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Aim: To screen five strains of lactic acid bacteria (LAB) isolated from rye sourdoughs for the potential production of antimicrobial substances. Methods and Results: Lactobacillus sakei KTU05‐06, Pediococcus acidilactici KTU05‐7, Pediococcus pentosaceus KTU05‐8, KTU05‐9 and KTU05‐10 isolated from rye sourdoughs were investigated for the production of bacteriocin‐like inhibitory substances (BLIS). The supernatants of analysed LAB inhibited growth of up to 15 out of 25 indicator bacteria strains as well as up to 25 out of 56 LAB strains isolated from rye sourdoughs. Moreover, these five LAB were active against ropes‐producing Bacillus subtilis and the main bread mould spoilage causing fungi –Aspergillus, Fusarium, Mucor and Penicillium. Lactobacillus sakei KTU05‐6 demonstrated the best antibacterial properties and is resistant towards heat treatment even at 100°C for 60 min. Conclusions: The use of LAB‐producing antibacterial substances may be a good choice as a co‐starter culture to ensure the stability of sourdoughs and to avoid the bacterial and fungi spoilage of the end product. Significance and Impact of the Study: The antimicrobial compounds designated as sakacin KTU05‐6, pediocin KTU05‐8 KTU05‐9, KTU05‐10 and AcKTU05‐67 were not identical to any other known BLIS, and this finding leads up to the assumption that they might be the novel.

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 gamma-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).

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction-modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.

Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.

Aim: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis. Methods and Results: Conductivity and pH were used to determine growth in RSM. A small increase in the generation time was found with increasing number of plasmids, while their size was unimportant. The introduction of a plasmid-encoding AbiS did only enhance the level of phage resistance significant when other plasmids encoding either AbiS1 or the restriction modification system LlaBIII was present. Conclusions: The earliest detection of growth was observed by measuring pH, rather than conductance. The plasmid-encoded AbiS system has a potential to be used as a phage resistance mechanisms in L. lactis during milk fermentations, especially when combined with other anti-phage mechanisms. Significance and Impact of the Study: This study widened the knowledge about the influence of plasmid introduction on the growth rate of L. lactis, which is important for the construction of new strains. The level of protection against 936 groups of phages was only significant when the mechanism was present together with the RM system LlaBIII.

Aims: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. Methods and Results: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 μl whey samples and the detection limit of the assay was 102–103 PFU ml−1 as opposed to the detection limit of 104 PFU ml−1 for conventional PCR performed on 1-μl whey samples. Conclusions: In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages. Significance and Impact of the Study: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.

The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM , encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P llaDIIM ) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P llaDIIR ) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P llaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR , which appears to be constitutively expressed.

The mixed TK5 starter culture was used in a Danish factory as the only starter for production of Cheddar cheese for more than 11 years before the factory experienced serious bacteriophage attacks with inhibition of the acid production in the curd. The cheese whey contained some phages from the beginning, and gradually new phages appeared able to infect an increasing number of isolates. Three bacteriophages jw30, jw31 and jw32 were isolated from the factory whey collected in 1989–94 and compared with lytic bacteriophages isolated in the period 1982–86 ( Josephsen et al., 1994 ) DNA hybridisation showed that the type phage P008 had high homology to the new phages jw30, jw31 and jw32 as had the phages isolated in 1982–84. The new phages had broader host ranges and higher burst sizes than the previously isolated phages, showing that the lytic phages had become more virulent with time.

The Lla DII 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 Pst I- Eco RI 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 Lla DII endonuclease was partially purified and found to recognize and cleave the sequence 5′-GC↓NGC-3′, where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu 4HI. Sequencing of the 2.4-kb Pst I- Eco RI 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 Lla DII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp 6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the Lla DII and Bsp 6I 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 Lla DII and Bsp 6I methylases with other m 5 C methylases showed that the protein primary structures possessed the same organization.

To cleave DNA, the single polypeptide restriction-modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5'-CTnGAyG-3' as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.

