARGVTPWEFGGVPDGFRAFAQRLERCAVLIPGC
GSAQEAGWLADAGWPVRAIDFAAQAVATAKAQLGAHADVVELADFFTYRPPFDVRWIYERAFLCALPPAR
RADYAAQMAALLPAGGLLAGYFFVTAKPKGPPFGIERAELDALLAPQFDLIDDWPVTDSLPVFEGHERWL
TWRRR
> Ralstonia_solanacearum_NP_518583
MAQPPVFTTRDAAAPAFWDERFSRDHMPWDAHGVPPAFRQFCEAQPAPLSTLIPGCGSAYEAGWLAERGW
PVAAIDFAPSAVASAQAVLGPHAGVVELADFFRFTPRQPVQWIYERAFLCAMPRRLWADYATQVARLLPP
GGLLAGFFVVVDGRAAAPSGPPFEITAQEQEALLSPAFERIADALVPENESIPVFAGRERWQVWRRRAD
> Ustilago_maydis_XP_762636
MTSSLSKDDQIQNLRRLFADSGVPNDPKAWDQAWIDSTTPWDANRPQPALVELLEGAHDADAKVPDVDGN
LIPVSQAIPKGDGTAVVPGCGRGYDARVFAERGLTSYGVDISSNAVAAANKWLGDQDLPTELDDKVNFAE
ADFFTLGTSKSLVLELSKPGQATLAYDYTFLCAIPPSLRTTWAETYTRLLAKHGVLIALVFPIHGDRPGG
PPFSISPQLVRELLGSQKNADGSAAWTELVELKPKGPETRPDVERMMVWRRS
> Hahella_chejuensis_YP_432621

S9

MDANFWHERWAENSIAFHQCEANPLLVAHFNRLDLAKGSRVFVPLCGKTLDISWLLSQGHRVVGCELSEM
AIEQFFKELGVTPAISEIVAGKRYSAENLDIIVGDFFDLTVETLGHVDATYDRAALVALPKPMRDSYAKH
LMALTNNAPQLMLCYQYDQTQMEGPPFSISAEEVQHHYADSYALTALATVGVEGGLRELNEVSETVWLLE
SR
> Brassica_oleracea_2_AAK69761
MAEVQQNSGNSNGENIIPPEDVAKFLPKTVDEGGWEKCWEDGVTPWDQGRATPLVVHLVESSSLPLGRGL
VPGCGGGHDVVAMASPERYVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVV
FCAIEPETRPAWAKAMYELLKPDGELITLMYPITDHDGGPPYKVAVSTYEDVLVPVGFKAVSIEENPYSI
ATRKGKEKLARWKKIN
> Burkholderia_ambifaria_YP_774669
MSEPKQPSTPGAADFATRDPGDASFWDERFARGVTPWEFGGVPEGFRAFAQRLGPCAVLIPGCGSAQEAG
WLAQAGWPVRAIDFAAQAVAAAKAQLGAHADVVEQADFFMYRPPFDVQWVYERAFLCALPPSLRAGYAAR
MAELLPAGALLAGYFFVTKKPKGPPFGIERAELDALLAPHFELIDDLPVTDSLAVFEGHERWLTWRRR
> Rhodoferax_ferrireducens_YP_522685
MAGPTTEFWQERFEKKETGWDRGSPSPQLLAWLASGALRPCRIAVPGCGSGWEVAELAQRGFDVVGLDYT
AAATTRTRALCDARGLKAEVLQADVLSYQPEKKFAAIYEQTCLCAIHPDHWIDYARQLHQWLEPQGSLWV
LFMQMIRPAATEEGLIQGPPYHCDINAMRALFPQKDWVWPKPPYARVSHPNLSHELALQLVRR
> Coccidioides_immitis_XP_001248254
MANEILRSAPNLSDRFKNLDGRNQGEVWDDLWKESRTPWDRGSHNPALEDALVEKRGFFGAPVFEDEPLR
RKKALVPGCGRGVDVFLLASFGYDAYGLEYSKTAVDVCLKEMEKYGEGGKVPPRDEKVGSGKVMFLEGDF
FKDDWVKEAGVEDGAFDLIYDYTFFCALNPALRPQWALRHRQLLAPSPRGNLICLEFPTTKDPAALGPPF
ASTPAMYMEHLSHPGEDIPYDDKGHVKSNPLQQPSDKGLERVAHWQPKRTHTVGMDDKGNVLDWVSIWRR
RD
> Synechococcus_elongatus_YP_172090
MTNAVNQAQFWEQRYQEGSDRWDLGQAAPVWRSLLAGTNAPAPGRIAVLGCGRGHDARLFAEQGFEVVGF
DFAPSAIAAAQALAQGTTAQFLQRDIFALPQEFAGQFDTVLEHTCFCAIDPDRRAEYVEVVRQILKPKGC
LLGLFWCHDRPSGPPYGCSLTELRDRFAQGWQEEQLESVTESVEGRRGEEYLGRWRRLD
