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Supplementary Material Supplementary Material for “The abundant and essential HU proteins in Deinococcus deserti and Deinococcus radiodurans are translated from leaderless mRNA” by Claire Bouthier de la Tour, Laurence Blanchard, Rémi Dulermo, Monika Ludanyi, Alice Devigne, Jean Armengaud, Suzanne Sommer, Arjan de Groot Contains: Table S1. Bacterial strains Table S2. Plasmids Table S3. Primers Fig. S1. Diagnostic PCR to verify HU gene deletion mutants of D. deserti. Fig. S2. Main differences in DdHU3 compared to residues conserved in DrHU, DdHU1 and DdHU2. 1 Table S1. Bacterial strains Strain Genotype or relevant characteristics Source or reference E. coli TOP10 F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Invitrogen ΔlacX74 recA1 araD139 Δ(ara, leu)7697 galU galK rpsL (StrR) endA1 nupG D. deserti RD19 As wild type strain VCD115 but streptomycin resistant (Vujicic-Zagar et (StrR) al., 2009) RD74 ΔDeide_2p01940Ωkan This work DelHU2 ΔDeide_3p00060Ωcat This work DelHU3 ΔDeide_00200Ωcat This work DelHU1HU3 ΔDeide_2p01940Ωkan ΔDeide_00200Ωcat This work DelHU2HU3 ΔDeide_3p00060Ωkan ΔDeide_00200Ωcat This work R1 ATCC 13939 Laboratory stock GY13320 DR_A0065::spaΩcat (Nguyen et al., D. radiodurans 2009) GY13792 Non-homogenotized ΔDR_A0065Ωcat (Nguyen et al., 2009) GY13918 R1/p11559 Laboratory stock GY13909 R1/p14001 Laboratory stock GY13344 R1/p13410 (DrHU(M1)) This work GY15954 As GY13344 but ΔDR_A0065Ωcat This work GY13368 R1/p13419 (DrHU(M2)) This work GY13370 As GY13368 but non-homogenotized ΔDR_A0065Ωcat This work GY15940 R1/p13434 (DrHU(M3)) This work GY15956 As GY15940 but non-homogenotized ΔDR_A0065Ωcat This work GY15942 R1/p13436 (DrHU(M1stop)) This work GY15952 As GY15942 but ΔDR_A0065Ωcat This work GY15944 R1/p13438 (DrHU(M1stop+mutM2)) This work GY15964 As GY15944 but non-homogenotized ΔDR_A0065Ωcat This work 2 GY13357 R1/p13414 (DdHU1(M1)) This work GY15955 As GY13357 but ΔDR_A0065Ωcat This work GY13358 R1/p13415 (DdHU1(M2)) This work GY13362 As GY13358 but non-homogenotized ΔDR_A0065Ωcat This work GY15941 R1/p13435 (DdHU1(M3)) This work GY15957 As GY15941 but ΔDR_A0065Ωcat This work GY15943 R1/p13437 (DdHU1(M1stop)) This work GY15953 As GY15943 but ΔDR_A0065Ωcat This work GY15949 R1/p13439 (DdHU1(M1stop+mutM2)) This work GY15959 As GY15949 but non-homogenotized ΔDR_A0065Ωcat This work GY15945 R1/p13440 (DdHU2) This work GY15958 As GY15945 but ΔDR_A0065Ωcat This work GY15947 R1/p13442 (DdHU3) This work GY15962 As GY15947 but non-homogenotized ΔDR_A0065Ωcat This work GY13345 R1/p13411 (DrHU-SPA(M1)) This work GY13369 R1/p13420 (DrHU-SPA(M2)) This work GY15969 R1/p13448 (DrHU-SPA(M3)) This work GY13359 