To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis.Conductivity and pH were used to determine growth in RSM. A small increase in the generation time was found with increasing number of plasmids, while their size was unimportant. The introduction of a plasmid-encoding AbiS did only enhance the level of phage resistance significant when other plasmids encoding either AbiS1 or the restriction modification system LlaBIII was present.The earliest detection of growth was observed by measuring pH, rather than conductance. The plasmid-encoded AbiS system has a potential to be used as a phage resistance mechanisms in L. lactis during milk fermentations, especially when combined with other anti-phage mechanisms.This study widened the knowledge about the influence of plasmid introduction on the growth rate of L. lactis, which is important for the construction of new strains. The level of protection against 936 groups of phages was only significant when the mechanism was present together with the RM system LlaBIII.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and φ645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:IS S1 , and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG , rgpE , ycbC , and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that φ645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:IS S1 integration in ycaG and rgpE , whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that φ645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and φ645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and φ645 that showed no homology in the C-terminal part.

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 φ7) 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.

Various strains of Escherichia coli, isolated from different patients, were screened for type II restriction endonuclease activity. In 1 out of 23 patients, a type II restriction endonuclease activity was found. The restriction endonuclease designated Eco1524I was purified to near homogeneity, based on hydroxyapatite and heparin sepharose chromatography. Eco1524I exhibited endonuclease restriction activity in the pH range from 6.0 to 10.0 (maximum level at pH 8.0) and required Mg2+ as divalent cation. The enzyme was stable till temperature 55 degrees C and pH range from 6.0 to 10.0. Eco1524I recognized the sequence 6-bp palindromic 5'AGG downward arrow CCT 3', producing blunt end and is found to be an isoschizomer of Stu I.

PART 1. CULTURED PRODUCTS: HISTORY, PRINCIPLES, AND APPLICATIONS Origin and History of Food Fermentations, K.S. Steinkraus Microorganisms, E.B. Hansen Starter Cultures and Fermented Products, J. Josephsen and Lene Jespersen Manufacture of Fermented Products, W-K. Nip Quality and Flavor of Fermented Products, G. Smit, J.T.M. Wouters, and W.C. Meijer PART II. SEMISOLID CULTURED DAIRY PRODUCTS Semisolid Cultured Dairy Products: Principles and Applications, D. Patel and M. Walker Yogurt, K.R. Nauth Sour Cream and Creme Fraiche, L. Meunier-Goddik Yogurt and Sour Cream: Operational Procedures and Processing Equipment, S. Clark and V.C. Plotka Fromage Frais, L. Meunier-Goddik Semisolid Cultured Dairy Products: Packaging, Quality Assurance, and Sanitation, Y. Zhao PART III. SOLID CULTURED DAIRY PRODUCTS Principles of Cheese Production, E.W. Nielsen Traditional Greek Feta, A. Polychroniadou-Alichanidou Cheddar Cheese, J.M. Banks and A.G. Williams Semihard Scandinavian Cheeses Made with Mesophilic DL-Starter, Y. Ardo Cheeses Made with Thermophilic Lactic Starters, S. Lortal Manufacture of Cheese: Operational Procedures and Processing Equipment, J.M.B. Kristensen, E.W. Nielsen, and J. Josephsen Packaging of Cheese, G. Mortensen, G. Bertelsen, and P.V. Nielsen Cheese Production: Quality Control and Sanitation, S. Lillevang PART IV. FERMENTED MEATS Meat Fermentation: Principles and Applications, D. Demeyer Dry-Cured Ham, F. Toldra Semidry Fermented Sausages, J.G. Sebranek Dry-Fermented Sausages, R. Talon, S. Leroy-Setrin, and S. Fadda Mold-Ripened Sausages, K. Incze Meat Products Processing: Operational Procedures and Processing Equipment, P. Bladini Fermented and Dry-Cured Meat: Packaging and Quality Control, F. Toldra, R. Gavara, and J.M. Lagaron Meat Processing Plant Sanitation, N.G. Marriott PART V. FERMENTED SOY PRODUCTS Fermented Soy Products: An Overview, K. Liu Soy Sauce: Manufacturing and Biochemical Changes, T-C. Huang and D-F. Teng Fermented Whole Soybeans and Soybean Paste, D-F. Teng, C-S. Lin, and P-C. Hsieh Fermented Tofu: Sufu and Stinky Tofu, D-F. Teng, C-S. Lin, and P-C. Hsieh Tempeh: The "Other" White Beancake, S. Tibbott PART IV. FERMENTED VEGETABLES Fermentation: Principles and Microorganisms, K-Y. Li Chinese Pickles: Leaf Mustard and Derived Products, R.Y-Y. Chiou Kimchi, K-Y. Park and H-S. Cheigh Jalapeno Pepper Preservation by Fermentation or Pickling, R.M.G. Cabrera Sauerkraut, Y.D. Hang Pickle Manufacturing in the United States: Quality Assurance and Establishment Inspection, Y.H. Hui PART VII. FERMENTED CEREAL FOODS Baker's Yeast, B. Poitrenaud Fermented Cereal-Based Functional Foods, H. Salovaara and L. Simonson Sourdough Bread, A...S. Hansen Fermented Doughs in Bread Production, F. Meuser and M. Valentin Packaging, Quality Control, and Sanitation of Bakery Products, P.V. Nielsen Kenkey: An African Fermented Maize Product, M. Halm, W.K. Amoa-Awua, and M. Jakobsen PART VIII. BEVERAGES Fermented Liquid Milk Products, V.V. Mistry Partially Fermented Tea, T. Kan, Y, Tsai, R. Chang. and W-K. Nip Whiskey, M. Henderson

