ptka epsb Figure S1. The PtkA kinase contributes to EPS production Shown is colony wrinkling on biofilm-inducing medium for strains NCBI 3610

Transcription

1 Figure S1 WT epsab ptka ptka epsb Figure S1. The PtkA kinase contributes to EPS production Shown is colony wrinkling on biofilm-inducing medium for strains NCBI 3610 (WT), BAE252 ( epsab), YC176 ( ptka), and BAE554 ( epsab ptka).

2 Figure S2 Figure S2. EpsB is autophosphorylated at tyrosines 225 and 227 Shown is the fragment spectra generated by collision-induced dissociation of the tryptic phosphopeptide precursor KSEHpYSpY at m/z (M+2H) 2+ leading to the identification of phosphotyrosine at position 225 and 227. Signals assigned to fragments of the b- and y-ion series are labeled with the observed mass. The addition of a phosphate moiety ( Da) to the specific tyrosine residues (163.1 Da) is demonstrated by the specific masses for the y1 and b6 ions for tyrosine 225 and for the specific mass differences between y2 and y3 as well as the b4 and b5 ions for tyrosine 227. The specific

3 masses are shown in the figure by a blue arrow between the ions labeled with Tyr-Po 4. The addition of two phosphate moieties to ion y3 and following y ions demonstrate that the peptide is phosphorylated at both tyrosine residues. (For details of methods see below)

4 His6-EpsB His6-EpsB~P epsh WT + epsh Figure S3 Figure S3. Cell-to-cell signaling The figure is an immunoblot showing the ability of the EpsAB kinase to sense EPS produced from another cell. A mutant blocked in EPS production ( epsh) that carries a fusion of the gene for His 6 -tagged EpsB to an IPTG-inducible promoter (BAE397) was grown alone or in co-cultivation with wild-type B. subtilis that does not carry a gene for His 6 -tagged EpsB. The cells were grown in MSgg medium for 24 hours to which IPTG was added. The upper row was probed with anti-his 6 antibodies and the lower row with anti-phosphotyrosine antibodies.

5 Figure S4 µg/ml EPS His6-EpsE His6-EpsE~P Figure S4. Exopolysaccharide-dependent phosphorylation of EspE The figure is an immunoblot showing the dose dependence of EpsE phosphorylation on purified EPS using cells that were mutant for epsh and contained an IPTG-inducible copy of epsab and a fusion of the gene for His 6 -tagged EpsE to a xyloseinducible promoter (BAE598). The cells were grown in LB medium to which IPTG and xylose were added and treated with increasing concentrations of EPS.

6 Figure S5 His6-EpsB time (min) + EPS - EPS Figure S5. Autophosphorylation destabilizes EpsB Shown is an immunoblot that displays the stabilization of EpsB in the presence of EPS. Cells were grown in LB until an OD 600 of 0.8 was reached. Then protein synthesis was stopped by the addition of 100 µg/ml chloramphenicol and samples were taken at indicated time points after the addition of chloramphenicol. The strains contained a copy of His 6 -EpsB integrated at amye. To increase expression of EpsB and to allow a high production of EPS all strains contained a sinr mutation, which allows constant expression of the eps operon. The +EPS (BAE541) strain was otherwise wild-type whereas the EPS strain carried an additional mutation in epsh (BAE542) to prevent the production of EPS. The blot was probed with anti-his 6 antibodies.

7 WT epsa amye::epsa amye::epsa Figure S6 His6-EpsB His6-EpsB~P Figure S6. EpsA is required for EpsB activation The figure shows that colony wrinkling (upper) and autophosphosphorylation (lower) depend on the extracellular domain of EpsA. The immunoblot is of His 6 -tagged EpsB. The blot was probed with anti-his 6 antibodies (upper row) and antiphosphotyrosine antibodies (lower row). The strains used were BAE318 (WT), BAE294 ( epsa), BAE295 ( epsa amye::epsa ) and BAE318 ( epsa amye::epsa), which all additionally contained the gene for His 6 -tagged EpsB.

