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SUPPLEMENTAL INFORMATION
To:
PiggyMac, a domesticated piggyBac transposase involved in programmed genome
rearrangements in the ciliate Paramecium tetraurelia
Baudry, Malinsky et al.
Baudry - Sup. Info. - 1
SUPPLEMENTARY MATERIALS AND METHODS Construction of GFP fusion transgenes
Plasmid FNOAF178AE04 carries gene GSPATT00016627001 (PGM) and part of upstream and downstream adjacent genes (bp 286,573-293,335 from scaffold_49, see http://www.genoscope.cns.fr/cgi-bin/ggb/paramecie/gbrowse/paramecie/). It was recovered from a plasmid library used by the Genoscope for shotgun sequencing of the MAC genome (Aury et al. 2006). Downstream adjacent sequences were removed by ClaI-XhoI restriction and insertion of a ClaI-XhoI linker (see Table S1), to give plasmid pBL49g-i. Upstream adjacent sequences were removed by inserting the BspHI-NcoI fragment of FNOAF178AE04 containing the 5’ part of PGM between the SpeI and NcoI sites of pBL49g-i (BspHI and SpeI cleaved ends were treated with the Klenow fragment of DNA polymerase I prior to NcoI-restriction). The resulting plasmid, pBL49g, contains the PGM coding sequence (including its introns), with 96 bp upstream of the ATG (carrying the putative endogenous promoter) and 54 bp downstream of the TGA stop codon (including the 3’ UTR and the polyadenylation site identified by cDNA sequencing performed by the Genoscope).
For the construction of in-frame GFP fusions, a GFP-coding fragment adapted to Paramecium codon usage (Nowacki et al. 2005) was inserted into the MscI or BsmBI sites of pPBL49g, to give plasmids pPBL49-GFP-M (N-ter fusion with GFP inserted between Pgm residues G100 and Q101) or pPBL49-GFP-B (C-ter fusion with GFP inserted between residues A889 and E890), respectively (plasmid sequences are available upon request). Construction of dsRNA-producing plasmids used in RNAi experiments
The RNAi plasmids used in silencing experiments were obtained as follows : PGM-1 : cloning of the 567-bp HindIII-NcoI fragment of gene PGM between the
HindIII and NcoI sites of plasmid vector L4440 (Timmons and Fire 1998). PGM-2 : a 349-bp fragment from PGM was amplified by PCR using primers PBL49-
09SpeI and PBL49-10SpeI (Table S1) and the Phusion high-fidelity DNA polymerase (Finnzymes). Following SpeI-restriction, the fragment was inserted into the XbaI site of L4440.
PtSPO11-1 : same cloning procedure as PGM-2, for a 545-bp fragment from PtSPO11 amplified using primers Spo11-02SpeI and Spo11-08SpeI (Table S1).
PtSPO11-2 : same cloning procedure for a 408-bp fragment from PtSPO11 amplified using primers Spo11-07SpeI and Spo11-10SpeI (Table S1).
The ND7 silencing vector was the same as in (Garnier et al. 2004). In all experiments, ND7 silencing was confirmed by the lack of trichocyst discharge (Skouri and Cohen 1997) in the presence of picric acid (not shown).
Baudry - Sup. Info. - 2
SUPPLEMENTARY FIGURE LEGENDS Figure S1. Alignment of piggyBac transposases from Paramecium tetraurelia and other organisms. piggyBac sequences associated with TIRs from Takifugu rubripes Pigibaku1, Ciona intestinalis CinPB3, Anopheles gambiae AgaPB1, Bombyx mori Yabusame-1, Heliothis virescens piggyBac and Trichoplusia ni piggyBac (Sarkar et al. 2003) are aligned with the piggyBac-like sequences derived from the macronuclear genomic sequences of Paramecium tetraurelia (GSPATG00016627001) and Tetrahymena thermophila (TTHERM_01107220 for Tetrahymena 1 and TTHERM_00309880 for Tetrahymena 2), using the Muscle web server at EBI (http://www.ebi.ac.uk/Tools/muscle/index.html). Conserved amino acids are highlighted using the BoxShade server (http://www.ch.embnet.org/software/BOX_form.html), with the DDD catalytic triad in red and the conserved C-rich domain in green. For the Paramecium Pgm protein, putative nuclear localization signals (mono- or bipartite) predicted by the PSORT II package (http://psort.ims.u-tokyo.ac.jp/form2.html) are boxed in blue or highlighted in yellow, respectively. The Paramecium Pgm protein sequence follows the Genoscope automatic annotation. In a blastp analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi), it was found to have strong similarity to the hypothetical proteins predicted by the original annotation of the two Tetrahymena genomic loci (e-values = 3e-63 and 3e-35 against TTHERM_01107220 and TTHERM_00309880, respectively) and to piggyBac-related transposases from other organisms (e-values = 9e-24; 6e-21; 9e-31; 2e-20; 7e-28 and 6e-29 against transposases from Ciona, Anopheles, Takifugu, Trichoplusia, Bombyx and Heliothis, respectively). We noticed that the translation of some intronic sequences from the original gene model (TTHERM_01107220) shows a good similarity to the Paramecium protein (including a stretch in intron 7 which appears to encode the second catalytic D): to maximize similarity between the two proteins, the 3' splice site of intron 5 was moved 39 nt upstream; the 5' splice site of intron 7 was moved 55 nt upstream and its length was changed from 68 to 53 nt: the predicted protein corresponding to the reannotated genomic sequence was called Tetrahymena 1. For Tetrahymena 2, only the C-terminal part of the protein encoded by exons downstream of intron 10 from the original gene model (TTHERM_00309880) was included in the alignement. We introduced one modification to the original annotation: intron 13 was shortened by 18 bp by using an alternative 3' splice