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Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk

Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk www.microbiology.univ.gda.pl e-mail: kaczorow@biotech.univ.gda.pl. Main fields of interest:. 1. Biology of bacterial DNA restriction and modification systems.

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Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk

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  1. Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk www.microbiology.univ.gda.pl e-mail: kaczorow@biotech.univ.gda.pl

  2. Main fields of interest: 1. Biology of bacterial DNA restriction and modification systems. 2. New approaches for DNA sequencing and analysis.

  3. Restriction-modification systems. What are they for?

  4. Table 1. Restriction-modification systems isospecific to HindIII Gram-positive bacteria Gram-negative bacteria Bse HI Bacillus stearothermophilus CPW5 Asp 3065I Alcaligenes sp. 3065 Bsm GII Bacillus stearothermophilus G170 Asp 52I Alcaligenes sp. 52 Bsp KT8I Bacillus sp. KT8 Bbr AI Bordetella bronchiseptica Bsp LAIII Bacillus sp. LAIII Bbr I Bordetella bronchiseptica 4994 Bst 170II Bacillus stearothermophilus D170 Bpe I Bordetella pertusis Bst FI Bacillus stearothermophilus FH58 32I RFL 32 Cfr Citrobacter freundii Z1I 14P 83I RFL 83 Bst Bacillus stearothermophilus Cfr Citrobacter freundii CI CI 231I RFL 231 Lla Lactococcus lactis subs. cremoris Cfr Citrobacter freundii Chu I Corynebacterium humiferum VIII 1585-68 Eco Escherichia coli Ssb I Streptomyces scabies 1219I RFL1219 Eco Escherichia coli 98I RFL98 Eco Escherichia coli Hin 1076III Haemophilus influenzae serotype b, 1076 Hin 173I Haemophilus influenzae 173 Hin 5III Haemophilus influenzae RFL5 Hin BIII Haemophilus influenzae Rb dIII Rd Hin Haemophilus influenzae Hin fII Haemophilus influenzae Rf Hin JCII Haemophilus influenzae JC9 Hin SAFI Haemophilus influenzae serotype b, SAF6 HsuI Haemophilus suis Mki I Moraxella kingae Spa PIV Spirulina platensis strain pacifica REBASE http://rebase.neb.com

  5. CH3 5’ A A G C T T 3’ 3’ T T C G A A 5’ 5’ A A G C T T 3’ 3’ T T C G A A 5’ CH3 M.HindIII R.HindIII toxin antytoxin The HindIII restriction-modification system is composed of two enzymes.

  6. Objectives: 1. how similar are genes encoding isospecific enzymes ? 2. is it possible to map their functional domains ? 3. do they recognize cognate sequence in the same way ? 4.what is their mode of action ?

  7. hindIIIR hindIIIM R-M System HindIII ( Haemophilus influenzae Rd) Nwankwo et al., Gene (1994) pHindIIIR pHindIIIM llaCIR llaCIM R-MSystem LlaCI (Lactococcus lactis subs. cremoris CI) Madsen i Josephsen, Biol.Chem. (1998) pLlaCIR pLlaCIR R-M System EcoVIII (Escherichia coli E1585-68) Mruk i Kaczorowski, Appl. Environ. Microbiol. (2003) Plasmid (2001) ecoVIIIR ecoVIIIM pEcoVIIIR pEcoVIIIM R-M System Csp231I (Citrobacter sp.RFL231) Mruk i Kaczorowski, unpublished csp231IC csp231IRcsp231IM pCsp231IC pCsp231IM pCsp231IR bstZ1IIR bstZ1IIM R-M System BstZ1II (Bacillus stearothermophilus 14 P) Mruk i Kaczorowski, unpublished pBstZ1IIR pBstZ1IIM

