Department of Microbiology

Faculty of Biology, Geography

and Oceanology

University of Gdańsk

www.microbiology.univ.gda.pl

e-mail: kaczorow@biotech.univ.gda.pl

1. Biology of bacterial DNA restriction and modification systems.

2. New approaches for DNA sequencing and analysis.

Main fields of interest:

Restriction-modification systems.

What are they for?

Table 1. Restriction-modification systems isospecific to HindIII

Gram-positive bacteria Gram-negative bacteriaBse HI Bacillus stearothermophilus CPW5 Asp 3065I Alcaligenes sp. 3065Bsm GII Bacillus stearothermophilus G170 Asp 52I Alcaligenes sp. 52Bsp KT8I Bacillus sp. KT8 Bbr AI Bordetella bronchisepticaBsp LAIII Bacillus sp. LAIII Bbr I Bordetella bronchiseptica 4994Bst 170II Bacillus stearothermophilus D170 Bpe I Bordetella pertusisBst FI Bacillus stearothermophilus FH58 Cfr 32I Citrobacter freundii RFL 32Bst Z1I Bacillus stearothermophilus 14P Cfr 83I Citrobacter freundii RFL 83Lla CI Lactococcus lactis subs. cremoris CI Cfr 231I Citrobacter freundii RFL 231Chu I Corynebacterium humiferum Eco VIII Escherichia coli 1585-68Ssb I Streptomyces scabies Eco 1219I Escherichia coli RFL1219

Eco 98I Escherichia coli RFL98 Hin 1076III Haemophilus influenzae serotype b, 1076 Hin 173I Haemophilus influenzae 173 Hin 5III Haemophilus influenzae RFL5 Hin BIII Haemophilus influenzae Rb HindIII Haemophilus influenzae Rd HinfII 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

The HindIII restriction-modification system is composed of two enzymes.

R.HindIII

5’ A A G C T T 3’

3’ T T C G A A 5’

CH3

5’ A A G C T T 3’

3’ T T C G A A 5’

CH3

M.HindIII

toxin antytoxin toxin antytoxin

1. how similar are genes encoding 1. how similar are genes encoding isospecific enzymes ?isospecific enzymes ?

2. is it possible to map their functional 2. is it possible to map their functional domains ?domains ?

3. do they recognize cognate sequence 3. do they recognize cognate sequence in the same way ?in the same way ?

4.4. what is their mode of action ?what is their mode of action ?

ObjectivesObjectives::

R-M System HindIII( Haemophilus influenzae

Rd)Nwankwo et al., Gene (1994)

R-M System LlaCI(Lactococcus lactis subs. cremoris

CI)Madsen i Josephsen, Biol.Chem.

(1998)

R-M System EcoVIII(Escherichia coli E1585-68)

Mruk i Kaczorowski, Appl. Environ. Microbiol. (2003)

Plasmid (2001)

ecoVIIIR ecoVIIIM

llaCIR llaCIM

hindIIIR hindIIIM

pHindIIIRpHindIIIM

pLlaCIR pLlaCIR

pEcoVIIIR pEcoVIIIM

R-M System Csp231I(Citrobacter sp.