site, resulting in the inclusion of 6 amino acids (GKTKFK) in a conserved region following the first catalytic D. Our new annotations are only predictions and need to be confirmed experimentally. Figure S2. Progression of autogamy in a culture of 51new starved in K. pneumoniae (time-course experiment presented in Figure 1). Progression of autogamy was monitored by DAPI staining of cells. V: vegetative cells; S: skein formation in old MAC; F: fragmented old MAC, no detectable new MACs; A: cells with two visible new developing MACs; C: post-autogamous cells with one new MAC and fragments of the old MAC. Time points are in hours. Figure S3. DAPI staining of 51new cells undergoing autogamy in PGM-silencing medium (a-d) or control ND7 silencing medium (e-h). Starved cells were fixed and stained at day 1 (a, e), day 2 (b, f), day 3 (c, g) and day 4 (d, h). Developing new MACs are marked with arrowheads. At day 4, 30 cells from each experiment were transferred to standard K. pneumoniae medium: 100% of PGM-silenced cells died while all ND7-silenced cells resumed normal vegetative growth. The RNAi construct used in this experiment was PGM-1.
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Figure S4. Time-course of autogamy in PGM and ND7-silenced 51∆A cells (same experiment as in Figures 3 and 4). A. Progression of autogamy in the two cultures (see legend to Figure S2 for details). In the ND7-silencing control, autogamy proceeded through the first post-autogamy cell division (see black bars in histograms). This was not observed reproducibly in all control time-courses (see Figures S2 or S5A) and is probably due to re-growth of residual bacteria at later time-points in this particular RNAi experiment. B. Northern blot validation of PGM silencing. The PGM probe used for hybridization is shown in Figure 2A. For quantification, the amount of full-size PGM mRNA was normalized relative to the 17S rRNA signal. Figure S5. Inhibition of DNA rearrangements at the G locus in PGM-silenced cells. A. Southern blot detection of excised and non-excised forms for IES 51G4404 during a time-course of autogamy in strain 51new silenced for ND7 or PGM (PGM-1 silencing construct). Hybridization of PstI-restricted genomic DNA was performed with PCR probe Gmac (grey line, see legend to Figure 3A). Autogamy stages at each time point are displayed on top (see legend to Figure S2 for details). Time points are in hours, except for D3 and D4 which refer to days of starvation (t = 0 and t = 10 were on day 1; t = 24 was on day 2). B. Southern blot detection of non-fragmented germline chromosomes amplified in the developing MACs (upper band) and of normal MAC telomeres (lower molecular weight smear) at the G51 locus during the same time-course experiment as in A. PstI-restricted genomic DNAs were probed with subtelomeric probe tel51G (grey line), a 505-bp fragment amplified by PCR (Table S1). The structure of the two hybridizing species is shown on the right (top: non-fragmented PstI restriction fragment, bottom: telomere addition region downstream of G). C. Analysis of germline DNA elimination downstream of the G gene in the 51∆A cell line. PstI-restricted genomic DNAs were probed first with probe tel51G, then with probe Tnp, a 2029-bp PCR fragment (Table S1) from a transposon located in the germline sequences downstream of the G gene (see diagram in B). The band carrying the G gene and its downstream sequences is indicated by an arrowhead. Other germline copies of the transposon are revealed with the Tnp probe and their elimination from the developing MACs is also blocked in PGM-silenced cells. Note that the timing of chromosome fragmentation is similar for the ND7 time-course experiments shown in B (51new) and C (51∆A), although autogamy was blocked before the first post-autogamy cell division in B and proceeded through the first division in C (see histograms on top of each figure). Figure S6. IES retention in PGM-silenced cells. A. Analysis of IES sm19-576 located in the SM19 η tubulin gene region (Ruiz et al. 2000). PCR primers were sm19-1 and sm19-4 (Table S1). PCR products were separated on 3% NuSieve gels in 1X Tris Borate EDTA and visualized by ethidium bromide staining. The MAC locus containing the rearranged SM19 gene is fully amplified in the parental MAC. Because of the high ploidy of parental MAC relative to new developing MACs at the time when IES excision takes place, only the excised (IES-) form is detected throughout autogamy in the ND7 control (left). In PGM-silenced cells, inhibition of IES excision results in accumulation of the non-excised (IES+) form (right). An additional PCR product (H) was shown to be a heteroduplex of IES- and IES+ strands, formed in vitro during the PCR. B. Analysis of IES 51A1835 excision in the 51∆A cell line. PCR primers were 51A1835-5’ and 51A1835-3’(3) (Table S1). Parental vegetative cells carry a macronuclear deletion of the A gene: only the MIC (IES+) form is detected in vegetative cells (V) and at the t = 0 time-
Baudry - Sup. Info. - 4
point. In the ND7 control (left panel), the newly formed excision junctions (lower band) can readily be amplified by PCR while none is detected in PGM-silenced cells (right panel). SUPPLEMENTARY REFERENCES Aury, JM, Jaillon, O, Duret, L, Noel, B, Jubin, C, Porcel, BM, Segurens, B, Daubin, V,
Anthouard, V, Aiach, N et al. 2006. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444(7116): 171-178.