8 WT epsab amye::epsb fusedab Figure S7 His6-EpsB fusedab - His6-EpsB fusedab ~P EPS dextran Figure S7. The receptor domain is required for EPS sensing The figure shows that colony wrinkling (upper) and EPS induced dephosphosphorylation (lower) depend on the extracellular domain of EpsA. Colony wrinkling shows the strains NCBI 3610 and BAE296 ( epsab fused ). The immunoblot is of His 6 -tagged EpsAB from epsh mutant cells in which expression of the chimeric and cytoplasmic EpsAB is upregulated by a mutation of sinr (BAE621) were grown in LB medium and treated with purified EPS (20µg) and dextran (0.1%). The blot was probed with anti-his 6 antibodies (upper row) and anti-phosphotyrosine antibodies (lower row).

9 Figure S8 epsa amye::epsa amye::epsabli amye::epsasau Figure S8. The receptor domain of the membrane-component senses EPS species-specific Shown is colony wrinkling on biofilm-inducing medium for strains BAE317 (epsa), BAE565 (epsawithreceptorfromepsafromb.licheniformis) (epsawithreceptorfromcapafroms.aureus). and BAE567

10 Supplemental materials and methods Strain construction Up- and downstream regions for the desired mutation site were amplified from 3610 chromosomal DNA using primer depsa_p1 and depsa_p2 for the upstream region of epsa, depsb_p1 and depsb_p2 for the upstream region of epsb, depsab_p1 and depsab_p2 for the upstream region of epsab, depse_p1 and depse_p2 for the upstream region of epse, depse_p3 and depse_p4 for the downstream region of epse, depsa_p3 and depsa_p4 for the downstream region of epsa, depsb_p3 and depsb_p4 for the downstream region of epsb and depsab_p3 and depsab_p4 for the downstream region of epsab. Primers P1 and P4 contained overlapping DNA sequence to the MCS of pminimad cut with EcoRI and BamHI and primer 2 and 3 contained overlapping sequences to each other. The two fragments were then joined in an isothermal assembly reaction with pminimad cut with EcoRI and HindIII and transformed into a reca + strain of E. coli, miniprepped, and transformed into 3610 under selection for MLS. Transformants were then grown at room temperature for 24 hours to promote plasmid excision and this cycle was repeated three times by reinoculating a new culture. Then, a fresh culture was shifted to 37 C to cure the plasmid. The deletion was then verified by sequencing. To sequence the constructs we used primers 210F-pMiniMAD2-seq and 210R-pMiniMAD2-seq. To create complementation constructs for epsa, epsb and epse we amplified fragments carrying the gene and its corresponding promoter from 3610 chromosomal DNA using epsa_pdg1730_p1 and epsa_pdg1730_p4 for epsa, epsb_pdg1730_p1

11 and epsb_pdg1730_p2 for the eps promoter and epsb_pdg1730_p3, epsb_pdg1730_p4 for epsb, epsab_pdg1730_p1 and epsab_pdg1730_p4 for epsab, epsehis_pdg1730_p1 and epsehis_pdg1730_p2 to create the eps promoter and epsehis_pdg1730_p3 and epsehis_pdg1730_p4 for epse. Primers P1 and P4 contained overlapping DNA sequence to the MCS of pdg1730 (Guérout-Fleury et al, 1996) cut with EcoRI and BamHI and primers 2 and 3 contained overlapping sequences to each other. To create a His 6 -tagged version EpsE we used primers epsehis_pdg1730_p1 and epsehis_pdg1730_p2 to create fragment 1 and epsehis_pdg1730_p3 and epsehis_pdg1730_p4 to create fragment 2 and for EpsB we used epsab_pdg1730_p1 and epsbhis_pdg1730_p2 for fragment 1 and epsab_pdg1730_p4 and epsbhis_pdg1730_p3 for fragment 2. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdg1730, whereas primers 2 and 3 contained an overlapping region encoding a his 6 -tag. The two fragments were then joined in an isothermal assembly reaction with pdg1730 cut with EcoRI and HindIII. To sequence the plasmid we used primers 007F-pDG1730-Check-F and 007R-pDG1730-Check-R. Integration of the plasmid was selected with 100 µg/ml of spectinomycin. To test if the plasmid integrated correctly into the designated amye-sites we tested the ability of these strains to metabolize starch on LB plates containing 1% starch, which depends on an intact amye gene. The plates were incubated for 24 hours and then treated with an iodine solution, which stains the starch, to screen for disrupted amye sites. To create inducible complementation constructs for epsab-his 6 we amplified fragments carrying the gene and its corresponding promoter from 3610 chromosomal DNA using epsab_pdr111_p1 and epsab_pdr111_p2 to create for epsab fragment 1