R1/p13416 (DdHU1-SPA(M1)) This work GY13360 R1/p13417 (DdHU1-SPA(M2)) This work GY15970 R1/p13449 (DdHU1-SPA(M3)) This work GY15981 R1/p13451 (DdHU2-SPA) This work GY15983 R1/p13453 (DdHU3-SPA) This work 3 Table S2. Plasmids Plasmid Description Source or reference pCR4Blunt-TOPO Cloning vector, AmpR, KanR Invitrogen pRD0 pUC19 with Ptuf::kan (BamHI-PstI fragment) (Dulermo et al., 2009) pSF0kan pUC19 with Ptuf::kan (BamHI-XbaI Laboratory stock fragment) pSF0cat pUC19 with DrPtuf::cat (BamHI-XbaI Laboratory stock fragment) p11559 Expression vector; Pspac, PtufA::lacI, SpcR (Mennecier et al., 2004) p14001 p11559 with spa cassette (DraI-XhoI Laboratory stock fragment); to express proteins with C-terminal SPA-tag p13410 p11559 with DR_A0065(M1) This work p13419 p11559 with DR_A0065(M2) This work p13434 p11559 with DR_A0065(M3) This work p13436 p11559 with DR_A0065(M1stop) This work p13438 p11559 with DR_A0065(M1stop+mutM2) This work p13414 p11559 with Deide_2p01940(M1) This work p13415 p11559 with Deide_2p01940(M2) This work p13435 p11559 with Deide_2p01940(M3) This work p13437 p11559 with Deide_2p01940(M1stop) This work p13439 p11559 with This work Deide_2p01940(M1stop+mutM2) p13440 p11559 with Deide_3p00060 This work p13442 p11559 with Deide_00200 This work p13411 p11559 with DR_A0065(M1)::spa This work p13420 p14001 with DR_A0065(M2) This work p13448 p14001 with DR_A0065(M3) This work p13416 p14001 with Deide_2p01940(M1) This work p13417 p14001 with Deide_2p01940(M2) This work p13449 p14001 with Deide_2p01940(M3) This work p13451 p14001 with Deide_3p00060 This work p13453 p14001 with Deide_00200 This work 4 Table S3. Primers (A) Primers for cloning of genes in p11559 or p14001 Primer sequencea Primer HUNde HUDra HUNde HU14Dra HU18Nde HUDra HU18Nde HUdes3Dra HU26Nde HUDra HU26Nde HUdes3Dra HUdes1Nde HUDra HUdes1Nde HUdes3Dra HUdes2Nde HUDra HUdes2Nde HUdes3Dra HUdes19Nde HUDra HUdes19Nde HUdes3Dra HU29Nde HUDra HUdes22Nde HUDra HU28Nde HUDra HUdes21Nde HUDra Dd3p00060NdeFW2 Dd3p00060DraRV Dd00200NdeFW2 Dd00200DraRV Dd3p00060NdeFW2 Dd3p00060DraRV2 Dd00200NdeFW2 Dd00200DraRV2 a Amplified gene GAGGCACAACATATGCCCCCCTTCACAGCCGCAAA GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGCCCCCCTTCACAGCCGCAAA GGACTTTAAACAAGAGCGCCGGGCCGGGAA GAGGCACAACATATGCTGCTCACCATGACGAAAAA GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGCTGCTCACCATGACGAAAAA GGACTTTAAACAGGTTGCCCTTGAGGGTGC GAGGCACAACATATGACGAAAAAGTCTACCAAGGCCCC GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGACGAAAAAGTCTACCAAGGCCCC GGACTTTAAACAGGTTGCCCTTGAGGGTGC GAGGCACAACATATGTTTCCGACCCCCTTCACAGC GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGTTTCCGACCCCCTTCACAGC GGACTTTAAACAGGTTGCCCTTGAGGGTGC