  8. R.HindIII MKKSALEKLLSLIENLTNQEFKQATNSLISFIYKLNRNEVIELVRS 46 R.LlaCI MNSEFEKLKGTGLQTVDTEKVTLDFFKMLKKISDKEFINILIT 43 R.EcoVIII MDLTNIEFINDAAINRRRYWTNEIVKLSGHFVNDSSRVEEEIIYEVSKSGSQALLDHLRL 60 R.Csp231I MMKVVKMVKIPPLAQDCDLDSSELSEFTPEEHFNKSITRWFSDHYASYSNRFESYEYIQNYIQNEHFNWSVAPNTNVIATKFSAHLRTLSLKDFSFLLCH 100 R.BstZ1I MNMVDAFYLMNYVDDQFKVNAQLTEEHERIANEFLKDIKKFDDKTFNKLLVS 52 R.BbrI MPVARHGVGERVVQCPPMLTTSDPLPHPLLHDGVVAAVVRLCADATLDFPARCARLEAAIRACTRDQIIEHLQH 74 : : : . . : P D/E D D - K R -- R R.HindIII IGIL P EAIKPSSTQEKLFSKAGDIVLAKAFQLLNLN - SKPLEQRGNAG D VIALSKEFNYGL VADAKSFRLSRT — - AKNQKDF - K VKALSEWRED ------ 136 R.LlaCI SGYI P DLYVADSKEETLFTKLCEALEVDWASRMGFE - ANAVTQKSSYE D VVIKIN -- NKII VSDTKSFRLGRSQQAPNVKDFVK PEDYSKWANRHSGQ -- 138 R.EcoVIII CTAI P ESYEHDSSEEKLYSKYTDALISECFKFFGLN - SIVLTERADAA D VEVVCD — - SYSF VADAKVFRLSRT — - AKNQKDF - K VQAMDGWRNT ------ 148 R.Csp231I TGYI P EIYKADSSQETLYSK LVEAMVNEWALRMKFTYSLLPTQKSSKE D ITISDG -- ENII VADAKSYRLGRSQAAPNVKDALK KGDITKWLAYYDQHKY 198 R.BstZ1I ATFI P DFYEPDSSRETLFSKLVEAMVTEWAIRMGYE - AAMQKEKASYE D VRIAIK -- DKLI VVDAKTFRLGRSQAAPNVKDFLK LEDIRKWCSRYKN --- 146 R.BbrI GGVI S ECIGHDSSEEKLLAKYSDVLLACALGELGFQ - ARVL AERSDAA D VYAECG — - DQRI VGDGKIFRLSRT — - ALNPKDY - K VPSLKKWRQGAGYS -- 166 :.: .*..*.* :* : : : . : :* * * * :**.* * * ** * * R.HindIII - KDYAVLTAPFFQYPTTKSQIFKQSLDE -- NVLLFSWEHLAILLQLDLEETNIFSFEQLWNFPKKQSKKT --- S VSDAENNFMRDFNKYFMDLFKIDKDT 230 R.LlaCI - KLGGLVVYPQLHEWTRKSDAHVYCSDKKNPILMLPYHYLAYFLERKDK - FNPKSLEKLWDYEKIFPEK ------ ADSRNDYWQKINNVILEITGDEKKE 230 R.EcoVIII - KDFAMVVCPIYQLPVKSSQIYQQAILR — - NVCVFTYTHLAVLVKYADI -- VGSDVRILLEEIFRSVILM --- NPSKDSVQYWTMINRTMLNYDKR IEKL 240 R.Csp231I NRIGGLVAFPSQHDWKNGSDFYLYLTDKNSPIIMLFYEHMAFMLLAGMD -- KNNLLDFYRNHKDIFPNEVF — - NKIGSRKIYFDKLEQYLLNCG --- KFK 291 R.BstZ1I - AIGGLVTYPCLHEWKNKSDAYTYCSTKDMPTVMLSYKHLAFLLDNKEN - FNTEKLIELWDYENIFPEKLPKNLKGGNKKPYWDAINKKLIEITNVGDKE 244 R.BbrI - ADYSVLLAPSFQYPLSRSQLYAQAIDN — - NVLLLSFEQLAFLVRHHQP --- GRDYRPLWEYAGTRAGQDT -- AAWKEARQYWHGLNQLVARLAGQTEQA 258 .:: * : *: . .. :: : :* :: : : ::. . . R.HindIII LNQLLQKEINFIEERSLIEKEYWK KQINIIKNF ------- TREEAIEALLKDINMSSKIETIDSFIKGIKSNDRLYL 300 R.LlaCI FKKFLNLAETKLYEFVEGRLKNLEYQKNIKIKKIEFEISSIPDSELRDKFLKYRQEIETQYIVTFQERIQKFRLTNNKESTTYSKFIDSSFDKS 324 R.EcoVIII WIDERIATSEGIYVLKKMAIEYLSSERGRILSM ------- SREEAVRALIKMNKIE SRIEQIKKVTDNNILSLK 307 R.Csp231I WSEFSETSDLIVSEKALRHSYR 313 R.BstZ1I YVKCLNRYDKIINQAVKEIINFLETIIINKKEEVAREIRKLSDKQIREAYEEYKISQETEEYQRILENVKAFRL 318 R.BbrI LRDSYAARRQLLQRQFDDEIAHWDAEAQRIRGL ------- SREQAIAELLVSKKIQARVDLMRGAQRQLTLQLDDARA 329 Comparison of the amino acid sequences of R.HindIII isoschizomers.