Garnier, O, Serrano, V, Duharcourt, S, and Meyer, E. 2004. RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia. Mol Cell Biol 24(17): 7370-7379.
Nowacki, M, Zagorski-Ostoja, W, and Meyer, E. 2005. Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia. Curr Biol 15(18): 1616-1628.
Ruiz, F, Krzywicka, A, Klotz, C, Keller, A, Cohen, J, Koll, F, Balavoine, G, and Beisson, J. 2000. The SM19 gene, required for duplication of basal bodies in Paramecium, encodes a novel tubulin, eta-tubulin. Curr Biol 10(22): 1451-1454.
Sarkar, A, Sim, C, Hong, YS, Hogan, JR, Fraser, MJ, Robertson, HM, and Collins, FH. 2003. Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences. Mol Genet Genomics 270(2): 173-180. Epub 2003 Aug 2029.
Skouri, F and Cohen, J. 1997. Genetic approach to regulated exocytosis using functional complementation in Paramecium: identification of the ND7 gene required for membrane fusion. Mol Biol Cell 8(6): 1063-1071.
Timmons, L and Fire, A. 1998. Specific interference by ingested dsRNA. Nature 395(6705): 854.
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Figure S1 : Alignment of piggyBac transposases from Paramecium tetraurelia and other organisms Paramecium 1 -----------------MFYNDEEEEDWGDKNEKDAEDEFQDLNPSEHKKRIPNNRPKLQPKPQKKKKEVQECEFAIDDP Tetrahymena1 1 ------------------MYDDGTIKKLGQKRTYEGQPEQYIVKTKKNDVYLVEKSKK------------------VRKA Tetrahymena2 1 YTSNIIIELILLIRNKSIQFEQYEIVSSEINLISTENHQNSVILTQIEEEFLRQN--LLQKEMSSLRVSSKNQENICQNQ Ciona 1 ------------------MFSATAAAEILMDA----GSDLSDLESDHDE------------------------------- Anopheles 1 -----------------MFISDKESTCQGPGRKHTYVADI-GTGPSKET---------------------------CRAS Takifugu 1 ---------------MAKRNSAQRALELILEDREVFDDDVEGEDSEEEVSEFEDH---------------------ISEN Trichoplus 1 ---------------MGSSLDDEHILSALLQS----DDELVGEDSDSEI---SDH---------------------VSED Bombyx 1 ---------------MDIERQEERIRAMLEEE----LSDYSDESSSEDE---TDH---------------------CSEH Heliothis 1 -----------MSKMASKRLSNTQIVDVLEDD-----EECIIDSPDEDE---------------------------VDAE Paramecium 64 IKRRQLANYQHVPVRLEAGEKMNINVQYDDGQVRKFGQQKAFEEKLIPISKSGTFIYEASTGKLERKNPGRP-------- Tetrahymena1 45 RKR----------------------KQFQEGED-----------AKKPLTEKEV---EKRWTEFSKENKMRQ-------- Tetrahymena2 79 TSKMKKILVNSLKNNQNNDEEIYYSDESETNQARRQKE----NDNSLPQDNHEN---QQNFNTQQQKKQNKQLVSFTQLS Ciona 28 --------------------------DDDVGS------------FIVPNVADIS---DSDSESSDDSLPPPP-------- Anopheles 36 STRQA--------------------TDSNAGRTP----------RVVPAKSKKD---QVDIQSKEAAKTNRT-------- Takifugu 45 SE-----------------------SDSDSEEDDEI--------EHQPVPKQRR---ATGPGRQQPSPGPEQ-------- Trichoplus 38 DVQ----------------------SDTEEAFIDEVH-------EVQPTSSGSEILDEQNVIEQPGSSLASN-------- Bombyx 38 EVN----------------------YDTEEERIDS---------VDVPSNSRQE---EANAIIANESDSDPD-------- Heliothis 38 VIQ----------------------SDHNSE---------------SQESANEI---DSEWSDTDNEPLSRL-------- Paramecium 136 REDTRSLFDDPKLQKMRNNYVPKLLQRITTNNTKEVDEEEGVQDPGVSWK--PVKSVHEVP--ESFYMRQVFDKSA---- Tetrahymena1 81 ---------------------------------QHKEDDLI----DDQWR--RIYSVDQVP--KSFYMKTKFD------- Tetrahymena2 