A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identi®ed and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Three hundred and thirty-five lactic acid bacteria were isolated from sour doughs and screened for antagonistic activity. Of these 145 showed activity against one or several of the indicator strains used in the screening. The antimicrobial activity of 18 isolates were due to a proteinaceous compound. These 18 isolates belonged to three different Lactobacillus species: Lactobacillus bavaricus, Lactobacillus curvatus and Lactobacillus plantarum. The spectrum of antimicrobial activity for the three species suggested that the inhibitory components were different. The inhibitory compound from Lact. bavaricus MI401 was chosen for further study. The proteinaceous nature, antimicrobial activity against closely-related species, heat resistance and sensitivity to alkaline treatment strongly indicated that this substance was a bacteriocin, which we designated bavaricin A. The bacteriocin was purified to homogeneity by ammonium sulphate precipitation, ion exchange, hydrophobic interaction and reverse-phase chromatography. The purification resulted in 193,000-fold increase in specific activity. SDS-PAGE of bavaricin A showed a molecular weight of 3500-4000 Da. By amino acid sequencing 41 amino acids were determined. Bavaricin A had a bactericidal mode of action and inhibited nine out of 10 Listeria monocytogenes. Lactobacillus bavaricus MI401 produced bavaricin A at temperatures from 4 degrees C to 30 degrees C. The production of active bavaracin A was inhibited at increasing sodium chloride concentration. In the presence of 3% sodium chloride at 4 degrees C no active bavaricin A could be detected. Nitrite (100 ppm) did not affect the production of active bavaricin A.

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.Lactococcus lactis is the most important bacterium used for starter cultures by the dairy industry. An important problem in industrial milk fermentation is infection of the starter bacteria by bacteriophages, which leads to bacterial lysis. The consequences of phage infection are fermentation delay, alteration of product quality, and in severe cases loss of the product. All these outcomes result in considerable economic loss to dairies (5,22). Industrial phage ecology is dominated by three phage species: the predominant species 936 and species c2 and P335, which also have considerable importance (5,22).The first step in phage infection is adsorption of the phage to the host cell. Despite the fact that little information concerning the adsorption process of lactococcal phages is available, it seems to be a two-step process that starts with reversible binding to specific carbohydrates exposed on the surface of the cell wall (30, 37, 39), which is followed by, at least in species c2, irreversible binding to a protein in the cell membrane (14,30,38). Information on receptor-binding proteins (RBPs) in phages infecting gram-positive bacteria is sparse compared to the information available for phages infecting gram-negative bacteria. However, recently, Duplessis and Moineau identified the first RBP genes for the Streptococcus thermophilus phages DT1 and MD4 (11). These authors repo...

LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).