> Ralstonia_eutropha_YP_724967
MSDPAKPVPTFATRNAADPAFWDERFEQGFTPWDQGGVPEEFRQFIEGRAPCPTLVPGCGNGWEAAWLFE
RGWPVTAIDFSPQAVASARQTLGPAGVVVQQGDFFAFTPQPPCELIYERAFLCALPPAMRADYAARVAQL
LPPGGLLAGYFYLGENRGGPPFAMPAEALDALLAPAFERLEDRPTAAPLPVFQGQERWQVWRRRSG
> Oryza_sativa_japonica_2_NP_001056843
MSSSAARVGGGGGRDPSNNPAVGRLRELVQRGDAADGWEKSWEAAVTPWDLGKPTPIIEHLVKSGTLPKG
RALVPGCGTGYDVVALASPERFVVGLDISSTAVEKAKQWSSSLPNADCFTFLADDFFKWKPSEQFDLIFD
YTFFCALDPSLRLAWAETVSGLLKPHGELITLIYLISDQEGGPPFNNTVTDYQKVLEPLGFKAILMEDNE
LAIKPRKGQEKLGRWKRFVPGSSL
> Burkholderia_multivorans_YP_001578763
MSDPKHAAAPAAASFETRDPGDASFWDERFARGMTPWEFGGVPAGFRAFASARPPCAVLIPGCGSAREAG
WLAQAGWPVRAIDFSAQAVAAAKAQLGAHADVVEQADFFAYRPPFDVQWIYERAFLCALPPARRADYAAT
MAALLPAQGLLAGYFFVADKQKGPPFGITRGELDALLGAHFELIDDAPVSDSLPVFEGHERWLAWRRR
> Cellulophaga_sp._ZP_01049440
MELTSTYWNNRYAEGSTGWDLKEVSPPIKAYLDQLENKELKILIPGGGYSYEAQYCWEQGFKNVYVVDFS
QLALENLKQRVPDFPSLQLIQEDFFTYDGQFDVIIEQTFFCALQPDLRPAYVAHMHTLLKAKGKLVGLLF
NFPLTEKGPPYGGSTTEYESLFSEHFDIQKMETAYNSVAARAGKELFIKMVKK
> Neosartorya_fischeri_XP_001266691
MSNDPRLLSSIPEFIARYKENYVEGWAELWNKSEGKPLPFDRGFPNPALEDTLIEKRDIIGGPIGRDAQG
NTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSDTAVQVCKEEQAKNGDKYPVRDAEIGQGKITFVQGD
FFKDTWLEKLQLPRNSFDLIYDYTFFCALDPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDTSLQGPP
WASTSEAYMAHLNHPGEEIPYDANRQCSIDPSKAPSPQGLERVAYWQPARTHEVGIVEGEVQDRVSIWRR
PN
> Tenacibaculum_sp._ZP_01053731
MIFDEQFWDNKYITNKTGWDLGQVSPPLKAYFDQLTNKDLKILIPGGGNSHEAEYLLENGFTNVYVIDIS
KLALTNLKNRVPGFPSSNLIHQNFFELNQTFDLVIEQTFFCALNPNLREEYVSKMHSVLNDNGKLVGLLF
DAKLNEDHPPFGGSKKEYTSLFRNLFTIEVLEECYNSIENRKGMELFCKFVK
> Arabidopsis_thaliana_3_NP_850403
MENAGKATSLQSSRDLFHRLMSENSSGGWEKSWEAGATPWDLGKPTPVIAHLVETGSLPNGRALVPGCGT
GYDVVAMASPDRHVVGLDISKTAVERSTKKFSTLPNAKYFSFLSEDFFTWEPAEKFDLIFDYTFFCAFEP
GVRPLWAQRMEKLLKPGGELITLMFPIDERSGGPPYEVSVSEYEKVLIPLGFEAISIVDNELAVGPRKGM

S10

EKLGRWKKSSTFHSTL
> Giberella_zeae_XP_390285
MATENPLEDRISSVPFAEQGPKWDSCWKDALTPWDRGTASIALHDLLAQRPDLVPPSQHQDHRGHPLRDA
TGAIQKKTALVPGCGRGHDVLLLSSWGYDVWGLDYSAAAKEEAIKNQKQAESEGLYMPVDGLDKGKIHWI
TGNFFAQDWSKGAGDDGKFDLIYDYTFLCALPPDARPKWAKRMTELLSHDGRLICLEFPSTKPMSANGPP
WGVSPELYEALLAAPGEEIAYNDDGTVHEDPCSKPWADALHRLSLLKPTRTHKAGMSPEGAVMDFLSVWS
R
> Legionella_pneumophila_YP_128074
MNKGQYFWNELWCEGRISFHKKEVNPDLIAYVSSLNIPAKGRVLVPLCGKSVDMLWLVRQGYHVVGIELV