152 SYNSIYSESETSQHEDQIIQNNQDLNSSIEESGEEEQKQDLKRNKGNFSKKLNLNDVNFKLCSDNKLLKET--LINIDLT Ciona 59 -------------------------------------------PPANVWK-------SSITQQSLNSFLGQ--------- Anopheles 75 ---------------------------LEGSLGKLAVGSVVEAPDGTQWKVVEVNNANGGIVAEVNVIRDV--------- Takifugu 83 -------------------------------ASQHGGEIWMSKNSEIEWS---SRPRKGPPHKAANVIRMQ--------- Trichoplus 81 -------------------------------RILTLPQRTIRGKNKHCWS--TSKSTRRSRVSALNIVRSQ--------- Bombyx 76 ------------------DDLPLSLVRQRASASRQVSGPFYTSKDGTKWY--KNCQRPNVRLRSENIVTEQ--------- Heliothis 70 -------------------------------ASEDSDNFYFSKNKCTKWA--KEPPRTSVRVRRHNIMRET--------- Paramecium 208 -SGPRSIDKS--KIKSEYDAFRLFFDNDIYNTIIKHTRERYQQKVEEQIYSYIHGMVHMGIRAKKPTLMQWEFTEYELEA Tetrahymena1 113 -KDLNRDFIPYNVPHSPYHLFRMFFDDRIYKLIIEETNRYKHQKYQEALL--------NLPPDKTLPKEAMDIDSIDLDA Tetrahymena2 230 YRKPQVKNIP--LESTPVQIFQKLWSDEIWKLITDETNKYSKQSFDLNQY--------NLDSQSLKKQKICFFSQDQIKR Ciona 80 -HGPSSHACV-SNDDSPLEYFRLIFTDELLTLVVEETNRYAAQYLQKTSL-----------SSKSRAHKWKPVDAEELSL Anopheles 119 -PGPSAYAKRYVVAGAVSSSWRLIIDNGILNQIRKCTE-----IEARRVL----------------QTKNWTLSFAELEA Takifugu 120 -PGPDMAVTHTRDIKS----FELFIPDSIQEIILDCTN-----LEGRRVF----------------GERWKELDQTQLHA Trichoplus 119 -RGPTRMCRN---IYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMT----------------GATFRDTNEDEIYA Bombyx 127 -AQVKNIARD---ASTEYECWNIFVTSDMLQEILTHTNSSIRHRQTKTAA---------ENSSAETSFYMQETTLCELKA Heliothis 108 -PGPKGRAKE---IQTISEAFFCMFSMDTVNLVLQQTNDYIKSIQEKFQR----------------ERDCKVLEYEELLA Paramecium 285 YFAVQIFFGIVRLS--NQRDYW-KSSARQKPIKKAETGRRKLRELAQEKMDRYAHWVTQRMSSIVSYEKFKTIRNCLNIS Tetrahymena1 184 YLSILIFMGVQRMK--SVKDYW-KRKNYINSE----------------------------IACIMKYSRFEQIDKHLHLA Tetrahymena2 300 FIICEILMGIQRLP--SFSDYF-SSDPLLSGG----------------------------LNRILGRENYQLLTRYLHIS Ciona 147 FLGLTLLTGVVHKRG-KLESYWIKNSMIETPY----------------------------FGKCMSRNRYQAITGFLHFN Anopheles 177 FLAILYIRGATESKGMEIDLMW-SEK-YGLPF----------------------------CKNVMSRNRFREIMKFLRFD Takifugu 174 YFGVLILAGVFRSKGESAESLW-DAE-TGREI----------------------------FRATMSLENFHIISRIIRFD Trichoplus 179 FFGILVMTAVRKDNHMSTDDLF-DRS--LSMV----------------------------YVSVMSRDRFDFLIRCLRMD Bombyx 194 LIALLYLAGLIKSNRQSLKDLW-RTDGTGVDI----------------------------FRTTMSLQRFQFLQNNIRFD Heliothis 168 YLGLLYMSGVLRSSHLNFKDLW-ATDGTGIEF----------------------------FQNTMSFNRFLFISRCVRFD Paramecium 362 GAEAL-KLKGRDPIWKIRDFLNQMNMRFAKYYYPGEFITIDEGMIPFAGKVQFKVYNPDKPTKWGIKEYLLCDASNTYTF Tetrahymena1 233 DNEDP--KIRSDPIGKVRQYMDYLNENFKKYYYPGEFLAIDEGMIPFNGKVAFKVYNPDKPDKFGIKEYVCCDSQNAYTL Tetrahymena2 349 DNQSR--MVHVDDHTKFKQFQSILNRNYQQFYVPSNYLAIDEGIIPFKGKTKFKVYCPQKPVKFGIKEYLFCD-YSGYTL Ciona 198 DNEKLAENIDNDKLYKVRPVYDLIVARWKALYNLGEHISIDEGMMKWRGRLGFRVYNKDKPIKYGIKSYILADSHSHYCW Anopheles 227 EKSTRSQRLQTDKFALISDVFSRFVSNCQTNYVPGPHISVDEQLFPSKTRCPFTQFMASKPDKYGQKYWMAVDVDSKYVV Takifugu 224 NZDDRPARWQRDKLGVIRTVWDKWVRRLPLLYNPGPNVTIDEQLMPFRGRCPFLQYLPSKPAKNGIKIWAACDATSSYAW Trichoplus 228 DKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMI Bombyx 245 DKSTRDERKQTDNMAAFRSIFDQFVQCCQNAYSPSEFLTIDEMLLSFRGRCLFRVYIPNKPAKYGIKILALVDAKNFYVV Heliothis 219 DKNTKSERLKTDKLAAVREFTDLMNNNFINNYCASENVTLDEQLPAFRGRFSGVVYMPNKPTKYGIKHYALVDSATFYLL Paramecium 441 QLRLYHGQTMWNNDFKQTMFVNEEDTQHRTMELVLQMCKDYEH-KAHKVVMDNYYSSWMLFRELRN--RGIGAVGTIRHN Tetrahymena1 311 ESQLYYGNH------ENEEFPEITLSK--TNEVVMNLLKDYEG-RFHKVVMDNYYNSPTLFYLMKQ--KSFGALGTMRIG Tetrahymena2 426 NLIIHSPHE--KQNIRDIYQPQTLQTQ----DIVNELIKDYQPLSGSILIMDNYYNSLNLIHDLNQ--QNIGVLGTVRPD Ciona 278 NLDMYHRVQ-------------KTLKE----TVSQILTSKCHF-LWHSLYMDNFYNSVSMSQMLLA--FQIHSVGTLRSN Anopheles 307 NIIPYLGKN------DERPAEERLGDF-----VVKKLVDPYLN-RGRNVTCDNFFTSLELAKFLKS--KKTSLVGTINKA Takifugu 304 NLQVYTGK-------PDGGAPEKNPRN-------ESCPRHVSGTQWTQHHMRHFFTSHKLGQELLK--RKLTIVGTIRKN Trichoplus 308 NGMPYLGRGTQTNGVPLGEY------------YVKELSKPVHG-SCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSN Bombyx 325 NLEVYAGKQ------PSGPYAVSNRPF----EVVERLIQPVAR-SHRNVTFDNWFTGYELMLHLLNE-YRLTSVGTVRKN Heliothis 299 KFEIYAGVQ------PEGPYRMPNDTV----SLVKRMTEPIWG-TGRNVTMDNWFTSVPLANILLKD-HQLTMVGTIRKN Paramecium 518 RTGLTKK---DLTSKHFQQIYNQYHYAYYLNQSNELMLMYFQGT-----SEKEIALISNFLDNSLNEQHMWDI-----SK Tetrahymena1 380 RLKLPPYL--LSQIMEVHQNKVLSY-----IQGDINLAIFFRGT-----NDKEICLMSNFIDQKKVKDDFWRVLAYVKED Tetrahymena2 498 RMNFTEEQKKMMKIQNFNKGETKSL-----TKDNIHIFLWRDK-------DKLVKMITNFLDNRKIVKSMKKT------- Ciona 338 RGEPREI---RTPPNQMKKGDIIAR-----QNQSVTVLAWKDKRVVKAISTKHDASVTTITRRQRRGGEXESV------- Anopheles 373 RREVPIC---VKKVKEKLYFTKAFK------SDDTTLTVYQGKT------KKNVVLLSSMHRDIRTGND--KK------- Takifugu 368 RSELPPQ---LLTSKNRPVKSSQFA-----YTADTSLVSYVPKK------GKNVVLMSTLHRDGRMCDQ--EH------- Trichoplus 375 KREIPEV---LKNSRSRPVGTSMFC-----FDGPLTLVSYKPKP------AKMVYLLSSCDEDASINESTG--------- Bombyx 393 KRQIPES---FIRT-DRQPNSSVFG-----FQKDITLVSYAPKK------NKVVVVMSTMHHDNSIDESTGEK------- Heliothis 367 KPEIPTC---FQPKRTRTEHSSLFG-----FQEDVTLCSYVPKK------SKAVLLISSMHNDNNIVES--EK-------
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Paramecium 585 QHYYVPHLKAPYMMYVYNKYKGGVDRRNSYVVKYRSRFPAKKWWQSVFERLFETAILNAYLIFRSYNPESSYRNKGQMRD Tetrahymena1 448 RDIFK---EQNLQSAYYNKYKGGVDRRNSYLAAYRNCRKNIKWYRPVFYRMLDNAIVNAFILY---NFNLSPKYKISQKE Tetrahymena2 559 ----GQIKTIPLMVDIYNKYAHSVDKRNQICQNYRIHKRSQKWWKCVFYRLLDTTLCNAYIIYKILNE--GKKSLLTHKD Ciona 403 --------EKPVCIADYNLHMSGVDQVDQMISYYPCHRKSLKWTKKVFFYFMTISVHNAYILYKS-------KSSAKSCK Anopheles 429 --------SKPETVAFYNSTKYGVDVVDQMCRKYSLKSASRRWSVHSFFNILDLAGINAWVLYKELTK-----ENISRRD Takifugu 425 --------HKPEIIMDYNATKGGVDNMDKLVTAYSCKRRTLRWPLVIFFDMLDISAYNAFVIWMALNPEWKRVKLQKRRL Trichoplus 432 ---------KPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSH-NVSSKGEKVQSRKK Bombyx 451 --------QKPEMITFYNSTKAGVDVVDELCANYNVSRNSKRWPMTLFYGVLNMAAINACIIYRT-NK----NVTIKRTE Heliothis 424 --------KKPEIILYYNSTKGGVDTNDQMCANYNVGRRTKRWPMVIFYHLLNVAGINAYVIFKN-----KIDHGISRRE Paramecium 665 FRINLMYQFAERYKSYEHQ-QEENGKNRFSYFAKIQ-----------------------PHTFIE-GEEIVKCSEC---- Tetrahymena1 522 FRIQLFKELACRYSPSKSQ--HAIKVQQFMKDKSAEVDKIPQYFEKAMKLHIMKNCDYSAHILVRSRTKKKSCIEC---- Tetrahymena2 633 