The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. The fragment still has the polymerase and 3'-5' exonuclease activity, but lacks the 5'-3' exonuclease activity of the holoenzyme (1). Radiolabeling of DNA fragments is most efficient with DNA fragments that contain recessed 3' ends (see Fig. 1, Chapter 39 ). The method described here is for generating 3' end-labeled DNA fragments that are suited for sequencing by the method of Maxam and Gilbert ( Chapter 51 ), but can in principle also be used when radioactively labeled DNA is required for other purposes (e.g., Southern hybridization). The principle of the method is shown in Fig. 1. The DNA fragment to be labeled is incubated in the presence of one or more deoxyribonucleotide triphosphates (one of which is labeled with (32)P in the a-phosphate group) and the Klenow fragment of DNA polymerase. As in the case with the kinase labeling of DNA ( Chapter 39 ), the DNA fragment is cleaved with a second restriction enzyme, to generate fragments of unequal size that are labeled only in one end.The fragments are subsequently separated on the basis of their different molecular weights and isolated ( chapter 7 and chapter 10 ). Fig. 1. Labeling of DNA with Klenow polymerase.

Labeling of DNA fragments at the 5' end with polynucleotide kinase is one of the methods currently employed to obtain highly labeled DNA for sequencing purposes. This end-labeled DNA is used in the partial chemical degradation method for sequencing DNA (see Chapter 51 and ref. 1).

A DNA-directed cell-free protein synthesizing system was originally developed by Zubay (1). The system contains a crude extract prepared from Escherichia coli. This extract contains the machinery necessary for the transcription and translation, i.e., ribosomes and RNA polymerase. To this system, it is necessary to add all 20 amino acids, all four ribonucleotide triphosphates, transfer RNA, an energy generating system, and various salts. The DNA template is incubated with this mixture for at least 30 min at 37°C before gene products are examined. The following method is essentially as described by Zubay (1), but with minor modifications as described by Valentin-Hansen et al. (2).

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LlaBIII, isolated from the naturally occurring plasmid pJW566 from Lactococcus lactis subsp. cremoris W56, encoded a restriction and modification (R/M) system. The plasmid pJK1 carrying the R/M system LlaBIII was transformed into L. lactis IL1403 with type I R/M system located on chromosome and the strain MG1614[pAW601] with AbiS gene on plasmid pAW601, respectively. The transformants obtained showed stacking resistance against bacteriophages. The plasmid pJK1 was transformed into industrial strains L. lactis SMQ86 and CHCC2281, the transformants showed the EOP of the bacteriophages decreased by 10(-3) and 10(-5), respectively. The results indicated that the R/M system LlaBIII could protect strains from bacteriophages in dairy fermentation.