EKAILQFVQEHQITVRENTIGQAKQYFTDNLNLWVTDIFALNSALIEPVDAIYDRAALVALPKKLRPAYV
DICLKWLKPGGSILLKTLQYNQEKVQGPPYSVSPEEIALSYQQCAKIKLLKSQKRIQEPNDHLFNFGISE
VNDSVWCIRKG
> Burkholderia_cenocepacia_AU_1054_YP_621990
MSDPKQPAAPSAADFATRDPGSASFWDERFARGVTPWEFGGVPDGFRVFAQRREPCAVLIPGCGSAQEAG
WLAQAGWPVRAIDFAAQAVAAAKAQLGAHADVVEQADFFQYRPPFDVQWVYERAFLCALPPGLRAGYAAR
MAELLPTGGLLAGYFFVVAKPKGPPFGIERAELDALLAPHFELLEDLPVTDSLAVFDGHERWLTWRRR
> Aspergillus_fumigatus_XP_751474
MSNDPRLVSSIPEFIARYKENYVEGWAELWDKSEGKPLPFDRGFPNPALEDTLIEKRDIIGDPIGRDAQG
NTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVKVCKEEQAKNGDKYPVRDAEIGQGKITYVQGD
FFKDTWWEKLQLPRNSFDLIYDYTFFCALDPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDTSLQGPP
WASTSEAYMAHLNHPGEEIPYDANRQCSIDPSKAPSPQGLERVAYWQPARTHEVGIVEGEVQDRVSIWRR
PN
> Microscilla_marina_ZP_01688206
MHTTLDKDFWSNRYQAQDTGWDAGSITTPIKAYVDQLEDKHLKILVPGAGNSHEAEYLHQQGFTNVTVID
IVQAPLDNLKSRSPDFPEAHLLQGDFFELVGQYDLIIEQTFFCALNPSLRESYVQKVKSLLKPEGKLVGV
LFCNVFLDRTEPPFGATEQQHQEYFLPHFIAKHFASCYNSIAPRQGAEWFICLIND
> Oceanicaulis_alexandrii_ZP_00951977
MTQASSDTPRSEDRSGFDWESRFQSDDAPWERQGVHPAAQDWVRNGEIKPGQAILTPGCGRSQEPAFLAS
RGFDVTATDIAPTAIAWQKTRFQTLGVMAEAIETDALAWRPETGFDALYEQTFLCAIHPKRRQDYEAMAH
ASLKSGGKLLALFMQKAEMGGPPYGCGLDAMRELFADTRWVWPDGEARPYPHPGLNAKAELAMVLIRR
> Burkholderia_cenocepacia_MC0-3_ZP_01560172
MSDPKQPAAPSAAEFATRDPGSASFWDERFARGVTPWEFGGVPDGFRAFAQRHEPCAVLIPGCGSAQEAG
WLAQAGWPVRAIDFAAQAVAAAKVQLGAHADVVEQADFFQYRPPFDVQWVYERAFLCALPPSLRADYAAR
MAELLPTGGLLAGYFFVVAKPKGPPFGIERAELDALLAPHFELLEDLPVTDSLAVFDGHERWLTWRRR
> Leptospirillum_sp._EAY56191
MPDKIFWNQRYLDKNTGWDLGQPAPPFVRLVEKGEFGPPGRVLIPGAGRSYEGIFLASRGYDVTCVDFAP
QAVREAREAARQAGVKLTVVEEDFFRLDPRTIGVFDYLVEHTCFCAIDPPMRQAYVDQSHALLAPGGLLI
GLFYAHGREGGPPWTTTEEEVRGLFGKKFDLLSLGLTDWSVDSRKGEELLGRLRRKNDRIE
> Mariprofundus_ferrooxydans_ZP_01453074
MTVWEERYQRGETGWDRGGVSPALTQLVDHLHLEARVLIPGCGRGHEVIELARLGFRVTAIDIAPSAIAH
LSQQLEQEDLDAELVNGDLFAYAPDHCFDAVYEQTCLCAIEPEQRADYEQRLHGWLKPEGVLYALFMQTG
IRGGPPFHCDLLMMRELFDASRWQWPEETGAVLVPHKNGRFELGHMLRRTGR
> Polaromonas_naphthalenivorans_YP_973752
MAGPTTDFWQARFDNKETGWDRGAPGPQLLAWLESGALQPCRIAVPGCGSGWEVAELARRGFEVVGIDYT
PAAVERTRALLAAQGLAAEVVQADVLAYQPHKPFEAIYEQTCLCALHPDHWVAYARQLQQWLKPQGSIWA
LFMQMVRPEATDEGLIQGPPYHCDINAMRALFPAQHWAWPRPPYAKVPHPNVGHELGLRLMLRQGR
> Synechococcus_sp._1_YP_001224608