  9. Molecular model of R.HindIII in complex with DNA(model was constructed by Janusz M. Bujnicki, Bioinformatics Laboratory, International Institute of Molecular and Cell Biology, Warsaw).

  10. Comparison of the amino acid sequences of M.HindIII isomethylomers III M.HindIII IYNSDSIFEIKKL SIHAI ---------------------------------------------------------------------------MIDC DSN 25 M.LlaCI IYNENSIDSIKKI SIHLI --------------------------------------------------------------------------MKIDT ETE 26 M.EcoVIII VTNGNSISLIKKL YAHLI ----------------------------------------------------------------MLNKIQLPDEIKKNT PSQ 36 M.Csp231I LINADSISELKKL SIDLI --------------------------------------------------------------------------MRKQL EDN 26 M.BstZ1I IILGDCTEYLSKL SVHMF MSNSSKNISLLNLKIDKLQKEISYLTFCLENDIKPDEYETEDIEMIIKSKKVEVSNLQSELDKILNNSRESDLTPYLNK PKN 100 : : :. :.*: : . : IV V VI M.HindIII ISDIPYGI YQEWVESWSNEWFRVL GSSVFVFAGRQF DYDDWDILHSNTNSALGGTSSAQHKT-SLFKRRGKPLNGWSEADKKRPQE KS AHRVVVAFENSG 124 M.LlaCI LSDIPYGI YQNWVESWAKEWFRVL GSSCFIFAGRRY SFADWDILHENTNSALGGSSPAQHKTGSGFKMRGKPINGWSEADRKIPLE KP AHRAIVGLENSG 126 M.EcoVIII LSDIPYGI YQQWCEEWATEWFRVL GASAIIFAGRRF GAEDWDVLHNNSNNAYLGSSPAQKNAGAIFKKRGKPINGWSEADKKIPLE KP SHRCICAMEDAG 136 M.Csp231I LSDIPYGI YYQWCQTWASEWLRVL GGTAFVFAGRRF GADDWDVLHKNTNTAYLGNSPAQKQAGNVFKRRGKPINGWSDADKKIPAE KP APRCIVALEDAG 126 M.BstZ1I LSDIPYGI YHDWVYSWAQQLFPVM GAPVLIFGARRT NLDEWDVLHNNTNSALLGASPAQEGK-SAFKRRGKPINGWNKEDRQINQA KE ISSAIAAMEKAG 199 :*******. :**:**.*:*.* * *.**. ** ****:***.. *:: * :* *: : : *:* *.. ::*..*: : .:*.:* VII VIII M.HindIII FTFKDMLSWEKDKAPHRAQRISCVFERRGD--------------------------------------------------------------------IA 156 M.LlaCI FTFRDMIGWNRSKATLKAQRISKVYERRND--------------------------------------------------------------------FE 158 M.EcoVIII FNLRDIIAWMRVKAPKRAQRLSCVYERRGD--------------------------------------------------------------------TL 168 M.Csp231I FNFRDMLSWIKPKATHRAQRLSIVYERRGM--------------------------------------------------------------------QD 158 M.BstZ1I FLLKDILAWEKSSAHHRSQDIFKVLVKRGNKYKLTEQSFNKMSDIEGLNVAAPTLESLLNIEFDKYDSFIKAVKNINPKLAKKYTYELLEIALADEKVKQ 299 * ::*::.* : .