12 and epsab_pdr111_p3 and epsab_pdr111_p4 for epsab fragment 2. Primers P1 and P4 contained overlapping DNA sequence to the MCS of pdr111 cut with EcoRI and HindIII. Primers P2 and P3 contained an overlapping region encoding a His 6 -tag. The two fragments were then joined in an isothermal assembly reaction with pdr111 cut with EcoRI and HindIII. Integration of the plasmid was selected with 100 µg/ml of spectinomycin. To test if the plasmid integrated correctly into the designated amye-sites we tested the ability of these strains to metabolize starch on LB plates containing 1% starch, which depends on an intact amye gene. The plates were incubated for 24 hours and then treated with an iodine solution, which stains the starch, to screen for disrupted amye sites. Site directed mutagenesis was done using isothermal assembly. For mutational analysis of the C-terminal tyrosine residues of epsb the epsab construct was amplified using primers epsab_pdg1730_p1 and epsbyf_p4 for tyrosine to phenylalanine substitutions and epsab_pdg1730_p1 and epsbye_p4 for tyrosine to glutamate substitutions. To create the catalytically inactive epsb variant we used epsab_pdg1730_p1 and epsbdxd_p2 for fragment 1 and epsbdxd_p3 and epsab_pdg1730_p4 for fragment 2. To create an epsa variant that lacked the receptor domain we used epsab_pdg1730_p1 and depsareceptor_p3 for fragment 1 and epsbdxd_p3 and depsareceptor_p4 for fragment 2. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdg1730, whereas primers 2 and 3 contained an overlapping region with the desired mutation. The fragments were then joined in an isothermal assembly reaction with pdg1730 cut with EcoRI and BamHI and the protocol was followed as described above. To create a chimeric kinase where the N-terminus of

13 EpsA is joined to EpsB we used primers EpsB_chim_P1 and EpsB_Chim_P2 for fragment 1 and primers EpsB_chim_P3 and EpsB_chim_P4 for fragment 2. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdg1730, whereas primers 2 and 3 contained an overlapping region containing a His 6 -tag and the N-terminal region of EpsA fused to EpsB. The fragments were then joined in an isothermal assembly reaction with pdg1730 cut with EcoRI and BamHI and the protocol was followed as described above. To create chimeric EpsA protein with B. licheniformis EpsA receptor we used primers epsab_pdg1730_p1 and EpsA_lich_P2 for fragment 1, primers Bli_P2_for and Bli_P3_rev for fragment 2 (from. B. licheniformis genomic DNA) and primers EpsA_lich_P3 and epsab_pdg1730_p4 for fragment 3. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdg1730, whereas primers Bli_P2_for and Bli_P3_rev contained an overlapping region with the end of the corresponding EpsA domain to allow fusion of the receptor to the rest of EpsA. The fragments were then joined in an isothermal assembly reaction with pdg1730 cut with EcoRI and BamHI and the protocol was followed as described above. To create chimeric EpsA protein with the CapA receptor we used 3610 genomic DNA and primers epsab_pdg1730_p1 and epsa_capreceptor_p2 to create fragment 1 and epsab_pdg1730_p4 and epsa_capreceptor_p3 to create fragment 3 and SH1000 genomic DNA and primers epsa_capreceptor_p2a and epsa_capreceptor_p3a to create fragment 2. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdg1730, whereas primers 2 and 3 and 2A and 3A contained an overlapping region with each other. Fragment 1 contained the N-terminal region of EpsA, fragment 2 contained the receptor region of CapA and fragment 3 contained the C-terminal region of EpsA and His 6 -tagged EpsB. The