The naturally occurring 12.1-kb plasmid, pEW104, in Lactococcus lactis ssp. cremoris W10 was found to confer decreased bacteriophage sensitivity to its host. Plasmid pEW104 encodes a non-classic restriction and modification (R/M) system, named LlaGI, consisting of only one single polypeptide. Analysis of the amino acid sequence revealed the presence of a catalytic motif and seven helicase-like motifs (DEAD-box motifs) characteristic of type I and III endonucleases, followed by four conserved methylase motifs characteristic of adenine-methylases. A comparison between LlaGI and the very similar R/M system, LlaBIII, suggests that the C-terminal region of LlaGI, apparently containing no known motifs, could possibly specify target DNA recognition. Conceivably, the LlaGI gene is included in the operon of the plasmid replication machinery. Finally, it is proposed that LlaGI represents a variant of the type I R/M systems.

A novel type I restriction-modification specificity subunit, S. LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

The interactions of lactococcal phages with bacterial hosts have been subjected to studies not only because of the ability of bacteriophages to disturb fermentation of industrial products but also because of the potential use of phage genetic elements in the study and genetic modification of lactococcal bacteria. Much of this work has been inspired by the findings and knowledge achieved from work with bacteriophage λ and its host, Escherichia coli. A model of the insertion of the bacteriophage λ genome into the chromosome of the host bacterium through site-specific recombination between the phage attachment site (attP) and the bacterial attachment site (attB) has been suggested (19). It has been shown that the insertion is mediated by an integrase protein (Int) encoded by bacteriophage λ (29, 67) as well as an integration host factor encoded by the host bacterium (41, 51). The mechanism of this insertion, as well as the excision of bacteriophage λ, is complicated but well characterized (for a review, see reference 45). Both the integrase-encoding genes (int) and the phage attachment sites have been identified and sequenced in five separate temperate lactococcal phages. The sequence information on the int gene of the φLC3 phage was the first published and revealed relationship with the Int family of site-specific recombinases (5, 47). Later, the int genes of Tuc2009, r1t, and BK5-T were also reported (11, 63, 64). The integrase proteins deduced from these sequences are found to be almost identical with φLC3 Int and could be called the φLC3 type of integrases. In addition, the cores of the attachment sites in these phages are identical. The Int protein of the lactococcal phage TP901-1 shows features relating to the resolvase-integrase family of site-specific recombinases (23, 58). Botstein (10) has proposed that the evolution of lambdoid phages in particular happens by exchange of genes organized in functional modules with homologous areas interspersed between the genes as recombination sites. Examples supporting this kind of recombination in lambdoid phages have been summarized by Campbell (20). In temperate lactococcal phages, the application of this model has been validated by the observation of different codon usage in lysin and holin genes of the phages φLC3 and Tuc2009 (4, 9); by the presence of moderate homology among genes of temperate Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus phages (17, 24, 26, 42, 43, 52, 60); and by the proposal of a conserved integration cassette of the phages BK5-T, φLC3, Tuc2009, and r1t (11, 12, 64). Additional examples supporting the Botstein evolutionary theory have not been presented for temperate lactococcal phages, although it is well established that lactococcal phages are able to evolve by acquisition of host chromosomal DNA (31, 50). In this paper we report on the characterization of a temperate phage, TPW22, from a bacterial isolate, Lactococcus lactis subsp. cremoris W22, isolated from the mixed starter culture TK5 (39). The integrase of this phage is identified and characterized.

The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L. lactis subsp. cremoris W15. The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat). Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L. lactis LM2301 and L. lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively. Increased plasmid copy number enhanced the level of phage restriction. Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation. IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR. The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction. The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon. It shows 24% identity to the HindIII endonuclease.

Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kb EcoRI fragment. This DNA fragment was sequenced ans a 1155-bp open reading frame, repB563, encoding a putative protein RepB563 of 385 amino acids was found. An AT-rich noncoding region, repA563, was found upstream of repB563. This segment included several direct and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled to repB563, RepB563 supplied in trans could support replication of a plasmid containing repA563 and a truncated repB563. This observation suggests that RepB563 is a trans-acting replication protein, and repA563 the cis-acting origin of replication, repA563, repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids. The repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison revealed highly conserved areas among this family of plasmids. In addition, variable domains emerged, presumably having a plasmid specific function, pVS40 and pC1305 were plasmids with replication proteins showing high homology to RepB563. Despite this homology, replication from repA563 could not be supported by the pVS40 or pC1305 replication protein supplied in trans. Likewise the pJW563 protein could not support replication from the pVS40 origin. pJW563 was found to be compatible with the pVS40 and pC1305 replicons. The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating pladmids. Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific in many cases rendering the plasmids compatible.