MTNVHLPQAWDARYQHGTDGWELGKAAPPLQAFLEHHPRAPQPEGTVLVPGCGRGHEAALLARLGFEVIG
LDFSSEAIREARRLHGEHPRLRWLQADLFDADALSGAGLASGSLSGVLEHTCFCAIDPSQRAHYRSTVDR
LLRAEGWLLGLFFCHPRPGGPPFGSDPEQLAASWAQIGFYPLIWEPARGSVAGRSEEWLGFWRKPEQRSA
> Flavobacteriales_bacterium_1_ ZP_01732881
MNYWEERYKKGETGWDAGTITTPLKEYIDQLTDKNLTILIPGAGNGHEFDYLIDNGFKNVFVVDIAITPL
ENIKKRKPKYSSHLINADFFSLTTTFDLILEQTFFCALPPEMRQRYVEKMTSLLNPNGKLAGLLFDFPLT
SEGPPFGGSKSEYITLFSNTFSIKTLERAYNSIKPRENKELFFIFETK
> Gramella_forsetii_YP_862431
MNKDFWSLRYQKGNTGWDIGNISTPLKEYIDHLHKKELKILIPGAGNSYEAEYLFEKGFKNIWICDIAKE
PIENFKKRLPEFPESQILNRDFFELKDQFDLILEQTFFCALPVNFRENYAKKVFELLKVNGKISGVLFDF
PLTPDGPPFGGSKEEYLAYFSPYFKINTFERCYNSINPRQGKELFFNFSKK

S11

> Brassica_oleracea_1_AAK69760
MAEEQQKAGHSNGENIIPPEEVAKFLPETVEEGGWEKCWEDGITPWDQGRATPLVVHLVDSSSLPLGRAL
VPGCGGGHDVVAMASPERFVVGLDISESALEKAAETYGSSPKAKYFTFVKEDFFTWRPNELFDLIFDYVV
FCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHDGGPPYKVAVSTYEDVLVPVGFKAVSIEENPYSI
ATRKGKEKLGRWKKIN
> Vitis_vinifera_3_CAO46360
MGLCVPSGRISGGVCGLLSGRSLTWAKNLGVSTTQLRMSNNGSSIESNPKVQKLNQIIGSDSAGGWEKSW
QQGHTPWDLGKPTPIIQHLHQTGTLPSGKTLVPGCGCGYDVVTIACPERFVVGLDISDSAIKKAKELSSS
LWNANHFTFLKEDFFTWNPTELFDLIFDYTFFCAIEPDMRSVWAKRMRHLLKPDGELLTLMFPISDHAGG
PPYKVSVADYEEVLHPMGFKAVSIVDNKMAIGPRKGREKLGRWKRTPSKSLL
> Burkholderia_sp._YP_370293
MSDPKQPKPNAPAAADFTTRDPGNASFWNERFERGVTPWEFGGVPEGFSVFAHRLELCAVLIPGCGSAQE
AGWLAEAGWPVRAIDFAAQAVAAAKAQLGAHAGVVEQADFFAYRPPFDVQWVYERAFLCALPPAMRADYA
ARMAELLPADGLLAGYFFLMAKPKGPPFGIERAELDALLTPHFELIEDLPVTDSLAVFEGHERWLTWRRR
> Flavobacteriales_bacterium_2_ZP_02183094
MISMKKNKLDSDYWEDRYTKNSTSWDIGYPSTPIRTYIDQLKDKSLKILIPGAGNSFEAEYLWNLGFKNI
YILDFAKQPLENFKKRLPDFPENQLLHIDFFKLDIHFDLILEQTFFCALNPSLREKYVEQMHQLLKPKGK
LVGLFFNFPLTKSGPPFGGSLTEYQFLFDKKFKIKILETSINSIKEREGKELFFIFESP
> Coprinopsis_cinerea_okayama_ XP_001831730
MADPNLAPEIRAKMQEIFKPDDRHSWDLLWKENITPWDAGDAQPSLIELIEESGLDFARKGRALVPGCGT
GYDAVYLASALGLQTIGMDISESAVEAANRYRDSSGVQGADRAIFQKADFFTYKVPDEERFDLIMDHTFF
CAIHPSLRPEWGQRMSELIKPGGYLITICFPMIPKVETGPPYYLRPEHYDEVLKETFEKVYDKVPTKSSE
NHKDKERMLVWKKK
> Flavobacterium_sp._ZP_01059895
MKTDLNKLYWEDRYQNQQTGWDIGSVSTPLKEYIDQIDDKNIQILVPGAGYGHEVRYLAQQGFKNVDVID
LSVSALTQLKKALPDTTAYQLIEGDFFEHHTSYDLILEQTFFCALEPDKRPDYAAHAASLLKDSGKISGV
LFNFPLTEKGPPFGGSSEEYKKLFSEYFNIKTLEACYNSIKPRLGNELFFIFEKSNQES
> Vibrio_alginolyticus_ZP_01260043