14 fragments were then joined in an isothermal assembly reaction with pdg1730 cut with EcoRI and BamHI and the protocol was followed as described above. To create chimeric EpsA protein with the CapA receptor we used 3610 genomic DNA and primers epsab_pdr111_p1 and epsa_capreceptor_p2 to create fragment 1 and epsab_pdr111_p4 and epsa_capreceptor_p3 to create fragment 3 and SH1000 genomic DNA and primers epsa_capreceptor_p2a and epsa_capreceptor_p3a to create fragment 2. Primers 1 and 4 contained overlapping DNA sequence to the MCS of pdr111 cut with EcoRI and HindIII, whereas primers 2 and 3 and 2A and 3A contained an overlapping region with each other. Fragment 1 contained the N-terminal region of EpsA, fragment 2 contained the receptor region of CapA and fragment 3 contained the C-terminal region of EpsA and His 6 -tagged EpsB. The fragments were then joined in an isothermal assembly reaction with pdr111 cut with EcoRI and HindIII and the protocol was followed as described above. Mass spectrometry Purified EpsB was denatured using 8 M Urea and proteins were reduced using 1mM TCEP for one hour. Then, free cysteines were alkylated with 5mM Iodoacetamide and incubated for 30 minutes in the dark. Afterwards, the urea concentration was decreased to below 1 M by the addition of 50 mm Tris/HCl ph 8 and 150 mm NaCl and proteins were subsequently digested with trypsin (1:20, Promega) for 12 h. Then, phospho-peptide enrichment was performed using Titansphere TiO2 beads (GL Science) according to a previously described protocol (Elsholz et al, 2012). Briefly, the peptide

15 mix was acidified using 1% TFA and solution was cleared by centrifugation for 5 minutes at 15,000 x g at room temperature. The supernatant was purified using a C18 Sep-Pak columns (Waters) and eluted with 50% acetonitrile and 0.1%TFA. Samples were lyophilized and resuspendend in 73% Acetonitrile, 25% lactic acid and 2% TFA, mixed with 30 µg beads and incubated at room temperature for 30 minutes. Beads were loaded onto a C18 spin column (Nest group) and washed three times with 80% acetonitrile and 2% TFA. Peptides were eluted with 50 µl 5% NH 4 OH followed by 50 µl 50% acetonitrile. Enriched peptides were separated and analyzed by LC-MS/MS using an EasynLCII HPLC system (Thermo Fisher Scientific) coupled directly to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) at the Harvard Mass Spectrometry and Proteomics Resource Laboratory, FAS Center for Systems Biology, Northwest Bldg. Room B247, 52 Oxford St, Cambridge, MA.

16 supplement)file)1a)strains) ) Strain Genotype ) Source or Reference PY79 Prototrophic derivative of B. subtilis subsp. subtilis 168 lab stock 3610 Wild B. subtilis isolate NCIB3610 RL4930 ywrk::tn917::amye::cat Camp & Losick 2009 RL3852 epsh::tet Kearns et al., 2005 RL3854 sinr::kan gift Y. Chai BAE250 epsa BAE251 epsb BAE252 epsab BAE260 epsa amye::epsa (spec) BAE261 epsb amye::epsb (spec) BAE262 epsab amye::epsab (spec) BAE294 epsab amye::his 6 -epsb (spec) BAE295 epsab amye::epsa His 6 -epsb (spec) BAE318 epsab amye::epsa-his 6 -epsb (spec) BAE320 epsab amye::epsa-epsb Y225F/Y227F (spec) BAE379 epsab amye::pspank-epsa-his6-epsb (spec) BAE388 sinr::kan epsb BAE397 epsab epsh::tet amye::pspank-epsa-his6-epsb (spec) BAE406 epsab amye::epsa-epsb Y225E/Y227E (spec) BAE450 epsab amye::epsa-his 6 -epsb Y225F/Y227F (spec) BAE451 epsab amye::epsa-his 6 -epsb D81A/D83A (spec) BAE476 epse BAE477 epse sinr amye::p eps -His 6 -epse (spec) BAE541 epsab amye::epsa-his 6 -epsb (spec) sinr::kan BAE542 epsab amye::epsa-his 6 -epsb (spec) sinr::kan epsh::tet BAE554 epsab ptka::tet BAE560 epse saca::p xyl -epse (mls) BAE561 epse epsab saca::p xyl -epse (mls) BAE562 epse epsh::tet saca-p xyl -His 6 -epse (mls)