* ::* : * :*. TRD X M.HindIII KWVGWRVANLRPLFEPILWFQKPYKTGSTLADNLIKHEVGAWN HVAQKPLNLMKLLIDLVT EQI NTN ENSLTHWNIQQGALNHSNILKVRITSEDKGY KE 256 M.LlaCI KLSEWRVGNLRPVFEPILWFTKPYKQGGTIADNMLKHGVGAYN HPTQKPLVLMKALIELTT NQI NAE ---LEKW--QTFSEKGDNYIEIPNLSSDRGL QE 253 M.EcoVIII KWNGWRVGNLQPTFEPILWFSKPYKIGGTIADNAIIHGVGAYN HPTQKPVALMKTLIELTT GQL NAE QDAFVAR-----NGKPENVITAGFSSNESGL QK 263 M.Csp231 KWNGWRIGNLRPIFEPIIWCFKPYKH--TIADNVLEHNLGAYN HDAQKPVKLLETLISLVT GQV ESL QFAFEEI-----TGHFNNTLEIGMSPKEGGL IP 251 M.BstZ1I KWKGWKLGNLAPYYEPIAWLFKPYDSVVTLTDNVLVNEVGAIN HDAQKPIELIKYLINIMT GQV DVE MNVGKIN-----GREPKNILKFDFTNEEEKV RE 394 : * *::.** * :*** * ***. *::** : : :** * . .* : .: * :***: *:: **.: * *: I II M.HindIII VLDPFAGSGTTL NRHFIGYEKNNGI LAAKEL YNIAVNRLGIEKNNCFYNKEKK 309 M.LlaCI VLDPFSGSGTTL NRHYLGFEIDKDY VAAKEL YNTSLNRLNTLL---------- 296 M.EcoVIII VIDPFSGSGSTL GRDYIGFEINPTY VAAKDL VETSIKRLNK------------ 304 M.Csp231 VLDPFAGSGSTA KRDFIMIEKDPNI IAALNL FSIMNDRLSKSKQTISLFD--- 301 M.BstZ1I ILDPFVGSGTTA NRNFIAFEINEHY VAAKEL HSLANKRLNQEIKKEHQIALEI 447 ::*** ***:* :** :* *.:: * : . .**.

  11. M.HindIII MIDCIYN--SDSIXEIKKLDSNSIHAIISDIPYGIDYDDWDILHSNTNSALGGTSSAQHK 58 R.HindIII MKKSALEKLLSLIENLTNQEFKQATNSLISFIYKLNRNEVIELVRSIGILPEAIKPSSTQ 60 * .. : . * ::.: : :. : .: * :: :: * . . . ..:. : M.HindIII TSLFKRRGKPLNGWSEADKKRPQEYQEWVESWSNEWFRVLKSGSSVFVFAGRQFAHRVVV 118 R.HindIII EKLFSKAGDIV--LAKAFQLLNLNSKPLEQRGNAGDVIALSKEFNYGLVADAKSFRLSRT 118 .**.: *. : ::* : : : : . . .*.. . :.*. : : . M.HindIII AFENSGFTFKDMLSWEKDKAPHRAQRISCVFERRGDIANTNKWVGWRVANLRPLFEPILW 178 R.HindIII AKNQKDFKVKALSEWREDKD-YAVLTAPFFQYPTTKSQIFKQSLDENVLLFSWEHLAILL 177 * ::..*..* : .*.:** : . . . . :: :. .* : . .** M.HindIII FQKPYKTGSTLADNLIKHEVGAWNENSLTHWNIQQGALNHSNILKVRITSEDKGYHVAQK 238 R.HindIII QLDLEETNIFXFEQLWNFPKKQSKKTSVSDAENNFMRDFNKYFMDLFKIDKDTLNQLLQK 237 . :*. ::* :. ::.*::. : : :. ::.: .:*. :: ** M.HindIII PLNLMK---LLIDLVTKEEQIVLDPFAGSGTTLLAAKELNRHFIGYEKNNGIYNIAVNRL 295 R.HindIII EINFIEERSLIEKEYWKKQINIIKNFTREEAIEALLKDIN-------MSSKIETIDSFIK 290 :*::: *: . *:: ::. *: . : *::* .. * .* M.HindIII GIEKNNCFYNKEKK 309 R.HindIII GIKSNDRLYL---- 300 **:.*: :* Comparison of the amino acid sequences of M.HindIII and R.HindIII.