GAGGCACAACATATGCTGCTCACCATGACGAAAAA GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGCTGCTCACCATGACGAAAAA GGACTTTAAACAGGTTGCCCTTGAGGGTGC GAGGCACAACATATGACGAAAAAGTCTGCGAAAGCCCC GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGACGAAAAAGTCTGCGAAAGCCCC GGACTTTAAACAGGTTGCCCTTGAGGGTGC GAGGCACAACATATGTGACCCCCCTTCACAGCCGCAAAGA GAT GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGTGATTCCGACCCCCTTCACAGCGGC GA GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGTGACCCTTCACAGCCGCAAAGAGAT ATGTCTAGCTGCTCACCATG GGACTTTAAATTACAGGTTGCCCTTGAGGG GAGGCACAACATATGTAACCGACCCCCTTCACAGCGGCGA GGGGATATGTCTAGCTGCTCACCAT GGACTTTAAATTACAGGTTGCCCTTGAGGG GGAGCATATGACGAAGAAAAATACAAAAGCGCC GGACTTTAAATTACAGCGTGCCCTTGAGGG GGAGCATATGGCCAAGAGCACCAAGC GGACTTTAAATTACAGGTTGCCTTTGAGGGTAC GGAGCATATGACGAAGAAAAATACAAAAGCGCC GGACTTTAAACAGCGTGCCCTTGAGGGTG GGAGCATATGGCCAAGAGCACCAAGC GGACTTTAAACAGGTTGCCTTTGAGGGTACTG final plasmid DR_A0065 (M1) p13410 DR_A0065(M1)::spa p13411 DR_A0065 (M2) p13419 DR_A0065 (M2) p13420 DR_A0065 (M3) p13434 DR_A0065 (M3) p13448 Deide_2p01940 (M1) p13414 Deide_2p01940 (M1) p13416 Deide_2p01940 (M2) p13415 Deide_2p01940 (M2) p13417 Deide_2p01940 (M3) p13435 Deide_2p01940 (M3) p13449 DR_A0065 (M1stop) p13436 Deide_2p01940 (M1stop) p13437 DR_A0065 (M1stop+mutM2) p13438 Deide_2p01940 (M1stop+mutM2) p13439 Deide_3p00060 p13440 Deide_00200 p13442 Deide_3p00060 p13451 Deide_00200 p13453 Restriction sites are indicated in bold (B) Primers to verify sequence of fragments cloned in p11559 or p14001 Primer p11559for p11559rev HH70 Primer sequence GAAGTTCAGAGCACGTTCCG TTTTCTCCGGCCGTTCGC GAGCAGATCTAGCACAAGAGCGGAAAGATG 5 (C) Primers for amplification of DNA fragments upstream and downstream of D. deserti genes to be deleted Primer sequencea Primer HuP2UpEcoRI HuP2UpBamHI HuDnHindIII5 HuDnHindIII3 Dd3p00060UpEco Dd3p00060UpBam Dd3p00060DnXba Dd3p00060DnHd3 Dd00200UpEco Dd00200UpBam Dd00200DnXba Dd00200DnHd3 a Amplified fragment GAGGAATTCGCCGGTTCCAGTAGCCCTGC CTTGGATCCGAAACAGCGAAGCCCGGCCA GTGAAGCTTGCCTGAGTACCGACCGGACG GTGAAGCTTGTGGCGCTGCTGGACTGGG GCGAATTCTTTACCGAAGCGTCCAGTCT TAGGATCCTCTGGAGCGAAGTTGTGTCT GGTCTAGACAAGGGCACGCTGTAATTCA GAAAGCTTTATCCTCGGTGTATGCCTGC GCGAATTCAGCCGTAGACTCGTACCATG TAGGATCCGGACTTTTGTGACGCTACCC GCTCTAGACCGTTTCAAGGTAGCCAGTA GGAAGCTTATGATGACTGCGGGCTGATA Upstream of Deide_2p01940 Downstream of Deide_2p01940 Upstream of Deide_3p00060 Downstream of Deide_3p00060 Upstream of Deide_00200 Downstream of Deide_00200 Restriction sites are indicated in bold (D) Primers used for diagnostic PCR of D. deserti or D. radiodurans gene deletion mutants Primer HUPXbaI HUGeneHd3 HuP2UpEcoRI HuBegin HuEnd HuDnHindIII3 HuP2UpUp TufBamComp2 KanPstComp HuP2DnDn Dd3p00060for Dd3p00060rev Dd3p00060UPfor