17 BAE563 epse epsab saca::pxyl-epse (mls) amye::epsaepsb (spec) BAE565 epsab epsh::tet amye::epsab.licheniformis his6-epsb (spec) BAE567 epsab epsh::tet amye::epsacapa his6- epsb (spec) BAE598 epse epsab epsh::tet saca::pxyl-epse (mls) amye::p spank epsa-epsb (spec) This study BAE619 epsab epsh::tet amye::p spank -epsa capa His 6 - epsb (spec) BAE621 epsab sinr::kan epsh::tet amye::epsabchimerickinase(spec) YC167 ptka::tet gift Y. Chai SH1000 Staphylococcus aureus Lab stock!!! Supplement!file!1B!primers! ) ) ) primer depsa_p1 depsa_p2 depsa_p3 depsa_p4 depsb_p1 depsb_p2 depsb_p3 depsb_p4 depsab_p1 depsab_p2 ) ) sequence AACAGCTATGACCATGATTACGCCAAGCTTGACGGCTGCGG GCAAATAG GCTGAGCTGCCGTGCGCTTTTTTCTTTGAAACTCATATTCTCA TTCAT ATGAATGAGAATATGAGTTTCAAAGAAAAAAGCGCACGGCA GCTCAGC CGTTGTAAAACGACGGCCAGTGAATTCCTGCGGCGGTTTTCG TCTCT AACAGCTATGACCATGATTACGCCAAGCTTGACGGCTGCGG GCAAATAG CCTTGCTTTCTTTTTTCTAAAGATCAC GTGATCTTTAGAAAAAAGAAAGCAAGGGCACTGGAACAATC CAATGCG CGTTGTAAAACGACGGCCAGTGAATTCTCCGGCAGTTCGTGT GCCAAG AACAGCTATGACCATGATTACGCCAAGCTTGACGGCTGCGG GCAAATAG CGCATTGGATTGTTCCAGTGC TTCTTTGAAACTCATATTCTCATTCAT

18 depsab_p3 depsab_p4 epsa_pdg17 30_P1 epsa_pdg17 30_P4 epsb_pdg17 30_P1 epsb_pdg17 30_P2 epsb_pdg17 30_P3 epsb_pdg17 30_P4 epsab_pdg1 730_P1 epsab_pdg1 730_P4 epsbhis_pdg 1730_P2 epsbhis_pdg 1730_P3 epsbyf_p4 epsbye_p4 epsbdxd_p2 epsbdxd_p3 EpsB_chim_P 1 EpsB_chim_P 2 EpsB_chim_P 3 EpsB_chim_P 4 depse_p1 depse_p2 depse_p3 GCACTGGAACAATCCAATGCG CGTTGTAAAACGACGGCCAGTGAATTCTCCGGCAGTTCGTGT GCCAAG AAACACACAAATTAAAAACTGGTCTGATCGGATCGACGGCT GCGGGCAAATAG TCGCCAGGGCTGCAGGAATTC AAACACACAAATTAAAAACTGGTCTGATCGGATCCATTGCC ATGCTTAGGCTCCGTTCC GTATTCATAGCCTTCAGCCTTCC GGAAGGCTGAAGGCTATGAATACGTGATCTTTAGA AAAAAGAAAGCAAGGCG TCGCCAGGGCTGCAGGAATTC CTAGTAGGAATAGTGTTCCGATTTTTTCATTTTC AAACACACAAATTAAAAACTGGTCTGATCGGATCCGACGGC TGCGGGCAAATAG TCGCCAGGGCTGCAGGAATTCCTAGTAGGAATAGTGTTCCGA TTTTTTCATTTTC TGCGCCATGATGATGATGATGATGTGATCCTCTCAT TCGCTTCACTCCCCGAAATG ATGAGAGGATCACATCATCATCATCATCATGGCGCAATCTTT AGAAAAAAGAAAGCAAGGCGA TCGCCAGGGCTGCAGGAATTCCTAGAAGGAAAAGTGTTCCG ATTTTTTC TCGCCAGGGCTGCAGGAATTC CTACTCGGACTCGTGTTCCGATTTTTTC GATTGATGGTCGGCTTTCTTAACACCAGCAGTACTTTCTTTTC GAAAAGAAAGTACTGCTGGTGTTAAGAAAGCCGACCATCAA TC CGCCTGGTAGGTCGGTGA ATTAGCTTGATATTTAGGTGATAAGACGAAAAA ATGAGAGGATCACATCATCATCATCATCATGGCGCATCA CCG ACC TAC CAG GCG TGCGCCATGATGATGATGATGATGTGATCCTCTCATCGGCTT GATCATCGGGCT AGC CCG ATG ATC AAG CCG AAA GTC GCA CCA AAA ACT GTA GTG AAT AACAGCTATGACCATGATTACGCCAAGCTTCCATTGCCATAC ACCGGTCG CATCACAGCTGCAGGCATAAAGG CGGTCCTGAGTTCATACGCTTTTC GAAAAGCGTATGAACTCAGGACCGCCTTTATGCCTGCAGCTG