MKQAPMINTQFWDDLFIRGTMPWDAQSTPQELKDYLDNSLHVGQSVFIPGCGAAYELSTFIQYGHDVIAM
DYSQEAVKMAQSALGNYKDKVVLGDVFNADFSHSFDVIYERAFLAALPRDMWSEYFSTVDKLLPSGGFLI
GFFVIDDDYCSRFPPFCLRSGELASFLEPTFELVKSSVVANSVEVFKGREQWMVWQKR
> Cryptococcus_neoformans_XP_566834
MAQASGDDNAWEERWAQGRTAFDQSAAHPVFVKFLKSDIARELGVPKSGKALVPGCGRGYDVHLLASTGL
DAIGLDLAPTGVEAARRWIGSQPSTSGKADILVQDFFTYDPLEKFDLIYDYTFLCALPPSLRQEWARQTT
HLANIAADTNPILITLMYPLPPSAKSGGPPFALSEEIYQELLKEQGWKMVWSEDIEEPTRMVGAPGGEKL
AVWKRI
> Algoriphagus_sp._ZP_01720187
MAELDEKYWSERYKSGLTGWDIGFPSTPIVQYLDQIVNKDVEILIPGAGNAYEAYYAFQSGFSNVHVLDI
SQEPLRNFKDKFPNFPSSNLHHGDFFEHHGSYNLILEQTFFCALNPSLRPKYVKKMSELLLKGGKLVGLL
FNKEFNSPGPPFGGGIKEYQKLFHNSFEIDVMEECYNSIPARAGSEAFIRLINSKG
> Vibrio_parahaemolyticus_NP_800656
MKSKDSPIINEQFWDALFFNGTMPWDRSQTPNELKHYLKRIADKTHSVFIPGCGAAYEVSHFVDCGHDVI
AMDYSAEAVNLAKSQLGQHQDKVMLGDVFNADFSREFDVIYERAFLAALPREIWGDYFAMIERLLPSNGL
LVGYFVISDDYRSRFPPFCLRSGEIEQKLEANFHLIESTPVTDSVDVFKGKEQWMVWQKK
> Psychrobacter_cryohalolentis_YP_581342
MENVNQAQFWQQRYEQDSIGWDMGQVSPPLKAYIDQLPEAAKNQAVLVPGAGNAYEVGYLHEQGFTNVTL
VDFAPAPIAAFAERYPNFPAKHLICADFFELSPEQYQFDWVLEQTFFCAINPSRRDEYVQQMASLVKPNG
KLIGLLFDKDFGRDEPPFGGTKDEYQQRFATHFDIDIMEPSYNSHPARQGSELFIEMHVKD
> Croceibacter_atlanticus_ZP_00949128
MTSNFWEQRYANNNTGWDLNTVSPPLKHYIDTLSNKTLFILIPGCGNAYEAEYLHNQGFENVFIVDLAEH
PLLEFSKRVPDFPKSHILHLDFFNLTQKFDLILEQTFFCALHPEQRLHYAHHTSKLLNSNGCLVGLFFNK
EFDKTGPPFGGNKKEYKNLFKNLFKIKKLENCYNSIKPRQGSELFFIFEKK
> Aspergillus_niger_3_XP_001388537
MTTPTDNKFKDAQAYLAKHQGDSYLKGWDLLWDKGDYLPWDRGFPNPALEDTLVERAGTIGGPIGPDGKR
RKVLVPGCGRGVDVLLFASFGYDAYGLECSAAAVEACKKEEEKVNNIQYRVRDEKVGKGKITFVQGDFFD
DAWLKEIGVPRNGFDVIYDYTFFCALNPELRPKWALRHTELLAPFPAGNLICLESPRHRDPLAPGPPFAS
PSEAYMEHLSHPGEEISYNDKGLVDADPLREPSKAGLERVAYWQPERTHTVGKDKNGVIQDRVSIWRRRD
> Ostreococcus_tauri_CAL52768

S12

MTTSSAPTRHTSMRVALAAPATVTRRLGTYKRVFDRRAMSTRAIDGAVTSNAGDFARQDGSTDWEGMWSR
GITKGAAFDCSRTEPAFQNALDAKEIAIGSGRALVPGCGRGYALASLARAGFGDVVGLEISETAKEACEE
QLKAESIPETARVEVVVADFFAYDPKEAFDAAYDCTFLCAIDPRRREEWARKHASLIKPGGTLVCLVFPV
GDFEGGPPYALTPEIVRELLAPAGFEEIELRETPAEMYARGRLEYLFTWRRRS
> Oryza_sativa_1_EAY99736
MDRALPLALSVSLWWLLVGDLGGRWTLEDDGGGGGVSRFGSWYRMCGWWWVWADWIIELGASSWGNLFGL
VLKRRKNEAVERDSSDGWEKSWEAAVTPWDLGKPTPIIEHLVKSGTLPKGRALGYDVVALASPERFVVGL
GISSTAVEKAKQWSSSLPNADCFTFLADDFFKWKPSEQFDLIFDYTFFCALDPSLRLAWAETVSGLLKPH