RFL231)Mruk i Kaczorowski,

unpublished

csp231IC csp231IR csp231IM

pCsp231IR pCsp231IMpCsp231IC

R-M System BstZ1II(Bacillus stearothermophilus

14 P)Mruk i Kaczorowski, unpublished

bstZ1IIR bstZ1IIM

pBstZ1IIR pBstZ1IIM

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 IGILPEAIKPSSTQEKLFSKAGDIVLAKAFQLLNLN-SKPLEQRGNAGDVIALSKEFNYGLVADAKSFRLSRT—-AKNQKDF-KVKALSEWRED------ 136 R.LlaCI SGYIPDLYVADSKEETLFTKLCEALEVDWASRMGFE-ANAVTQKSSYEDVVIKIN--NKIIVSDTKSFRLGRSQQAPNVKDFVKPEDYSKWANRHSGQ-- 138 R.EcoVIII CTAIPESYEHDSSEEKLYSKYTDALISECFKFFGLN-SIVLTERADAADVEVVCD—-SYSFVADAKVFRLSRT—-AKNQKDF-KVQAMDGWRNT------ 148 R.Csp231I TGYIPEIYKADSSQETLYSKLVEAMVNEWALRMKFTYSLLPTQKSSKEDITISDG--ENIIVADAKSYRLGRSQAAPNVKDALKKGDITKWLAYYDQHKY 198 R.BstZ1I ATFIPDFYEPDSSRETLFSKLVEAMVTEWAIRMGYE-AAMQKEKASYEDVRIAIK--DKLIVVDAKTFRLGRSQAAPNVKDFLKLEDIRKWCSRYKN--- 146 R.BbrI GGVISECIGHDSSEEKLLAKYSDVLLACALGELGFQ-ARVLAERSDAADVYAECG—-DQRIVGDGKIFRLSRT—-ALNPKDY-KVPSLKKWRQGAGYS-- 166 :.: .*..*.* :* : : : . : :* * * * :**.* * * ** * * R.HindIII -KDYAVLTAPFFQYPTTKSQIFKQSLDE--NVLLFSWEHLAILLQLDLEETNIFSFEQLWNFPKKQSKKT---SVSDAENNFMRDFNKYFMDLFKIDKDT 230 R.LlaCI -KLGGLVVYPQLHEWTRKSDAHVYCSDKKNPILMLPYHYLAYFLERKDK-FNPKSLEKLWDYEKIFPEK------ADSRNDYWQKINNVILEITGDEKKE 230 R.EcoVIII -KDFAMVVCPIYQLPVKSSQIYQQAILR—-NVCVFTYTHLAVLVKYADI--VGSDVRILLEEIFRSVILM---NPSKDSVQYWTMINRTMLNYDKRIEKL 240 R.Csp231I NRIGGLVAFPSQHDWKNGSDFYLYLTDKNSPIIMLFYEHMAFMLLAGMD--KNNLLDFYRNHKDIFPNEVF—-NKIGSRKIYFDKLEQYLLNCG---KFK 291 R.BstZ1I -AIGGLVTYPCLHEWKNKSDAYTYCSTKDMPTVMLSYKHLAFLLDNKEN-FNTEKLIELWDYENIFPEKLPKNLKGGNKKPYWDAINKKLIEITNVGDKE 244 R.BbrI -ADYSVLLAPSFQYPLSRSQLYAQAIDN—-NVLLLSFEQLAFLVRHHQP---GRDYRPLWEYAGTRAGQDT--AAWKEARQYWHGLNQLVARLAGQTEQA 258 .:: * : *: . .. :: : :* :: : : ::. . . R.HindIII LNQLLQKEINFIEERSLIEKEYWKKQINIIKNF-------TREEAIEALLKDINMSSKIETIDSFIKGIKSNDRLYL 300 R.LlaCI FKKFLNLAETKLYEFVEGRLKNLEYQKNIKIKKIEFEISSIPDSELRDKFLKYRQEIETQYIVTFQERIQKFRLTNNKESTTYSKFIDSSFDKS 324 R.EcoVIII WIDERIATSEGIYVLKKMAIEYLSSERGRILSM-------SREEAVRALIKMNKIESRIEQIKKVTDNNILSLK 307 R.Csp231I WSEFSETSDLIVSEKALRHSYR 313 R.BstZ1I YVKCLNRYDKIINQAVKEIINFLETIIINKKEEVAREIRKLSDKQIREAYEEYKISQETEEYQRILENVKAFRL 318 R.BbrI LRDSYAARRQLLQRQFDDEIAHWDAEAQRIRGL-------SREQAIAELLVSKKIQARVDLMRGAQRQLTLQLDDARA 329

Comparison of the amino acid sequences of R.HindIII isoschizomers.

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).

Comparison of the amino acid sequences of M.HindIII isomethylomers

M.HindIII ---------------------------------------------------------------------------MIDCIYNSDSIFEIKKLDSNSIHAI 25M.LlaCI --------------------------------------------------------------------------MKIDTIYNENSIDSIKKIETESIHLI 26M.EcoVIII ----------------------------------------------------------------MLNKIQLPDEIKKNTVTNGNSISLIKKLPSQYAHLI 36M.Csp231I --------------------------------------------------------------------------MRKQLLINADSISELKKLEDNSIDLI 26M.BstZ1I MSNSSKNISLLNLKIDKLQKEISYLTFCLENDIKPDEYETEDIEMIIKSKKVEVSNLQSELDKILNNSRESDLTPYLNKIILGDCTEYLSKLPKNSVHMF 100 : : :. :.*: : . :