19 depse_p4 epsehis_pdg 1730_P1 epsehis_pdg 1730_P2 epsehis_pdg 1730_P3 epsehis_pdg 1730_P4 epse_saca_p 1 epse_saca_p 2 epsab_pdr11 1_P1 epsab_pdr11 1_P2 epsab_pdr11 1_P3 epsab_pdr11 1_P4 depsareceptor _P3 depsareceptor _P4 epsa_caprece ptor_p2 epsa_caprece ptor_p2a epsa_caprece ptor_p3a epsa_caprece ptor_p3 EpsA_lich_P2 EpsA_lich_P3 Bli_P2_for Bli_P3_rev TGATG CGTTGTAAAACGACGGCCAGTGAATTCCAGAGACGGCATGA CAAATACATCG CGGTACAAACGGATAATTCTTCCAATC TGCGCCATGATGATGATGATGATGTGATCCTCTCATGTATTC ATAGCCTTCAGCCTTCCCG ATGAGAGGATCACATCATCATCATCATCATGGCGCAATGAA CTCAGGACCGAAAGTTTC TCGCCAGGGCTGCAGGAATTCCTATTCATGCTTGACAAGCCC TTCC CTAAAAATCAAAGGGGGAAATGGGATCCCGGTTTTCTGATC AAAATCGGTGAC GAGTGCGGCCGCCCGCGGCTATTCATGCTTGACAAGCCCTTC TGTGTAATTGTGAGCGGATAACAATTAAGCTTATGAATGAGA ATATGAGTTTCAAAGAATTATATG TGCGCCATGATGATGATGATGATGTGATCCTCTCATTCGCTT CACTCCCCGAAATG ATGAGAGGATCACATCATCATCATCATCATGGCGCAATCTTT AGAAAAAAGAAAGCAAGGCGA TGATGACCTCGTTTCCACCGAATTAGCTTGCATGCCTAGTAG GAATAGTGTTCCGATTTTTTCATTTTC GACAATCGCATAAATTCTTTGAAACTCATATTC GAATATGAGTTTCAAAGAATTTATGCGATTGTCAGGCTGCGA AATATGGTCATGGCTTTTGGC AATGACCTTAAATTGCACAAAACCCAT ATGGGTTTTGTGCAATTTAAGGTCATTGACAAATATACTGCT TCTACTCAAATATTAG CAGCAGCGCCAAAAGCCATCCAACAATAATGCTGATAACTA AGTT ATGGCTTTTGGCGCTGCTG ATGGGTTTTGTGCAATTTAAGGTCATT GTC TAT CAG GCA TCG ACG CAG GCAGCGCCAAAAGCCATGACCATATT CAC CTG TCA GAC CGA ATT ACA AAA TG GGA AGG CTG AAG GCT ATG AAT AC ATG AAA GAA AAT ATT GAT TTT AGA GAA CTG ATT GC CCGTGCGCTTTTGCATGTATC

20 Receptor_set A_for Receptor_set A_rev CapA_setA_I TA_for CapA_setA_I TA_rev 007F3 pdg17303 Check3F! 007R3 pdg17303 Check3R! 210F3 pminimad23 seq! 210R3 pminimad23 seq!! ATCGATAAAGTCCAAAAAGAACGCC CGACGATGACGATAAGGATCGATGGGGATCCTCACCGACCT ACCAGGCGTCGAC CTTTGTTAGCAGCCGGATCAAGCTTCGAATTC GACCATATTT CGCAGCCTGGCCG CGA CGA TGA CGA TAA GGA TCG ATG GGGATCC AAA TAT ACT GCT TCT ACT CAA ATA TTA GTG CTTTGTTAGCAGCCGGATCAAGCTTCGAATTC TACTTTTACAGCATTATCATGTGCTGAGG ATCATACCACCAGTGATTATGCCG ATTCATTGTTTTAAAAATATCTCTTGCCAG AACAGCTATGACCATGATTACGCCA CGTTGTAAAACGACGGCCAGTG