GELITLIYLVTEESIYSFVYFSIEDVMVLIISYCAERISYYRSVTKKEDHHSIIQSPILLRCPFRNHSYQ
KVLEPLGFKAILMEDNELAIKPRKAISAFRTSEQPSLAAQDVTE
> Oryza_sativa_japonica_1_ABF99844
MASAIVDVAGGGRQQALDGSNPAVARLRQLIGGGQESSDGWSRCWEEGVTPWDLGQRTPAVVELVHSGTL
PAGDATTVLVPGCGAGYDVVALSGPGRFVVGLDICDTAIQKAKQLSAAAAAAADGGDGSSSFFAFVADDF
FTWEPPEPFHLIFDYTFFCALHPSMRPAWAKRMADLLRPDGELITLMYLVINRRYQHV
> Polaribacter_irgensii_ZP_01117536
MNLSADAWDERYTNNDIAWDLGEVSSPLKAYFDQLENKEIKILIPGGGNSHEAAYLFENGFKNIWVVDLS
ETAIGNIQKRIPEFPPSQLIQGDFFNMDDVFDLIIEQTFFCAINPNLRADYTTKMHHLLKSKGKLVGVLF
NVPLNTNKPPFGGDKSEYLEYFKPFFIIKKMEACYNSFGNRKGRELFVILRSK
> Aspergillus_niger_1_XP_001389353
MTDQSTLTAAQQSVHNTLAKYPGEKYVDGWAEIWNANPSPPWDKGAPNPALEDTLMQRRGTIGNALATDA
EGNRYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSGAAVQACRQEEKESTTSAKYPVRDEEGDFFKDDW
LEELGLGLNCFDLIYDYTFFCALSPSMRPDWALRHTQLLAPSPHGNLICLEYPRHKDPSLPGPPFGLSSE
AYMEHLSHPGEQVSYDAQGRCRGDPLREPSDRGLERVAYWQPARTHEVGKDANGEVQDRVSIWRRR
> Aspergillus_nidulans_XP_663698
MSSPSQQPIKGRLISHFENRPTPSHPKAWSDLWDSGKSSLWDRGMPSPALIDLLESYQDTLLHPFEIDIE
DEEDSSDAGKTRKRKRALVPGCGRGYDVITFALHGFDACGLEVSTTAVSEARAFAKKELCSPQSGNFGRR
FDRERARHIGVGKAQFLQGDFFTDTWIENESTGLDQGRTENGKFDLVYDYTFLCALHPAQRTRWAERMAD
LLRPGGLLVCLEFPMYKDPALPGPPWGVNGIHWELLAGGDTGQGKFTRKAYVQPERTFEVGRGTDMISVY
ERK
> Gloeobacter_violaceus_NP_923764
MPSEESSGVDQPAFWEYRYRGGQDRWDLGQPAPTFVHLLSGSEAPPLGTVAVPGCGRGHDALLFAARGYK
VCGFDFAADAIADATRLALRAGAAATFLQQDLFNLPRPFAGLFDLVVEHTCFCAIDPVRREEYVEIVHWL
LKPGGELVAIFFAHPRPGGPPYRTDAGEIERLFSPRFKITALLPAPMSVPSRRGEELFGRFVRA
> Anaeromyxobacter_dehalogenans_YP_466408
MGTSYRLAYLIGFTPWEDQPLPPELSALVEGLRARPPGRALDLGCGRGAHAVYLASHGWKVTGVDLVPAA
LAKARQRATDAGVDVQFLDGDVTRLDTLGLSPGYDLLLDAGCFHGLSDPERAAYARGVTALRAPRAAMLL
FAFKPGWRGPAPRGASAEDLTSAFGPSWRLVRSERARESRLPLPLRNADPRWHLLEAA
> Lentisphaera_araneosa_ZP_01876851
MRTKGNEKAESWDKIYREGNPGWDIKKPAPPFEDLFKQNPSWLKAGSLISFGCGGGHDANFFAQNDFNVT
AVDFASEAVKLARSNYPQLNVIQKNILELSPEYDEQFDYVLEHTCFCAVPLDHRRAYMESAHAILKAGAY
LFGLFYRFDPPDQDGPPYSLSLEDLEDAYSGLFTLEENAIPKRSHGRRTQRERFIVLKKI
> Psychrobacter_arcticus_YP_265081
MGNVNQAEFWQQRYEQDSIGWDMGQVSPPLKVYIDQLPEAAKEQAVLVPGAGNAYEVGYLYEQGFTNITL
VDFAPAPIKDFAERYPDFPADKLICADFFDLLPKQHQFDWVLEQTFFCAINPARRDEYVQQMARLLKPKG
QLVGLLFDKDFGRNEPPFGGTKEEYQQRFSTHFDTEIMEQSYNSHPARQGSELFIKMRVKD
> Mycobacterium_vanbaalenii_YP_952715
MDLTPRLSRFDEFYKNQTPPWVIGEPQQAIVELEQAGLIGGRVLDVGCGTGEHTILLARAGYDVLGIDGA