Dd3p00060DNrev Dd00200for Dd00200rev Dd00200UPfor Dd00200DNrev A B Primer sequence to check: CATCTAGAGCGGGTGGGTACCGTGGAATC TAAAGCTTGTGACCCCCTGCAAAAGGACGC GAGGAATTCGCCGGTTCCAGTAGCCCTGC CTCTCAGCGGCCACAGCACC CAAGCAGACCGCCGCCCGTA GTGAAGCTTGTGGCGCTGCTGGACTGGG GTGATAAACGCGTTGGCCGTGTCG CTCAGCACGGCAATTACAAGGATCC CTTGACGAGTTCTTCTGACTGC GATGTTCGACCCGGCCGACC GCATTTATGGGGTGTGGTCC CACGCTGACCACTTCTTCAC CCGTTTTCTGGTGTCATGCA CTGTACGTAGCCAGGGTAGG CCCAGCTGATCGAAATGGTC ATCTTCTCACTAGTGCCGGG GTACTCAATCTGCAGGCCAC TAGTGCAGCGCGTTTTACAG TTGAGGCCCTGGCCTTAATG GGTGCTCAATGACGACTTGG Deide_2p01940 gene replacement absence Deide_2p01940 absence Deide_2p01940 double crossover at correct locus (5’ of Deide_2p01940) double crossover at correct locus (3’ of Deide_2p01940) absence Deide_3p00060 Deide_3p00060 gene replacement absence Deide_00200 Deide_00200 gene replacement DR_A0065 gene replacement (E) Primers for 5’-RACE Primer DT88 DT89 DRA0065RC0 DRA0065RC1 DRA0065RC2 Dd2p01940RC0 Dd2p01940RC1 Dd2p01940RC2 Primer sequence GAAGAGAAGGTGGAAATGGCGTTTTGG CCAAAACGCCATTTCCACCTTCTCTTC CGGCGGGAATCTGGATCTTT ATGGCTTCTTCGCTCTGCTT TCAGCCACCATTTCGACCAG CTGGATCTTCTCGCTGGTGC CTTGACGCTCAGGGTTCCG TTCCTCGCTCTGCTTCTTGG Use Anchor oligonucleotide Anchor-specific primer Reverse transcription First-round PCR (together with primer DT89) Second-round PCR (together with primer DT89) Reverse transcription First-round PCR (together with primer DT89) Second-round PCR (together with primer DT89) 6 Fig. S1. Fig. S1. Diagnostic PCR to verify HU gene deletion mutants of D. deserti. (a) Schematic drawing of the PCR products to verify replacement of the gene encoding DdHU1, DdHU2 or DdHU3 by the chloramphenicol or kanamycin resistance cassette. If the D. deserti HU gene of interest can be entirely deleted, the smaller PCR fragment (with the HU gene) will be absent and replaced by a larger PCR product (with the antibiotic resistance cassette). L and R indicate the primers, and these primer pairs correspond to primers HUPXbaI & HUGeneHd3 for DdHU1, Dd3p00060UPfor & Dd3p00060DNrev for DdHU2, Dd00200UPfor & Dd00200DNrev for DdHU3. (b) Agarose gel showing the PCR results. Indicated at the top are the tested strain, including the wild-type (wt) control strain, and the HU-encoding gene whose replacement is verified. The lane at the left contains marker DNA fragments (the length of some of these, in bp, is indicated). Additional PCRs with primers specific for HU genes or antibiotic resistance cassettes confirmed the gene replacement results observed in this figure (data not shown). 7 Fig. S2. Fig. S2. Main differences in DdHU3 compared to residues conserved in DrHU, DdHU1 and DdHU2. 8
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