M.HindIII ISDIPYGIDYDDWDILHSNTNSALGGTSSAQHKT-SLFKRRGKPLNGWSEADKKRPQEYQEWVESWSNEWFRVLKSGSSVFVFAGRQFAHRVVVAFENSG 124M.LlaCI LSDIPYGISFADWDILHENTNSALGGSSPAQHKTGSGFKMRGKPINGWSEADRKIPLEYQNWVESWAKEWFRVLKPGSSCFIFAGRRYAHRAIVGLENSG 126M.EcoVIII LSDIPYGIGAEDWDVLHNNSNNAYLGSSPAQKNAGAIFKKRGKPINGWSEADKKIPLEYQQWCEEWATEWFRVLKPGASAIIFAGRRFSHRCICAMEDAG 136M.Csp231I LSDIPYGIGADDWDVLHKNTNTAYLGNSPAQKQAGNVFKRRGKPINGWSDADKKIPAEYYQWCQTWASEWLRVLKPGGTAFVFAGRRFAPRCIVALEDAG 126M.BstZ1I LSDIPYGINLDEWDVLHNNTNSALLGASPAQEGK-SAFKRRGKPINGWNKEDRQINQAYHDWVYSWAQQLFPVMKEGAPVLIFGARRTISSAIAAMEKAG 199 :*******. :**:**.*:*.* * *.**. ** ****:***.. *:: * :* *: : : *:* *.. ::*..*: : .:*.:*

M.HindIII FTFKDMLSWEKDKAPHRAQRISCVFERRGD--------------------------------------------------------------------IA 156M.LlaCI FTFRDMIGWNRSKATLKAQRISKVYERRND--------------------------------------------------------------------FE 158M.EcoVIII FNLRDIIAWMRVKAPKRAQRLSCVYERRGD--------------------------------------------------------------------TL 168M.Csp231I FNFRDMLSWIKPKATHRAQRLSIVYERRGM--------------------------------------------------------------------QD 158M.BstZ1I FLLKDILAWEKSSAHHRSQDIFKVLVKRGNKYKLTEQSFNKMSDIEGLNVAAPTLESLLNIEFDKYDSFIKAVKNINPKLAKKYTYELLEIALADEKVKQ 299 * ::*::.* : .* ::* : * :*.

M.HindIII NTNKWVGWRVANLRPLFEPILWFQKPYKTGSTLADNLIKHEVGAWNENSLTHWNIQQGALNHSNILKVRITSEDKGYHVAQKPLNLMKLLIDLVTKEEQI 256M.LlaCI NAEKLSEWRVGNLRPVFEPILWFTKPYKQGGTIADNMLKHGVGAYN---LEKW--QTFSEKGDNYIEIPNLSSDRGLHPTQKPLVLMKALIELTTQENQI 253M.EcoVIII NAEKWNGWRVGNLQPTFEPILWFSKPYKIGGTIADNAIIHGVGAYNQDAFVAR-----NGKPENVITAGFSSNESGLHPTQKPVALMKTLIELTTQKGQL 263M.Csp231 ESLKWNGWRIGNLRPIFEPIIWCFKPYKH--TIADNVLEHNLGAYNQFAFEEI-----TGHFNNTLEIGMSPKEGGLHDAQKPVKLLETLISLVTIPGQV 251M.BstZ1I DVEKWKGWKLGNLAPYYEPIAWLFKPYDSVVTLTDNVLVNEVGAINMNVGKIN-----GREPKNILKFDFTNEEEKVHDAQKPIELIKYLINIMTREGQV 394 : * *::.** * :*** * ***. *::** : : :** * . .* : .: * :***: *:: **.: * *:

M.HindIII VLDPFAGSGTTLLAAKELNRHFIGYEKNNGIYNIAVNRLGIEKNNCFYNKEKK 309M.LlaCI VLDPFSGSGTTLVAAKELNRHYLGFEIDKDYYNTSLNRLNTLL---------- 296M.EcoVIII VIDPFSGSGSTLVAAKDLGRDYIGFEINPTYVETSIKRLNK------------ 304M.Csp231 VLDPFAGSGSTAIAALNLKRDFIMIEKDPNIFSIMNDRLSKSKQTISLFD--- 301M.BstZ1I ILDPFVGSGTTAVAAKELNRNFIAFEINEHYHSLANKRLNQEIKKEHQIALEI 447 ::*** ***:* :** :* *.:: * : . .**.

III

IV V VI

TRD X

I II

VII VIII

M.HindIII MIDCIYN--SDSIXEIKKLDSNSIHAIISDIPYGIDYDDWDILHSNTNSALGGTSSAQHK 58R.HindIII MKKSALEKLLSLIENLTNQEFKQATNSLISFIYKLNRNEVIELVRSIGILPEAIKPSSTQ 60 * .. : . * ::.: : :. : .: * :: :: * . . . ..:. :

M.HindIII TSLFKRRGKPLNGWSEADKKRPQEYQEWVESWSNEWFRVLKSGSSVFVFAGRQFAHRVVV 118R.HindIII EKLFSKAGDIV--LAKAFQLLNLNSKPLEQRGNAGDVIALSKEFNYGLVADAKSFRLSRT 118 .**.: *. : ::* : : : : . . .*.. . :.*. : : .