PTAVEQARRNAEAQGVDARFELADALHLGPDPTYDTIVDSALFHIFDDADRATYVRSLHAATRPGSVVHL
LALSDSGRGFGPEVSEHTIRAAFGAGWEVEALTETTYRGVVIDAHTEALNLPAGTVVDEPAWSARIRRL
> Aspergillus_niger_2_XP_001390073
MSEAPNPPVQGRLISHFADRRAEDQGSGWSALWDSNESVLWDRGSPSIALVDVVEQQQDVFFPYTRDGRR
KKALVPGCGRGYDPVMLALHGFDVYGLDISATGVSEATKYATSEMQSPQDVKFIAGDFFSSEWESQALQD
GDKFDLIYDYTFLCALHPDLRRKWAERMSQLLHPGGLLVCLEFPMYKDTSLPGPPWGLNGVHWDLLARGG
DGITNITKEEEDEDSGIQLSGQFRRAQYFRPIRSYPSGKGTDMLSIYVRR
> Methanosarcina_acetivorans_NP_617055
MFWDEVYKGTPPWDIDHPQPAFQALIESGEIRPGRALDIGCGRGENAIMLAKNGCDVTGIDLAKDAISDA
KAKAIERHVKVNFIVGNVLEMDQLFTEDEFDIVIDSGLFHVITDEERLLFTRHVHKVLKEGGKYFMLCFS
DKEPGEYELPRRASKAEIESTFSPLFNIIYIKDVIFDSLLNPGRRQAYLLSATKS

S13

> Rhodococcus_sp._YP_709003
MVDAPRFPYPGSPPVHGPDDLYVTPPPWDIGRAQPVFVALAEGGAIRGRVLDCGCGTGEHVLLAAGLGLD
ATGVDLAATALRIAEQKARDRGLTARFLHHDARRLAELGERFDTVLDCGLFHIFDPDDRAAYVDSLRDVL
VPGGRYLMLGFSDQQPGDWGPHRLTRDEITTAFDDGWTIDSLESATLEVTLDPAGMRAWQLAATRTWPHP
IERECSAPC
> Flavobacterium_psychrophilum_YP_001296326
MKKIDQKYWQNRYQTNDIAWDTGKITTPIKAYIDQIEDQSIKILIPGCGNGYEYEYLIKKGFYNSFVADY
AQTPIDNLKKRIPNCNANQLLISDFFELEGSYDLIIEQTFFCALNPELRVKYAQKMLSLLSPKGKIIGLL
FQFPLTEAGPPFGGSKEEYLKLFSTNFNIKTIETAYNSIKPREGNELFFIFTKK
> Synechococcus_sp._2_ZP_01080043
MQLDGASSAPTLTARDWDARYRQGTDRWELGMAAPPLQAFLEQHPLAPKPTGTVLVPGCGRGHEAALLAR
LGFDVVGLDFSVEAIREARRLQGEHENLRWLQADLFNGAALDRAGLGAHSLSGVVEHTCFCAIDPSQRDH
YRSTVDRLLEPGGWLLGVFFCHDRPGGPPYGSDAEQLAASWSQIGFTGVIWEPAQGSVAQRSDEWLGLWR
KPSQADNEAIPAGSR
> Alkaliphilus_metalliredigens_YP_001319591
MNDKLDQEVILNQEDLLNMLDSLLEKWDEEWWNEFYSDKGKPIPFFVNAPDENLVTYFDKYFDDIGRALD
VGCGNGRNSRFIASRGYDVEGLDFSKKSIEWAKEESKKTGDIALYVNDSFFNINRELSSYDLIYDSGCLH
HIKPHRRSQYLEKVHRLLKPGGYFGLVCFNLKGGANLSDHDVYKKSSMAGGLGYSDIKLKKILGTYFEIV
EFREMRECADNALYGKDICWSILMRRLAK
> Halorhodospira_halophila_YP_001002952
MSGDPDPRRAPWEARWREGRTGWDRGGVSPTLEAWLSAGVIPGRRVLVPGAGRGYEVEALARRGYKVTAV
DIAAEACQQLRDGLDAAGVEARVVQADLLAWQPDTPFDAVYEQTCLCALDPADWPAYEQRLYGWLRPGGV
LLALFMQTGASGGPPFHCALPEMATLFDSERWQWPAEPPRQWPHPSGRWEEAVRLLRR
> Mycobacterium_smegmatis_YP_886428
MDTTPTRELFDEAYESRTAPWVIGEPQPAVVELERAGLIRSRVLDVGCGAGEHTILLTRLGYDVLGIDFS
PQAIEMARENARGRGVDARFAVGDAMALGDLGDGAYDTILDSALFHIFDDADRQTYVASLHAGCRPGGTV
HILALSDAGRGFGPEVSEEQIRKAFGDGWDLEALETTTYRGVVGPVHAEAIGLPVGTQVDEPAWLARARR
L
> Plesiocystis_pacifica_ZP_01912520
