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Appl Environ Microbiol, July 1998, p. 2424-2431, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Characterization of the Lactococcal Plasmid-Encoded Type II Restriction/Modification System, LlaDII

Annette Madsen and Jytte Josephsen*

Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark

Received 28 October 1997/Accepted 3 April 1998

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GCdown-arrow NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stem-loop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.

* Corresponding author. Mailing address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 4, DK-1958 Frederiksberg C, Denmark. Phone: 45 35 28 32 32. Fax: 45 35 28 32 14. E-mail: jyj{at}kvl.dk.

Appl Environ Microbiol, July 1998, p. 2424-2431, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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