M.HindIII AFENSGFTFKDMLSWEKDKAPHRAQRISCVFERRGDIANTNKWVGWRVANLRPLFEPILW 178R.HindIII AKNQKDFKVKALSEWREDKD-YAVLTAPFFQYPTTKSQIFKQSLDENVLLFSWEHLAILL 177 * ::..*..* : .*.:** : . . . . :: :. .* : . .**

M.HindIII FQKPYKTGSTLADNLIKHEVGAWNENSLTHWNIQQGALNHSNILKVRITSEDKGYHVAQK 238R.HindIII QLDLEETNIFXFEQLWNFPKKQSKKTSVSDAENNFMRDFNKYFMDLFKIDKDTLNQLLQK 237 . :*. ::* :. ::.*::. : : :. ::.: .:*. :: **

M.HindIII PLNLMK---LLIDLVTKEEQIVLDPFAGSGTTLLAAKELNRHFIGYEKNNGIYNIAVNRL 295R.HindIII EINFIEERSLIEKEYWKKQINIIKNFTREEAIEALLKDIN-------MSSKIETIDSFIK 290 :*::: *: . *:: ::. *: . : *::* .. * .*

M.HindIII GIEKNNCFYNKEKK 309R.HindIII GIKSNDRLYL---- 300 **:.*: :*

Comparison of the amino acid sequences of M.HindIII and R.HindIII.

DNA sequencing by primer walking.

Known sequence (vector)

3’ 5’

Universal primer

New primer

New primer

5’ 3’

Analyzed DNA

INDEXING STRAND INDEXING STRAND (24-mer)(24-mer)

PRIMER STRAND PRIMER STRAND (20-MER)(20-MER)

GGTACAGGATGCGAAGACAA

CCATGTC CT ACGC TTC T GT T NNNN

Indexer structure

5’ - - OH - 3’

3’ - - P - 5’

R.FokI cuts DNA in a precise distance from its recognition site.

Specific sequence

Cut site (9 nt)

Cut site (13 nt)

5’ NNGGATGGGATGNNNNNNNNNNNNNN 3’3’ NNCCTACCCTACNNNNNNNNNNNNNNNNNN 5’

Enzyme Specific sequence Average fragment length (bp)

AarI CACCTGC (4/8) 8192Alw26I (BsmAI) GTCTC (1/5) 512BbsI (BpiI) GAAGAC (2/6) 2048BbvI (BseXI) GCAGC (8/12) 512BsmFI GGGAC (10/14) 512BspMI ACCTGC (4/8) 2048BtgZI GCGATG (10/14) 2048Eco31I GGTCTC (1/5) 2048Esp3I CGTCTC (1/5) 2048FokI GGATG (9/13) 512SfaNI GCATC (5/9) 512

AceIII CAGCTC (7/11) 2048Bbr7I GAAGAC (7/11) 2048BbvII GAAGAC (2/6) 2048Sth132I CCCG (4/8) 128StsI GGATG (10/14) 512

DNA sequencing by indexer walkingType IIS restriction endonucleases

(www.rebase.neb.com/rebase)(www.rebase.neb.com/rebase)

Commercially availableCommercially available

Commercially unavailableCommercially unavailable

DNA fragmentation and cloning

1. DNA sequencing using primer REV

2. Nucleotide sequence analysis

3. Partial digestion with class-IIS ENase

Genomic DNA

DNA amplification

Primer REVERSE (REV)

FORWARD (FR)

Insert (4-5 kb)

FR

REV

DNA sequencing by indexer walking

Ligation of specific indexer

1. DNA amplification

2. DNA sequencing

Consecutive steps of DNA sequencing by indexer walking

Streptavidin coated paramagnetic particles

AAAG

FR TTTC

REV

Purification of biotin labeled DNA fragment

INDEXER 1

TTTC

primer AAAG

FR

INDEXER 1

AAAG

FR TTTC

primer

INDEXER 2

GCAA

FR CGTT

primer

AAAG

INDEXER 1

CGTT TTTC

primerGCAA

FR

Removed DNA fragment5. Ligation of the next indexer

3. Nucleotide sequence analysis

4. Partial digestion with type-IIS ENase

Recent publications: 1. Furmanek, B., Gromek, K., Sektas, M and Kaczorowski, T.: Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688. FEMS Microbiology Letters 196 (2001) 171-176. 2. Mruk, I., Sektas, M. and Kaczorowski, T.: Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68that carries genes of the EcoVIII restriction-modification system. Plasmid 46 (2001) 128-139. 3. Mruk, I. and Kaczorowski, T.: Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs. Applied and Environmental Microbiology 69 (2003) 2638-2650. 4. Mruk, I., Cichowicz, M. and Kaczorowski, T. Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into biology of type II restriction-modification systems. Microbiology 149 (2003) in press.

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

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