MRVIVPGAGVGHDALAWAQAGHEVVALDFAPAAVARLRERAAEAGLTIEAHVADVTNPGPALNDGLGGRF
DLVWEQTCLCAITPELRGAYLAQARSWLTPDGSMLALLWNTGNEGGPPYDMPPELVERLMTGLFVIDKFA
PVTGSNPNRREHLYWLRPEPT
> Arabidopsis_thaliana_1_ NP_181920
MAEEQQNSSYSIGGNILPTPEEAATFQPQVVAEGGWDKCWEDGVTPWDQGRATPLILHLLDSSALPLGRT
LVPGCGGGHDVVAMASPERFVVGLDISDKALNKANETYGSSPKAEYFSFVKEDVFTWRPNELFDLIFDYV
FFCAIEPEMRPAWGKSMHELLKPDGELITLMYPMTDHEGGAPYKVALSSYEDVLVPVGFKAVSVEENPDS
IPTRKGKEKLARWKKIN
> Burkholderia_mallei_YP_102027
MKDRLMSQGDGVTNEANQPEAAGQAAGDAQPASPAGPAHIANPANPANPPALPSFSPPAAASSSASSAAP
FSSRDPGDASFWDERFEQGVTPWDSARVPDAFAARHARVPVLIPGCGSAYEARWLARAGWPVRAIDFSAQ
AVAAARRELGEDAGLVEQADFFTYAPPFVPQWIYERAFLCAIPRSRRADYARRMAELLPPGGFLAGFFFI
GATPKGPPFGIERAELDALLCPHFALVEDEPVADSLPVFAGRERWLAWRRS
> Saccharopolyspora_erythraea_YP_001106907
MDDELAESQRAHWQDTYSAHPGMYGEEPSAPAVHAAGVFRAAGARDVLELGAGHGRDALHFAREGFTVQA
LDFSSSGLQQLRDAARAQQVEQRVTTAVHDVRHPLPSADASVDAVFAHMLLCMALSTEEIHALVGEIHRV
LRPGGVLVYTVRHTGDAHHGTGVAHGDDIFEHDGFAVHFFPRGLVDSLADGWTLDEVHAFEEGDLPRRLW
RVTQTLPR
> Vibrio_sp_ZP_01477195
MKQAPTINQQFWDNLFTQGTMPWDAKTTPQELKAYLENALHSGQSVFIPGCGAAYELSSFIQYGHDVIAM
DYSEQAVKMAQSTLGKHKDKVVLGDVFNADSTHSFDVIYERAFLAALPRDQWPEYFAMVDKLLPRGGLLI
GYFVIDDDYHSRFPPFCLRSGELEGYLEPVFKLVESSVVANSVEVFKGRERWMVWQKSCRI
> Vitis_vinifera_1_CAN68137
MASPDNTKPKARSSESVTGQRRGRRPSDRHWPCVGEESGSFYNTIADGERQYQHRIELRASKNKPSSWEE
KWQQGLTPWDLGKATPIIEHLHQAGALPNGRTLIPGCGRGYDVVAIACPERFVVGLDISDSAIKKAKESS
SSSWNASHFIFLKADFFTWNPTELFDLIIDYTFFCAIEPDMRPAWASRMQQLLKPDGELLTLMFPISDHT
GGPPYKVSIADYEKVLHPMRFKAVSIVDNEMAIGSRKKKYPLKPDLSLFGFVDRPKRAYEARSEEFRISD
WVCGWMGLCVPSGRISGGVCGLLSGRSLTWAKNLGVSTTQLRMSNNGSSIESNPKVQKLNQIIGSDSAGG
WEKSWQQGHTPWDLGKPTPIIQHLHQTGTLPSGKTLVPGCGCGYDVVTIACPERFVVGLDISDSAIKKAK

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EISDHAGGPPYKVSVADYEEVLHPMGFKAVSIVDNKMAIGPRKGREKLGRWKRTPSKSLL
> Arabidopsis_thaliana_2_NP_001078053
MAEEQQNSDQSNGGNVIPTPEEVATFLHKTVEEGGWEKCWEEEITPWDQGRATPLIVHLVDTSSLPLGRA
LVPGCGGGHDVVAMASPERFVVGLDISESALAKANETYGSSPKAEYFSFVKEDVFTWRPTELFDLIFDYV
FFCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHVGGPPYKVDVSTFEEVLVPIGFKAVSVEENPHA
IPTRQREAGKVEEDQLIPKKEILLFGKSVICVIYKE

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.

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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.

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