Several restriction-modification (R-M) systems have been identified in Lactococcus lactis. Most of the systems have been plasmid encoded and function as phage-resistance mechanisms. At least five different type-II R-M systems, Lla AI, Lla BI, Lla CI, Lla DI and Lla EI, were identified in isolates from a mixed Cheddar starter culture. Lla AI and Lla BI recognized the DNA sequences 5′-↓GATC-3′ and 5′-C↓TRYAG-3′, respectively. The genes coding for the Lla AI and Lla BI R-M systems have been cloned and sequenced. The Lla AI R-M system had two genes coding for methyltransferases (MTases) and one gene coding for a restriction endonuclease (ENase). The MTases showed high homology to the MTases from Dpn II. The Lla BI R-M system had one gene coding for a MTase and one gene coding for an ENase.

Two type-II restriction endonucleases have been purified from Lactococcus lactis subsp. cremoris W9 and W56, the strains isolated from a mixed Cheddar starter. Their characterization showed that LlaAI was an isoschizomer of MboI from Morexella bovis with the cleaving sequence, 5'/GATC-3', being sensitive to methylation of the adenine residue; LlaBI was an isoschizomer to SfcI from Streptococcus faceium with the cleaving sequence, 5'-C/TRYAG-3'. Both LlaAI and LlaBI restriction-modification (R-M) systems are encoded by the plasmids, respectively, pFW094 and pJW563, protecting the harboring strain against phage attack.

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.

Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2. The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events. Analysis of cotransformants showed that three different R/M plasmids could be combined in L. lactis MG1363. The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased. Some plasmid combinations were unstable suggesting replicon incompatibility.

Streptococcus lactis subsp. cremoris W56 (S. cremoris W56) is a strain partially resistant to phage attack. Derivatives which had lost either plasmid pJW563 or pJW566 no longer expressed the restriction and modification systems encoded by these plasmids. Genetic evidence for the correlation between the plasmids and the R/M systems was obtained by transformation. In addition, a third R/M system was discovered among the transformants and was shown to be encoded by pJW565. Thus, genetic evidence for at least 3 distinct R/M systems encoded by plasmids in S. cremoris W56 is presented. One of the R/M-systems showed stronger restriction of the isometric phage p2 than of the prolate phage c2. The other two systems restricted both classes of phages with equal efficiencies.

The nucleotide sequence of the gene encoding the K88ac fimbrial subunit has been determined and the amino acid sequence was derived. In comparison with the two other, previously determined sequences of the K88ab and K88ad sequences, the most striking features of the K88ac protein sequence are the insertion of a lys residue at position 104 and the deletion of three amino residues at positions 165. The differences between the three sequences are discussed with respect to possible structure-functions relationships and antigenic determinants.

Rat erythrocytes, when either injected i.v. into chickens, or incubated in vitro with chicken serum, will be lysed within minutes. This lysis is complement dependent but does not involve specific antibodies since Ig-free bursectomized chickens behave in exactly the same way as normal chickens with high levels of haemagglutinating antibodies. The lysis is mediated by activation of complement component C3 via the alternative complement pathway and various observations lead us to doubt the functional existence in this lytic reaction of the classical complement pathway.

Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage lambda (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming lambda phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.

The development of bacteriophages lytic for Lactococcus lactis subsp. cremoris was followed for five years in a Danish Cheddar cheese factory, during which time the factory used the mixed Cheddar starter TK5 exclusively. All isolated lytic bacteriophages were small, isometric-headed phages; the host range, burst size and restriction endonuclease digestion pattern differed among these phages. The virulence of the phages was, in general, low. On the basis of DNA hybridization, the majority of the phages could be divided into two groups with a low degree of homology between them, but with a high degree of homology within each group. The first group consisted mainly of the phages that appeared between February 1982 and July 1984, while the second group consisted of phages isolated from January to April of 1986. The type phage P008 hybridized to the first group, while the type phage P335 hybridized to the second group. All phages from one group showed some DNA-DNA homology with the phages from the other group. The results indicate that new phages appear with time and that a ‘new’ phage may develop in a cheese plant from existing lytic phages or from lysogenic strains.