FRIKIVEELIQSLNESTAQLTKSLSHNRGIIYSLIDI----------NKVRKQGVQQEIIAHFLERQNQVGLCSLC---- Ciona 468 TLYSFILTLVSQLCQQDRL-QPQID-----DENLAG-----------PPPKSPRIDS--TKRLKGGFKKHVIALYPPTKK Anopheles 496 FLFKLGEELAEEYVENKSA-NANLP-----------------------------MTN--AGGSRRRYKVQTSCH------ Takifugu 497 FLEDLGKALVRPQIERRKH-IPRTPASAAMVRRIQKENAGALSTQPTEPQSAEPEVN--VZXVVNSSNKKKRCEVCGPKM Trichoplus 502 FMRNLYMSLTSSFMRKRLE-APTLK--RYLRDNISNI----------LPNEVPGTSD--DSTEEPVMKKRTYCTYCPSKI Bombyx 518 FIRSLGLSMIYEHLHSRNK-KKNIP--TYLRQRIEKQ----------LGEPSPRHVN--VPG---RYVR---CQDCPYKK Heliothis 491 FLKHLAVDLVKVHQQTRSN-IPQLP--RAVQKRLKRN----------AEVQDPGSTS--RGGPSTSYKR---CHICPRSK Paramecium 716 GNETKVFCQECTILKAEVVGLCHEKDTIKCQRFHEFMDFE---------------------------------------- Tetrahymena1 596 KQLTLFSCSTCSNMFKMRIPLC-QSGFNQCYDFHASKTYEEVVEDMFTKRRKDGRKLDIEIKKKKKKLVKLSDVYDSILK Tetrahymena2 699 KQATFFTCESC-NYGNKKIALC-PV---NCHKEHMKKVYN---------------------------------------- Ciona 529 KAAAQRPCRAC------MKNGCRKDTILL--------------------------------------------------- Anopheles 538 EGKSANECFTC------NRPVCKKCTKRISYVCVSCEPGSDEAM------------------------------------ Takifugu 574 DRKTQYTCIKC------KKYICNTHTVKLWPSCVV--------------------------------------------- Trichoplus 567 RRKANASCKKC------KKVICREHNIDMCQSCF---------------------------------------------- Bombyx 577 DRKTKRSCNAC------AKPICMEHAKFLCENCAELDSSL---------------------------------------- Heliothis 553 DKKIRFMCAKC------HHHICHDHSTMICDKCID--------------------------------------------- Paramecium 756 ---------------LDKNKEVIDKRKGKDPYKPNFLEKLNQRTNAKGNQSSQKKESPLVNLLNKINDQIKQEARVKQEV Tetrahymena1 675 PKQSNQNPIQVKTKQILQKSVTIDHYTSNKAFLEDDMYSENQPNSTELNDQSTRMKTNQIMSSSNSNSIQGSKKKKFKVI Tetrahymena2 734 -------------------------------------------------------------------------------- Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis -------------------------------------------------------------------------------- Paramecium 821 KREDNTNKQTTYIIPEVKSEDESSTDSDIYIQRTTNQRLIEIHQKIEQMSMCSEEFLNGSVANSQENFAERDENDQFNDN Tetrahymena1 755 KKEKNLKTQKTKSCDAIFSSEKIKKKYTDEINEFFDSKASIRLTNFFQESQRLQNLLKESQENLSKKFVVLDQTSDEDFK Tetrahymena2 734 -------------------------------------------------------------------------------- Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis -------------------------------------------------------------------------------- Paramecium 901 NGNDDNQFQFPQQRAQ------------------QIDD-----------------------------------------D Tetrahymena1 835 NNNTDEDLQWINTTLEKINQKKIEPNEKLSPGLGIINKKQEAEEVIEVLSSSSTLNEEKVRTKYQEIPFTFGSILEVEQE Tetrahymena2 734 ----------------------------------LIDK------------------------------------------ Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis -------------------------------------------------------------------------------- Paramecium 922 DEQRNSKNEEQQKDFIKKMMEFADDEGSENEEIQ----YPEDEADHFYQ------------------------------- Tetrahymena1 915 KEESDDEVAKEYKKVSKRIEEFNEDSIHSKQQLEDSNFFPQKAGVIFYSMSELDKKKEQVISDQNKEQNQIIVKQEHRSD Tetrahymena2 -------------------------------------------------------------------------------- Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis -------------------------------------------------------------------------------- Paramecium 967 ---------------------------QLLQQEEQAIKYQQKKQLQQQLEEESERSNISHKSKKQQKLEQKFIETS---- Tetrahymena1 995 QKKKNSYPLQLYDCENVLNNAIDFSDYSFSSSDEEQFVYNKNKNKQQELNHESDTNNPQLQYKGKIKNNKFKIDESFDES Tetrahymena2 -------------------------------------------------------------------------------- Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis --------------------------------------------------------------------------------
Baudry - Sup. Info. - 7
Paramecium 1016 --------------------------------------------------MRGIKQSQIQSNSEIGQDLQKIISASQDLN Tetrahymena1 1075 NLKIERPFDLIPFTFQNDAMQTSQNFVVIDDDSDVEFQNINSQIINQSSIISESQKLEFEGFIDNPQPTKKRIISCQDMK Tetrahymena2 -------------------------------------------------------------------------------- Ciona -------------------------------------------------------------------------------- Anopheles -------------------------------------------------------------------------------- Takifugu -------------------------------------------------------------------------------- Trichoplus -------------------------------------------------------------------------------- Bombyx -------------------------------------------------------------------------------- Heliothis -------------------------------------------------------------------------------- Paramecium 1046 Q--ISKQITESKGDNQNSQSDQ------------------ Tetrahymena1 1155 RKIIIKKNKEPKNDNQNIIYINHQRFKIVKLKTTIQIDSD Tetrahymena2 ---------------------------------------- Ciona ---------------------------------------- Anopheles ---------------------------------------- Takifugu ---------------------------------------- Trichoplus ---------------------------------------- Bombyx ---------------------------------------- Heliothis ----------------------------------------
Baudry - Sup. Info. - 8
51new in K. pneumoniae
V S F A C
100
80
60
40
20
0
%
Figure S2: Progression of autogamy in a culture of 51new starved in K. pneumoniae
V -6 -3 0 5 10 20 30 4015
Baudry - Sup. Info. - 9
Figure S3: Progression of autogamy in PGM and ND7 silenced cells
PGM
ND7
a b c d
e f g h
Day 1 Day 2 Day 3 Day 4
Baudry - Sup. Info. - 10
full sizePGM
mRNA
17S
VkV 0 5 1015 203040 V 0 5 10 15 203040
B ND7 PGM
arbi
trary
units
V 0 5 1015 203040 V 0 5 10 15 203040
ND7 PGM100%
50%
0%
AVSFAC
Figure S4: Time-course of autogamy in PGM and ND7-silenced 51∆A cellsBaudry - Sup. Info. - 11
V 0 5 10 15 20 30 40 V 0 5 10 15 20 30 40
PGMND7100%
50%
0%
tel5
1Gpr
obe
Tnp
prob
e
A
12421020
bp
IES
Gmac
0 10 25 D3 D4VK 0 10 25 D3 D4
PGMND7100%
50%
0%
VSFAC
tel51G
C
0 10 25 D3 D4VK 0 10 25 D3 D4
B G
G
Figure S5: Inhibition of DNA rearrangements at the G locus in PGM-silenced cells
Tnp
Baudry - Sup. Info. - 12
PGMND7
V 0 5 10 15 20 30 40
MAC development
mic
MAC
parentalcell
sexualprogeny
bp
396298
506H
A
Figure S6: IES retention in PGM-silenced cells
V 0 5 10 15 20 30 40
PGM
V 0 5 10 15 20 30 40
ND7
MAC development
∆mic
MAC
parentalcell