  12. Known sequence (vector) Analyzed DNA 3’ 5’ 5’ 3’ Universal primer New primer New primer DNA sequencing by primer walking.

  13. PRIMER STRAND (20-MER) 5’ - GGTACAGGATGCGAAGACAA - OH - 3’ 3’ - CCATGTCCTACGCTTCTGTTNNNN - P - 5’ INDEXING STRAND (24-mer) Indexer structure

  14. Cut site (9 nt) 5’ NNGGATGNNNNNNNNNNNNNN 3’ 3’ NNCCTACNNNNNNNNNNNNNN 5’ Cut site (13 nt) Specific sequence R.FokI cuts DNA in a precise distance from its recognition site.

  15. DNA sequencing by indexer walking Type IIS restriction endonucleases Enzyme Specific sequence Average fragment length (bp) Commercially available AarI CACCTGC (4/8) 8192 Alw26I ( BsmAI) GTCTC (1/5) 512 BbsI ( BpiI) GAAGAC (2/6) 2048 BbvI ( BseXI) GCAGC (8/12) 512 BsmFI GGGAC (10/14) 512 BspMI ACCTGC (4/8) 2048 BtgZI GCGATG (10/14) 2048 Eco31I GGTCTC (1/5) 2048 Esp3I CGTCTC (1/5) 2048 FokI GGATG (9/13) 512 SfaNI GCATC (5/9) 512 Commercially unavailable AceIII CAGCTC (7/11) 2048 Bbr7I GAAGAC (7/11) 2048 BbvII GAAGAC (2/6) 2048 Sth132I CCCG (4/8) 128 StsI GGATG (10/14) 512 (www.rebase.neb.com/rebase)

  16. FR DNA fragmentation and cloning Genomic DNA Insert (4-5 kb) REV DNA amplification FORWARD (FR) Primer REVERSE (REV) 1. DNA sequencing using primer REV 2. Nucleotide sequence analysis 3. Partial digestion with class-IIS ENase DNA sequencing by indexer walking

  17. Streptavidin coated paramagnetic particles Purification of biotin labeled DNA fragment TTTC FR REV AAAG FR AAAG FR GCAA INDEXER 2 FR CGTT primer GCAA INDEXER 1 TTTC primer Ligation of specific indexer INDEXER 1 FR TTTC primer AAAG 1. DNA amplification 2. DNA sequencing 3. Nucleotide sequence analysis 4. Partial digestion with type-IIS ENase INDEXER 1 TTTC CGTT AAAG primer Removed DNA fragment 5. Ligation of the next indexer Consecutive steps of DNA sequencing by indexer walking

  18. Financial support: Polish Committee for Scientific Research KBN 6P04B-027-18 2000-2003 KBN 6P04B-003-12 1997-2000 KBN IA-1433/99 1999 KBN IA-0589/2001 2001

  19. Lab members: Beata Furmanek-Blaszk Anna Kaczorowska Tadeusz Kaczorowski Iwona Mruk Józef Nieradko Marian Sęktas Łucja Adamczyk Jadwiga Pomorska Magdalena Cichowicz Agnieszka Dekowska Katarzyna Gromek Anna Kossobucka

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