sexualprogeny
∆
bp396298220
B
V 0 5 10 15 20 30 40
Baudry - Sup. Info. - 13
Tabl
e S1
. Olig
onuc
leot
ides
use
d in
this
stu
dy
Nam
eS
eque
nce
(5' t
o 3'
)ap
plic
atio
nC
laI-X
hoI l
inke
r UC
GA
TATA
AA
TTTA
TATA
ATT
ATC
ATG
AC
TTTT
TTA
TTG
AG
TTG
CC
laI-X
hoI l
inke
rC
laI-X
hoI l
inke
r RTC
GA
GC
AA
CTC
AA
TAA
AA
AA
GTC
ATG
AA
TAA
TTTT
AA
AA
TTTA
TAT
Cla
I-Xho
I lin
ker
PB
l49-
09S
peI
GG
AC
TAG
TTC
TTTA
AA
TGA
AC
AA
CA
TATG
TGG
Gfe
edin
g in
sert
PG
M-1
PB
l49-
10S
peI
GG
AC
TAG
TCC
GTT
TTC
CTC
TTG
TTG
ATG
TTC
ATA
Gfe
edin
g in
sert
PG
M-1
PB
l49-
01TA
GA
GG
CA
TTG
GA
GC
AG
TGG
GA
AC
TATT
AG
PG
M h
ybrid
izat
ion
prob
eP
Bl4
9-02
AA
GTT
TCTC
TAA
GA
AA
TTTG
GC
TTG
TATG
GP
GM
hyb
ridiz
atio
n pr
obe
Spo
11-0
2Spe
IG
GA
CTA
GTA
TGA
AC
TTA
CC
ATT
ATT
GC
ATG
AA
GA
AA
TGfe
edin
g in
sert
PtS
PO
11-1
Spo
11-0
8Spe
IG
GA
CTA
GTG
CA
TTTC
ATT
TATC
AA
GTG
AA
AA
TAC
AC
AG
feed
ing
inse
rt P
tSP
O11
-1S
po11
-07S
peI
GG
AC
TAG
TAA
AA
CA
CTG
TCTT
GA
TTA
CA
GG
AA
AA
GG
feed
ing
inse
rt P
tSP
O11
-2S
po11
-10S
peI
GG
AC
TAG
TAC
ATA
ATT
TGTA
ATA
CG
AA
TTC
GG
ATA
GG
feed
ing
inse
rt P
tSP
O11
-2S
po11
-02
ATG
AA
CTT
AC
CA
TTA
TTG
CA
TGA
AG
AA
ATG
PtS
PO
11 h
ybrid
izat
ion
prob
eS
po11
-03
TCA
ATA
TTA
TAG
ATC
AC
AA
TCTT
AA
CG
TTA
CP
tSP
O11
hyb
ridiz
atio
n pr
obe
51G
20m
ac_u
pG
CTG
TTA
GG
AA
GC
CA
AA
GC
TGG
TTG
CA
AA
GG
mac
hyb
ridiz
atio
n pr
obe
51G
21m
ac_R
ATC
AC
AA
GTC
TTTC
GTA
CG
CA
TGA
TGTT
CC
Gm
ac h
ybrid
izat
ion
prob
e17
Sex
t_N
coI
AC
CC
GTG
AC
TGC
CA
TGG
TAG
TCC
AA
TAC
A17
S rD
NA
ybr
idiz
atio
n pr
obe
51A
2591
-03
ATT
CTA
AA
ATT
AA
CA
AA
AA
TAC
ATT
ATT
TTA
CIE
S 5
1A25
91 h
ybrid
izat
ion
prob
e51
A25
91-0
4TT
GA
GA
AG
TTA
AG
AA
ATA
AA
ATG
ATG
GIE
S 5
1A25
91 h
ybrid
izat
ion
prob
ete
l51G
-01u
pG
ATG
TTA
GA
CA
TATT
AA
GA
GTT
TGTT
AG
AC
tel5
1G h
ybrid
izat
ion
prob
ete
l51G
-02d
own
CTT
AA
GA
ATA
AA
GC
TATG
CC
AA
AA
GG
AA
GG
tel5
1G h
ybrid
izat
ion
prob
eZF
1TC
TAG
AA
ATC
ATG
AG
AG
CG
AA
AG
AC
AA
TCT
Tnp
hybr
idiz
atio
n pr
obe
ZF15
AA
TGA
ATT
CA
AA
ATA
TCTC
GA
GC
AG
ATG
CT
Tnp
hybr
idiz
atio
n pr
obe
sm19
-1C
TAA
GA
AA
ATG
AG
CA
AA
TAG
TGG
AA
TAA
AT
PC
R a
roun
d IE
S s
m19
-576
sm19
-4G
AC
AA
GA
TCC
TATA
TATT
CA
TTTA
CA
TTTG
PC
R a
roun
d IE
S s
m19
-576
51A
1835
-5'
TAA
TGTA
TTG
ATA
AG
GC
TTG
CTC
TAC
AG
CC
PC
R a
roun
d IE
S 5
1A18
3551
A18
35-3
'(3)
GTA
GTA
CA
AG
ATT
TTTC
GA
CA
CA
AG
TTG
AG
PC
R a
roun
d IE
S 5
1A18
35
Baudry - Sup. Info. - 14
Table S2: Details of all survival tests following autogamy
exp. n° progeny PGM-1 PGM-2 PtSPO11-1 PtSPO11-2 ND7 Klebsiella1 wt 4 0 29 30
sick 3 1 0 0dead 23 29 1 0
2 wt 0 0 30sick 0 0 0dead 30 30 0
3 wt 0 30sick 0 0dead 30 0
4 wt 0 1 48 47sick 5 2 0 0dead 19 21 0 1
5 wt 3 17 23sick 0 0 1dead 21 7 0
6 wt 2 1 1 29 29sick 2 2 6 0 0dead 26 27 23 1 1
7 wt 3 0 5 30 30sick 1 1 6 0 0dead 26 29 19 0 0
8 wt 0 5 28 29sick 2 3 0 0dead 28 22 2 1
9 wt 0 1 28 29sick 1 4 0 0dead 29 25 2 1
10 wt 0 30sick 0 0dead 30 0
11 wt 0 12sick 0 0dead 12 0
Total wt 4 5 8 9 311 217sick 3 12 12 12 0 1dead 125 187 124 63 13 4total 132 204 144 84 324 222
Baudry - Sup. Info. - 15
