FEBS Lett, 1993 Sep 6, 330(1), 23 - 7 Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis; Kuipers OP et al.; The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A . This strain simultaneously secreted nisin A and a protein of approximately 6 kDa . Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two-dimensional 1H-NMR showed that it consists of the unmodified leader peptide of subtilin, without the N-terminal methionine residue, linked to a fully matured nisin Z part . The hybrid protein and its main tryptic fragment {ITPQ}-nisin Z, showed at least 200-fold lower antimicrobial activities than nisin Z against three different indicator strains. Mol Gen Genet, 1993 Sep, 240(3), 428 - 34 Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous alpha-amylase; van Asseldonk M et al.; The ups45 gene encodes the major extracellular protein from Lactococcus lactis . The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide . This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L . lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide . In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus alpha-amylase, encoded by the amyS gene . Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct alpha-amylase secretion . In L . lactis the shorter signal peptides did not result in secretion of alpha-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms. FEMS Microbiol Rev, 1993 Sep, 12(1-3), 179 - 206 The physiology and biochemistry of the proteolytic system in lactic acid bacteria; Pritchard GG et al.; The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source . Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci . Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane . Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo . This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria. J Bacteriol, 1993 Sep, 175(18), 6002 - 9 Analysis of a region from the bacteriophage resistance plasmid pCI528 involved in its conjugative mobilization between Lactococcus strains; Lucey M et al.; A 10-kb HindIII fragment of pCI528 cloned into the nonconjugative shuttle vector pCI3340 could be transferred by conjugative mobilization from Lactococcus lactis subsp . lactis MG1363, whereas other HindIII fragments of pCI528 or the vector alone were nonmobilizable . Subcloning of this 10-kb region identified a 4.4-kb BglII-EcoRI fragment which contained all the DNA essential for transfer . Sequence analysis of a 2-kb region within this 4.4 kb-segment revealed a region rich in inverted repeats and two potential overlapping open reading frames, one of which demonstrated homology to mobilization proteins of two nonconjugative staphylococcal plasmids. J Bacteriol, 1993 Sep, 175(17), 5510 - 9 Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp . lactis C2; Geller BL et al.; A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp . lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain . Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive . A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment . The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases . The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb . Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb . The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region . Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter . The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames . The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein) . pip is 2,703 bases in length . Potential promoters start 206 and 212 bases upstream of the open reading frame . A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon . The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426 . Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus . The amino terminus has characteristics of a signal sequence . The putative protein would have a 650-residue, central polar domain. J Bacteriol, 1993 Sep, 175(17), 5438 - 44 Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis; Molenaar D et al.; Lactococcus lactis subsp . lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength . These pools are created by specific transport systems . A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively . The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH . A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity . This transport system is also stimulated by high osmolarity . The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity . The accumulated proline can be exchanged for betaine . Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM) . The proline transport system therefore probably also transports betaine . The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM . The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven . At low osmolarities, proline uptake is low but significant . This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM . These data indicate that proline uptake in L . lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed. J Dairy Sci, 1993 Sep, 76(9), 2455 - 67 The contribution of lactococcal starter proteinases to proteolysis in cheddar cheese; Law J et al.; The contribution of the lactococcal proteinase to proteolysis and flavor development in Cheddar cheese was investigated using the starter strains Lactococcus lactis ssp . lactis UC317, its proteinase-negative derivative FH041, and variants of UC317 modified in proteinase production, location, and specificity . Lactococcus lactis ssp . lactis FH041 was transformed by electroporation with plasmids pCI3601, pCI3602, or pNZ521 . Plasmids pCI3601 and pCI3602 harbor the cloned proteinase genes of L . lactis ssp . lactis UC317 on a high copy number vector and, as such, encode an increased concentration of cell wall-associated and secreted enzymes, respectively . Plasmid pNZ521 contains the cloned proteinase genes from Lactococcus lactis ssp . cremoris SK11 . Assessment of proteolysis and flavor development in Cheddar cheese made with these strains revealed that starter proteinases are required for the accumulation of small peptides and free amino acids in Cheddar cheese . Proteolysis was not enhanced by an approximately threefold increase in concentration of the lactococcal proteinase . The strain in which the proteinase remained attached to the cell wall appeared to contribute more to proteolysis than the strain that secreted the enzyme . Water-soluble peptides unique to Lactococcus lactis ssp . cremoris SK11 and L . lactis ssp . lactis UC317 were detected by PAGE and HPLC, respectively . Sensory evaluation showed that the flavors of all cheeses made with proteinase-positive starters were similar, but cheeses made with proteinase-negative starters lacked flavor. Appl Environ Microbiol, 1993 Sep, 59(9), 3076 - 82 Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp . cremoris IMN-C12; Sahlstrom S et al.; A peptidase from the cell wall fraction of Lactococcus lactis subsp . cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration . The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu . The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate . Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect . Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+ . The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity . The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively. Biosci Biotechnol Biochem, 1993 Sep, 57(9), 1503 - 7 Structural organization of a cryptic plasmid, pMA1, from Microcystis aeruginosa f . aeruginosa Kützing; Tominaga H et al.; The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f . aeruginosa Kutzing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed . The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens's plasmid, pFTB14, B . subtilis BAA1's pBAA1, and B . coagulans's pBC1, respectively, when conservative amino acid substitutions were included . Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates . The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp . PCC 6803 . Novel features of pMA1 are discussed. J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 2009 - 17 Physiological and genetic regulation of rRNA synthesis in Lactococcus; Beresford T et al.; The macromolecular composition of Lactococcus was regulated by growth rate in the same general way as that of less fastidious bacteria such as Escherichia coli and Salmonella typhimurium . The ratios of RNA:DNA and RNA:protein increased approximately threefold over a 13.5-fold increase in growth rate, whereas the ratio of DNA:protein remained approximately constant . Using reporter genes fused to a DNA fragment of a cloned lactococcal rRNA operon, promoter activity was located upstream of the 16S rRNA structural gene . This DNA fragment had some characteristics typical of a rrn promoter in E . coli . Two consensus promoter sequences P1 and P2 were located 296 and 157 bp, respectively, upstream of the start of the 16S rRNA gene . Between P2 and the start of the 16S rRNA gene, sequences were identified with typical anti-termination motifs characteristic of E . coli rrn promoter regions . A putative transcription terminator sequence was identified downstream of the 5S rRNA gene and putative primary RNA transcript processing sites at both ends of the lactococcal rRNA operon were also noted. FEMS Microbiol Lett, 1993 Aug 15, 112(1), 49 - 53 Rapid isolation of genes from bacterial lambda libraries by direct polymerase chain reaction screening; Griffin HG et al.; A method for the direct screening of bacterial lambda libraries by polymerase chain reaction technology has been developed . This technique permits the identification and isolation of specific DNA sequences without the need for any filter hybridisation or radioactive probing . This strategy has been used to isolate a gene encoding lactate dehydrogenase from a Lactococcus lactis lambda library. Eur J Biochem, 1993 Aug 15, 216(1), 281 - 91 Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis . Requirement of expression of the nisA and nisI genes for development of immunity; Kuipers OP et al.; The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was characterized . This fragment was previously shown to direct nisin-A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively {van der Meer, J . R., Polman, J., Beerthuyzen, M . M., Siezen, R . J., Kuipers, O . P . & de Vos, W . M . (1993) J . Bacteriol . 175, 2578-2588} . Further sequence analysis revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA . The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7-RNA-polymerase system . This resulted in the production of radiolabelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI) . The nisT gene product was not detected, possibly because of protein instability . The deduced amino acid sequence of NisI contained a consensus lipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein . Expression of nisI in L . lactis provided the cells with a significant level of protection against exogenously added nisin, indicating that NisI plays a role in the immunity mechanism . In EDTA-treated E . coli cells, expression of nisI conferred up to a 170-fold increase in immunity against nisin A compared to controls . Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10-fold less resistant to nisin A than the wild-type strain . A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids . Transcription analyses of several L . lactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription. J Biol Chem, 1993 Aug 5, 268(22), 16361 - 8 Structure, organization, and expression of the lct gene for lacticin 481, a novel lantibiotic produced by Lactococcus lactis; Piard JC et al.; The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein . The transcription of the lct gene was analyzed, and its promoter was mapped . DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids . Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 51-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics . The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481 . Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in beta-methyllanthionine formation, and a 4th threonine residue is dehydrated . This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry. J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1785 - 93 Cloning, nucleotide sequence and expression in Streptomyces lividans and Escherichia coli of pabB from Lactococcus lactis subsp . lactis NCDO 496; Arhin FF et al.; A gene (pabB) encoding the aminase activity of p-aminobenzoate (PABA) synthase in Lactococcus lactis subsp . lactis was cloned in pIJ41 and expressed in Streptomyces lividans strains defective in PABA biosynthesis . Expression of the gene was associated with a 1.2 kb deletion between the aph promoter and the cloning site in pIJ41 . Subcloning in pBR322 and expression in Escherichia coli AB3295 of the cloned L . lactis DNA fragment localized the pabB-complementing gene in a 1.9 kb segment . The nucleotide sequence of this segment contained a 1410 bp open reading frame encoding a 470-amino-acid polypeptide of 50937 Da . The deduced amino acid sequence showed substantial similarity to those reported for PabB and TrpE from several organisms . Synonymous codon usage reflected the low G + C content in the genomic DNA of L . lactis subsp . lactis, and therefore differed markedly from the preferred usage in the S . lividans host . The cloned heterologous pabB DNA was expressed in amounts that allowed accumulation of excreted PABA in cultures of S . lividans transformants. J Bacteriol, 1993 Aug, 175(16), 5168 - 75 Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains; Simons G et al.; Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon . LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene . However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate . Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose . Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate. Can J Microbiol, 1993 Aug, 39(8), 767 - 74 Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2; Ward LJ et al.; Approximately 80% of the genome of the prolate-headed lactococcal bacteriophage c2 was cloned into shuttle vectors pSA3 and pFX3 in Escherichia coli and transferred to Lactococcus lactis . A 1.67-kilobase EcoRV fragment containing the gene for the phage lysin was identified and the position and orientation of the phage lysin gene in the physical map of the phage were determined . The phage lysin was expressed in E . coli and its sequence was determined and compared with the sequences of other bacteriophage lytic genes . The sequence was similar, but not identical, to that of the related lactococcal phage m13, having a number of silent substitutions and an apparent deletion that altered the carboxy terminus of the protein . Possible alternative translation initiation codons for the lysin gene and two possible alternative mechanisms for access of the lysin enzyme to the cell wall are discussed . An open reading frame upstream of the putative lysin gene was found to be 177 base pairs longer than that reported for phage m13 . A codon usage table for the lysin genes of several phages as well as for reported gene sequences from L . lactis and lactococcal bacteriophages is presented. Lett Appl Microbiol, 1993 Aug, 17(2), 92 - 6 The regulation of expression of the Lactococcus lactis lactose operon; Griffin HG et al.; Translational gene fusions between the Escherichia coli beta-galactosidase (lacZ) gene and the Lactococcus lactis lactose operon were constructed such that transcription from the lactose operon promoter could be assessed by measuring beta-galactosidase activity . The level of beta-galactosidase activity was up to 2.5-fold lower when MG5267 cells, which contain a chromosomal copy of the lactose operon, were grown in glucose compared to those grown in lactose . A greater degree of repression was seen in cells containing the multi-copy plasmid-encoded repressor than in those with only the single-copy chromosomal gene, indicating that the repressor protein is at least partly responsible for the reduction in expression when the cells are grown in glucose (i.e . in the absence of inducer) . However, the beta-galactosidase activity was found to be 5.5-fold lower in glucose than in lactose in cells which lacked a fully functional lactose operon . The decrease in expression was shown to be due to glucose repression . The levels of expression when the cells were grown in glucose were considerably higher for MG5267 than for MG1363 suggesting perhaps that a product of the chromosomally-encoded operon in MG5267 has a positive effect on transcription. Biochem Biophys Res Commun, 1993 Jul 15, 194(1), 65 - 71 The primary structure of phosphofructokinase from Lactococcus lactis; Xiao Q et al.; The primary amino acid sequence of phosphofructokinase (EC2.7.1.11) from Lactococcus lactis, obtained by Edman analysis of peptides obtained from proteolytic digestions, is MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIF PLTSASVGDKIGRGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVI GGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDAL DKIRDTSSSHNRTFVVEVMGRNAGDIALNAGIAAGADDISIPELEFKFEN VVNNINKGYEKGKNHHIIIVAEGVMTGEEFATKLKEAGYKGDLRVSVLGH IQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGT AEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNL. J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1503 - 9 Association of the lactococcin A immunity factor with the cell membrane: purification and characterization of the immunity factor; Nissen-Meyer J et al.; The physicochemical characteristics of the lactococcin A immunity protein, as deduced from its gene sequence, were used to devise a procedure for its purification . The protein was purified from cell extracts by cation-exchange and reverse-phase chromatography . As judged from the amino acid composition and amino acid sequencing, the immunity protein is not post-translationally processed by cleavage at its N- or C-terminus . Consequently, the absorption coefficient at 280 nm, the isoelectric point, and the molecular mass of the immunity protein may be calculated to be, respectively, 8.2 x 10(3) M-1 cm-1, 10.2 and 11,163 Da from the amino acid sequence predicted from the nucleotide sequence . The immunity protein is a major cell protein component--one cell may contain (to an order of magnitude) 10(5) molecules--and it is in part associated with the cell membrane, as judged by immunoblot analysis of membrane vesicle-associated proteins . Exposing lactococcin-A-sensitive cells to an excess of the immunity protein did not affect the lactococcin-A-induced killing of the cells, indicating that the immunity protein does not protect cells by simply binding to lactococcin A, nor to externally exposed domains on the cell surface . Exposing immune-positive cells to antiserum against the immune protein did not sensitize the cells to lactococcin A, suggesting that the immunity protein in fact does not act extracellularly. J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1495 - 501 The use of bacterial luciferase genes as reporter genes in Lactococcus: regulation of the Lactococcus lactis subsp . lactis lactose genes; Eaton TJ et al.; Lactose metabolism is an important industrial trait in dairy lactococci . In Lactococcus lactis, lactose is taken up via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and is subsequently metabolized via the glycolytic and tagatose 6-phosphate pathways . Genes for the lactose-specific PEP-PTS proteins, phospho-beta-galactosidase and tagatose 6-phosphate pathway enzymes are encoded by a single 8 kb operon, lacABCDFEGX, and there is a divergently transcribed lacR repressor gene . Transcriptional fusions of both the lac operon promoter and the lacR promoter to the luxAB genes of Vibrio fischeri were used to investigate the regulation of expression of both promoters . In vivo bioluminescence assays demonstrated that lacR negatively regulates the lac operon and also autoregulates itself . Induction of transcription occurred for both promoters during growth on lactose: sevenfold for lacR and fivefold for the lac operon . The lacR promoter was demonstrated to be a particularly strong promoter, being approximately four times more efficient than the lac operon promoter . Both promoters provide good potential for the inducible expression of foreign proteins in Lactococcus. J Bacteriol, 1993 Jul, 175(14), 4383 - 90 Gene inactivation in Lactococcus lactis: branched-chain amino acid biosynthesis; Godon JJ et al.; The Lactococcus lactis subsp . lactis strains isolated from dairy products are auxotrophs for branched-chain amino acids (leucine, isoleucine, and valine), while most strains isolated from nondairy media are prototrophs . We have cloned and sequenced the leu genes from one auxotroph, IL1403 . The sequence is 99% homologous to that of the prototroph NCDO2118, which was determined previously . Two nonsense mutations and two small deletions were found in the auxotroph sequence, which might explain the branched-chain amino acid auxotrophy . Nevertheless, the leu genes from the auxotroph appear to be transcribed and regulated similarly to those from the prototroph. Lett Appl Microbiol, 1993 Jul, 17(1), 25 - 8 The nucleotide sequence for the replication region of pVS40, a lactococcal food grade cloning vector; von Wright A et al.; The replication region of a limited host range lactococcal vector, pVS40, was located within a 2359 bp EcoRI--ClaI fragment . Within this fragment a sequence for a 1197 bp reading frame coding for a 46,826 Da protein together with a putative ribosomal binding site and the -10 and -35 promoter regions could be detected . Immediately upstream from the promoter were three complete and one nearly complete successive direct repeats (TATAGCGTATGAAAAAACTGTG), suggesting an origin of replication . The protein has considerable homologies to certain lactococcal plasmid replication proteins. J Bacteriol, 1993 Jul, 175(14), 4391 - 9 Gene inactivation in Lactococcus lactis: histidine biosynthesis; Delorme C et al.; Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine . Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains had this requirement . Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor . This indicates that adaptation to milk often results in histidine auxotrophy . The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested . A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames {ORFs}) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol . Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive . Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6 . In addition, several mutations were detected in the promoter region of the operon . Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain . The mutations detected account for the histidine auxotrophy of the analyzed strain . Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy. J Bacteriol, 1993 Jun, 175(11), 3628 - 35 High-efficiency gene inactivation and replacement system for gram-positive bacteria; Biswas I et al.; A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system . The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C . A nested set of L . lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb . Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome . We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement . Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy . More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene . Between 1 and 40% of cells underwent replacement recombination when no selection was applied . Chromosomal insertions and deletions were obtained in this way . These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector . This procedure is applicable to numerous gram-positive bacteria. Mol Microbiol, 1993 Jun, 8(6), 1155 - 62 Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge; Wells JM et al.; To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen . A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L . lactis . The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium . When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC . Mice immunized subcutaneously with L . lactis expressing TTFC were protected from lethal challenge with tetanus toxin . These results show for the first time that L . lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form. J Appl Bacteriol, 1993 Jun, 74(6), 629 - 36 Improved cloning vectors and transformation procedure for Lactococcus lactis; Wells JM et al.; Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1 . All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli . By cloning the E . coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E . coli . The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria . One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L . lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells . This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v . Although growth of L . lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency . Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength . These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L . lactis. J Dairy Sci, 1993 Jun, 76(6), 1514 - 9 B-cell mitogen produced by slime-forming, encapsulated Lactococcus lactis ssp . cremoris isolated from ropy sour milk, viili; Kitazawa H et al.; A substance, active as a B-cell mitogen, was isolated from the slime products produced by Lactococcus lactis ssp . cremoris KVS20 . The mitogenic substance was prepared by anion-exchange chromatography and gel filtration chromatography and then purified by proteinase digestion and HPLC . Chemical analysis determined that the mitogenic substance was a phosphopolysaccharide and consisted of rhamnose, glucose, galactose, and phosphorus . The activity of the mitogenic substance was higher than that of the slime products . The optimal concentration for the activity was approximately 120 micrograms/ml . The mitogenic substance also had substantial mitogenic activity to spleen cells from C3H/HeJ mice, which are resistant to lipopolysaccharide . The findings indicated that a B-cell mitogen different from lipopolysaccharide is produced from L . lactis ssp . cremoris KVS20. Gene, 1993 May 15, 127(1), 121 - 6 Cloning and sequencing of the Lactococcus lactis subsp . lactis groESL operon; Kim SG et al.; The operon (groESL) coding for the Lactococcus lactis subsp . lactis heat-shock proteins GroEL and GroES, has been isolated and its complete nucleotide (nt) sequence determined . A set of degenerate PCR primers, deduced from amino acids which are conserved in a number of prokaryotic GroELs, were synthesized and used to amplify a 957-bp fragment . This PCR fragment was used as a probe to isolate a 5.0-kb EcoRI chromosomally derived fragment . A region of this 5.0-kb EcoRI fragment was sequenced and revealed that the groES gene was located 5' to groEL . This sequence was then used to design a set of inverse PCR primers and a 2.5-kb HindIII fragment was cloned which contained the region 5' to groEL . The complete nt sequence of the groESL operon was determined from overlapping fragments . It revealed that the groESL operon was preceded by a stem-loop structure and the promoter appears similar to most L . lactis subsp . lactis and other Gram+ bacterial promoters . Northern analysis demonstrated that the groESL operon is under tight regulation and a dramatic induction of mRNA synthesis occurs within 15 min after heat shock. J Biolumin Chemilumin, 1993 May-Jun, 8(3), 147 - 52 Application of in vivo bioluminescence to the study of ionophoretic action; Simpson WJ; Ionophores (carbonyl cyanide m-chlorophenylhydrazone; valinomycin; and the hop-derived compounds colupulone, trans-isohumulone and trans-humulinic acid) reduced the rate of dodecanal-dependent light emission from IuxA/B-transformed cells of Lactococcus lactis subsp . diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose . This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non-destructive technique in real time. J Biolumin Chemilumin, 1993 May-Jun, 8(3), 141 - 5 Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis subsp . diacetylactis; Simpson WJ; High levels of constitutive aldehyde-dependent light emission were obtained from nongrowing cells of Lactococcus lactis subsp . diacetylactis F712 transformed with IuxA/B when they were suspended in buffered solutions . Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate . The major factor controlling light emission in such cells appears to be the intracellular pH value . Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission. J Bacteriol, 1993 May, 175(9), 2578 - 88 Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis; van der Meer JR et al.; Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome . A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L . lactis, resulting in the production of an extracellular nisin precursor peptide . This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it . This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide . Deletion studies showed that the nisR gene is essential for the production of this intermediate . The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators . The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor . Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin . A similar activation was obtained with whole cells but not with membrane-free extracts of L . lactis strains carrying Tn5276 in which the nisA gene had been inactivated . The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide. J Bacteriol, 1993 May, 175(9), 2541 - 51 Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase; Llanos RM et al.; The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported . The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively . The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh . Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host . The identity of ldh was established previously by the same approach (R . M . Llanos, A . J . Hillier, and B . E . Davidson, J . Bacteriol . 174:6956-6964, 1992) . Each of the genes is preceded by a potential ribosome binding site . The operon is expressed in a 4.1-kb transcript . The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon . The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons . This degree of bias suggests that the operon is highly expressed . The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L . lactis . For this reason, we have called the operon the las (lactic acid synthesis) operon. J Dairy Sci, 1993 May, 76(5), 1243 - 52 Copy number and location of insertion sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria; Polzin KM et al.; Genomic DNA from 49 lactococcal strains was screened by Southern hybridization for the presence and relative copy number of lactococcal insertion sequence ISS1: ISS1 was found in 47 of 49 strains giving 1 to 20 hybridizing bands per strain . Southern hybridizations of undigested plasmid DNA from 17 lactococcal strains probed with ISS1 and IS981 showed that ISS1 was present on plasmids in all 17 strains, whereas IS981 was present on plasmids in 14 of the 17 strains . Both insertion sequences were present primarily on larger plasmids (> 25 kb), and some plasmids contained copies of both insertion sequences . When probed with ISS1, Southern hybridizations of DNA isolated from Lactococcus lactis ssp . lactis ML3 frozen stock culture and from isolated colonies showed that the stock culture consisted of a mixture of cells having different ISS1-hybridizing bands, indicating that stock cultures may contain cells with varying locations of ISS1 sequences . The number of copies and their widespread distribution among lactococcal strains establish that insertion sequences will contribute significantly to genotypic and phenotypic events that may affect the industrial performance and stability of lactococcal strains. Appl Environ Microbiol, 1993 May, 59(5), 1430 - 6 Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp . cremoris Wg2; Tan PS et al.; The mode of action of purified aminopeptidase N from Lactococcus lactis subsp . cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed . The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry . Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed . The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein . The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture. J Chromatogr, 1993 Apr 23, 636(1), 63 - 8 Monitoring tripeptidase activity using capillary electrophoresis . Comparison with the ninhydrin assay; Mulholland F et al.; Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp . lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made . Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic . The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data . The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities . The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme. J Bacteriol, 1993 Apr, 175(7), 2087 - 96 Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase; Mierau I et al.; The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L . lactis subsp . cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced . pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L . lactis . The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found . By means of gene disruption, a pepO-negative mutant was constructed . Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L . lactis in milk. J Bacteriol, 1993 Apr, 175(7), 2052 - 9 Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis; Kunji ER et al.; Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined . The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E . J . Smid, A . J . M . Driessen, and W . N . Konings, J . Bacteriol . 171:292-298, 1989) . The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues . The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L . lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides . This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine . The oligopeptide transport system has a lower activity than the di-tripeptide transport system . Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate . The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate. Microbiologia, 1993 Apr, 9(1), 63 - 8 Phenotypic and phylogenetic evidence for a close relationship between Lactococcus garvieae and Enterococcus seriolicida; Domenech A et al.; Cultural, biochemical and protein profiling studies were performed on L . garvieae strains isolated from diseased rainbow trout and on the fish pathogen Enterococcus seriolicida ATCC 49156 . The results, confirmed by 16 rRNA sequence analyses, indicate that E . seriolicida ATCC 49156 should be reclassified in the genus Lactococcus . Contrary to previous reports, both L . garvieae and E . seriolicida were found to be beta-haemolytic. Appl Environ Microbiol, 1993 Mar, 59(3), 777 - 85 ScrFI restriction-modification system of Lactococcus lactis subsp . cremoris UC503: cloning and characterization of two ScrFI methylase genes; Davis R et al.; Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp . cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli . No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome . Neither clone encoded the cognate endonuclease . The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5) . The amino acid sequence contained the highly characteristic motifs of an m5C methylase . Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm. J Bacteriol, 1993 Mar, 175(6), 1745 - 55 Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage phi LC3 and construction of integration-negative phi LC3 mutants; Lillehaug D et al.; The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp . cremoris, were identified and characterized . The phi LC3 phage attachment site, attP, was mapped and sequenced . DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration . The attB core sequence is located within the C-terminal part of an open reading frame of unknown function . The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP . The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family . Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed. Eur J Biochem, 1993 Mar 1, 212(2), 417 - 22 In vitro pore-forming activity of the lantibiotic nisin . Role of protonmotive force and lipid composition; Garcera MJ et al.; Nisin is a lantibiotic produced by some strains of Lactococcus lactis subsp . lactis . The target for nisin action is the cytoplasmic membrane of Gram-positive bacteria . Nisin dissipates the membrane potential (delta psi) and induces efflux of low-molecular-mass compounds . Evidence has been presented that a delta psi is needed for nisin action . The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein . Nisin-induced efflux of carboxyfluorescein was observed in the absence of a delta psi from liposomes composed of Escherichia coli lipids or dioleoylglycerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios . The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios . Both delta psi (inside negative) and delta pH (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values . Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory . It is concluded that a delta psi is not essential, but that the total protonmotive force stimulates the action of nisin. Mol Microbiol, 1993 Mar, 7(6), 957 - 65 Two genes present on a transposon-like structure in Lactococcus lactis are involved in a Clp-family proteolytic activity; Huang DC et al.; The lactose-protease plasmid pUCL22 of Lactococcus lactis subsp . lactis strain CNRZ270 contained two inverted copies of IS 1076 flanking a region of 3.7 kb . This internal region was sequenced and found to contain two large open reading frames, ORF1 and ORFP in opposite orientations . ORF1 consists of 2289 bp; the deduced 763-amino-acid sequence is similar to the ATPases of the ClpA family . It contains two well-conserved consensus ATP-binding sites . It was named ClpL . ORFP consists of 930 bp encoding a protein of 310 amino acids . No similarity with any known protein was found in GenBank data for ORFP . Increased ATP-dependent proteolytic activity was detected in extracts from Escherichia coli cells expressing the clpL and ORFP genes. Protein Eng, 1993 Feb, 6(2), 201 - 6 Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression; van Rooijen RJ et al.; Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate . A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer . Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine . The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose . Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR . Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response . The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate. J Appl Bacteriol, 1993 Feb, 74(2), 134 - 42 Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus pentosaceus; Skytta E et al.; The broad-spectrum antibacterial activity exhibited by three Pediococcus strains isolated from beer was preliminarily characterized . Factors affecting the production rate of bacterial inhibitors were screened and the effects of simultaneous cultivation of Lactococcus and Pediococcus on the production of inhibitory substances were studied . The antibacterial activity against a range of Gram-negative test organisms was not affected by catalase or proteolytic enzymes and was extremely thermotolerant . Production of the inhibitors was maximal between pH 6 and pH 7 . A growth medium containing unhopped end-fermented wort was beneficial for the production of inhibitors, particularly by the Pediococcus damnosus strain, and anaerobic growth conditions were preferable . The antagonistic activity against the Gram-negative test organism Salmonella infantis could be demonstrated after an incubation period of only 2 d if the Pediococcus and Lactococcus strains were incubated simultaneously as a mixed population. Biochemistry, 1993 Jan 12, 32(1), 310 - 8 15N- and 13C-labeled media from Anabaena sp . for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis; Sailer M et al.; A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described . Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp . ATCC 27899 cells grown on sodium {13C}bicarbonate and/or sodium {15N}nitrate as sole carbon and nitrogen sources . Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation . NMR analyses of {13C,15N}nisin A using H{13C}MQC, H{13C}MBC, 2D INADEQUATE, and H{15N}MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments . The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis . Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H{13C}MQC to unlabeled and {13C}leucocin A afforded the complete 1H and 13C assignment . Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions. Plasmid, 1993 Jan, 29(1), 70 - 3 Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis; Ward AC et al.; Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis . In L . lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid . Transconjugants of strain HID113 carrying this plasmid were unstable . Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome. Appl Environ Microbiol, 1993 Jan, 59(1), 330 - 3 Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp . cremoris AM2; Chapot-Chartier MP et al.; A gene coding for an aminopeptidase (PepC) from Lactococcus lactis subsp . cremoris AM2 was cloned by complementation of an Escherichia coli mutant lacking aminopeptidase activity . The nucleotide sequence was determined . A portion of the predicted amino acid sequence of PepC (436 amino acids) showed strong homology to the active site of cysteine proteases . No signal sequence was found, indicating an intracellular location of the enzyme. Appl Environ Microbiol, 1993 Jan, 59(1), 213 - 8 Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis; de Vos WM et al.; Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known . They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M . Mulders, I.J . Boerrigter, H.S . Rollema, R.J . Siezen, and W.M . de Vos, Eur . J . Biochem, 201:581-584, 1991) . The nisin variants were purified to apparent homogeneity, and their biological activities were compared . Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria . However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A . This was observed with all tested indicator strains . These results suggest that nisin Z has better diffusion properties than nisin A in agar . The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product . In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L . lactis strains analyzed. Anal Biochem, 1993 Jan, 208(1), 49 - 56 Molecular analysis of lipid macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic acids; Fischer W; Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(alpha 1-2)Glc(alpha 1-3)acyl2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate)lipoteichoic acid of Staphylococcus aureus with Glc(beta 1-6)Glc(beta 1-3)acyl2Gro as the sole lipid moiety . Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids . Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain . A similar fractionation was observed within the single peak of lipoteichoic acid III . With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased . Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed . Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition . The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans . Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup. J Dairy Sci, 1993 Jan, 76(1), 1 - 19 Transposable elements in Lactococci: a review; Romero DA et al.; Genetic studies have identified the presence of transposable elements within the genus Lactococcus, which includes industrially important microorganisms used in the production of fermented dairy products . Three insertion sequences have been fully characterized in addition to several reports of transpositionlike events . The three insertion sequence elements, ISS1, IS904, and IS981, exhibit the physical and genetic properties characteristic of known insertion sequences . They are closely related to insertion sequences isolated from a wide variety of microorganisms . In lactococci, insertion sequence elements are associated with lactose and sucrose metabolism, proteinase activity, nisin production and immunity, conjugal transfer determinants, and bacteriophage resistance, which are attributes significant for growth in a milk environment . The characteristics, involvement in lactococcal evolution, and recent developments as tools for genetic engineering of the lactococcal elements are discussed. Crit Rev Biochem Mol Biol, 1993, 28(3), 235 - 57 The MIP family of integral membrane channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the proteins; Reizer J et al.; The major intrinsic protein (MIP) of the bovine lens fiber cell membrane was the first member of the MIP family of proteins to be sequenced and characterized . It is probably a homotetramer with transmembrane channel activity that plays a role in lens biogenesis or maintenance . The polypeptide chain of each subunit may span the membrane six times, and both the N- and C-termini face the cell cytoplasm . Eighteen sequenced or partially sequenced proteins from bacteria, yeast, plants, and animals have now been shown to be members of the MIP family . These proteins appear to function in (1) metazoan development and neurogenesis (MIP and BIB), (2) water transport across the human erythrocyte membrane (ChIP), (3) communication between host plant cells and symbiotic nitrogen-fixing bacteria (NOD), (4) transport across the tonoplast membrane during plant seed development (alpha-TIP), (5) water stress-induced resistance to desiccation in plants (Wsi-TIP), (6) suppression of a genetic growth defect on fermentable sugars in yeast (FPS1), and (7) transport of glycerol across bacterial cell membranes (GlpF) . One other sequenced member of the MIP family (ORF1 of Lactococcus lactis) has no known physiological function . The biochemical functions of the eukaryotic proteins are not well established . Computer analyses have revealed that the first and second halves of all MIP family proteins probably arose by a tandem, intragenic, duplication event . Thus, the primary structure of putative transmembrane helices 1 to 3 is similar to that of putative transmembrane helices 4 to 6 even though they are of opposite orientation in the membrane . Among the most conserved residues in these two repeated halves are a membrane-embedded glutamate (E) in helices 1 and 4, an asparagine-proline-alanine (NPA) sequence in the loops between helices 2 and 3 (cytoplasmically localized) and helices 5 and 6 (extracellularly localized), and a glycine within helices 3 and 6 . Statistical analyses suggest that the two halves of these proteins have evolved to serve distinct functions: the first half is more important for the generalized or common functions of these proteins, while the second half of these proteins is more differentiated to provide specific or dissimilar functions of the proteins . The apparent origin of MIP family proteins by duplication of a three-spanner precursor protein suggests an evolutionary origin distinct from other transport proteins with six transmembrane spanners.(ABSTRACT TRUNCATED AT 400 WORDS) DNA Seq, 1993, 4(3), 211 - 4 The cos region of lactococcal bacteriophage phi LC3; Birkeland NK et al.; The nucleotide sequence of the DNA regions flanking the cohesive end site of the temperate lactococcal bacteriophage phi LC3 was determined . Four pairs of sequence repeats of 8 to 13 base-pairs in length were revealed close to the cohesive ends . These repeats possibly represent signal sequences involved in phage DNA maturation and packaging . The start of an open reading frame was localized only 42 nucleotides from the left end of the phi LC3 phage double-stranded region, implying that transcription probably proceeds across the cos site . Compilation of the characteristics of the DNA termini of bacteriophages with complementary cohesive DNA ends suggests that phages of Gram-negative bacteria generally carry 5'-protruding single-strands whereas those of Gram-positive hosts carry 3'-protruding ends. Biosci Biotechnol Biochem, 1993 Jan, 57(1), 88 - 92 Genetic and molecular analysis of the rpoD gene from Lactococcus lactis; Araya T et al.; A gene of Lactococcus lactis ATCC19435, the product of which is homologous with the principal sigma factors of Escherichia coli and Bacillus subtilis, was cloned and sequenced . The deduced amino acid sequence of the 340-residue protein and the upstream open reading frame of the cloned gene showed a homology to B . subtilis sigma 43 factor (the rpoD product) and DNA primase (the dnaE product), respectively, suggesting that L . lactis also has the rpoD operon . Surprisingly, introduction of the cloned L . lactis rpoD gene into a rpoD temperature-sensitive mutant of E . coli caused partial complementation. J Biol Chem, 1992 Dec 15, 267(35), 25078 - 85 Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis; Liu W et al.; Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively . Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes . Although subtilin and nisin share considerable structural homology, subtilin has a greater tendency than nisin to undergo spontaneous inactivation . This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days . It was hypothesized that the R group carboxyl of Glu4 in subtilin participates in the chemical modification of the adjacent DHA5 . Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of subtilin to Ile, in order to eliminate this carboxyl-group participation . The DHA5 of this mutant subtilin (E4I-subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural subtilin, and the rate of loss of biological activity dropped by a like amount . These results suggest that an intact DHA5 is critical for subtilin activity against bacterial spore outgrowth . A double mutant of subtilin, in which the DHA5 residue of E4I-subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth . The specific activity of E4I-subtilin was 3-4-fold higher than natural subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity . These are the first mutants of subtilin that have been reported, and E4I-subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis . In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural subtilin gene is replaced by the mutant gene at the normal location in the chromosome . This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell. FEBS Lett, 1992 Dec 14, 314(2), 139 - 42 Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis; Chich JF et al.; The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified . The enzyme was labeled by {3H}DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC . The main radiolabeled peptide was sequenced . Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine . A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area . The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid. J Biol Chem, 1992 Dec 5, 267(34), 24340 - 6 Engineering dehydrated amino acid residues in the antimicrobial peptide nisin; Kuipers OP et al.; The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges . Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering . Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L . lactis as a host . Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR . Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used . In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis . This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties. Microb Releases, 1992 Dec, 1(3), 165 - 71 Colonization of the digestive tract of germ-free mice by genetically engineered strains of Lactococcus lactis: study of recombinant DNA stability; Gruzza M et al.; The ability of genetically engineered Lactococcus lactis strains to become established in the digestive tract (DT) of germ-free mice was examined together with the stability of their genetic markers . Seven L . lactis strains were genetically modified by insertion of genetic markers on different replicons: chloramphenicol resistance gene cat was carried by self-transmissible plasmid pIL205, a derivative of plasmid pIP501; erythromycin resistance gene erm, originating from pAM beta 1, was inserted into non-transmissible plasmids pIL252 and pIL253 of low and high copy number respectively; erm gene from plasmid pMS1.5B was inserted into the chromosome . All strains carried a common wild-type plasmid pIL9 involved in lactose fermentation . It was observed that the DT of mice was rapidly and efficiently colonized with either the inoculated parental strain or with its derivatives or with both of them, but plasmid-free derivatives were always at dominant levels . Both plasmids pIL9 and pIL205 were lost, but the parental strains and the plasmid-lacking derivatives were at codominant levels, indicating that there is an equilibrium between plasmid loss and plasmid transfer in the DT . Strains that carried non-transmissible and low copy number plasmid pIL252 were rapidly eliminated from the DT, which in turn was colonized with the respective pIL252-less derivatives; this is probably due to the high segregational instability of pIL252.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1992 Nov 2, 121(1), 55 - 61 Transcriptional regulation of the Tn5276-located Lactococcus lactis sucrose operon and characterization of the sacA gene encoding sucrose-6-phosphate hydrolase; Rauch PJ et al.; The Lactococcus lactis sucrose operon was located on the conjugative transposon Tn5276 and the nucleotide sequence of the sacA gene, encoding sucrose-6-phosphate hydrolase, and its surrounding regions was determined . Northern blot analysis showed that the sucrose operon contains two divergent transcriptional units of 3.2 and 3.6 kb, the expression of which is considerably higher in cells grown on sucrose than in cells grown on glucose . This was confirmed by primer extension studies which demonstrated that transcription is initiated at two sucrose-inducible promoters with a back-to-back organization . The 3.2-kb transcriptional unit includes the sacB gene which most probably encodes the sucrose-specific enzyme II of the phosphotransferase system, and may contain the gene encoding fructokinase . The 3.6-kb transcriptional unit includes genes sacA and sacR . The protein encoded by the sacR gene is likely to be involved in the regulation of the sac operon expression, since its deduced N terminus is homologous to helix-turn-helix DNA-binding domains found in several regulatory proteins. Appl Environ Microbiol, 1992 Nov, 58(11), 3730 - 43 Biosynthesis of the lantibiotic nisin: genomic organization and membrane localization of the NisB protein; Engelke G et al.; Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of peptide-antibiotics containing lanthionine bridges (lantibiotics) (N . Schnell, K.-D . Entian, U . Schneider, F . Gotz, H . Zahner, R . Kellner, and G . Jung, Nature {London} 333:276-278, 1988) . Nisin is ribosomally synthesized, and its structural gene, nisA, encodes a prepeptide that is posttranslationally modified, revealing the active lantibiotic (C . Kaletta and K.-D . Entian, J . Bacteriol . 171:1597-1601, 1989) . Adjacent to nisA, the additional genes nisB, nisT, and nisC were identified . Over their entire sequences, these genes were homologous to genes recently identified as important for the biosynthesis of lantibiotics, that is, subtilin from Bacillus subtilis ATCC 6633 and epidermin from Staphylococcus epidermidis Tu 3298 . Genes nisB, nisT, and nisC corresponded to open reading frames of 993, 600, and 418 amino acid residues, respectively . The nisT open reading frame is homologous to proteins of the HlyB (hemolysin B protein of Escherichia coli) subfamily . Proteins of this subfamily are responsible for the secretion of a variety of compounds, including large polypeptides, polysaccharides, and anti-drug tumors, indicating that NisT may be involved in nisin transport . Northern (RNA) blot analysis revealed a 0.3-kb transcript for the nisA structural gene, and the transcriptional start point of the nisA gene was determined by primer extension . Additionally, a mRNA of at least 3 kb was identified by using a hybridization probe specific to nisB . Antibodies were raised against the NisB protein, and Western blot (immunoblot) analysis revealed a molecular weight of about 115 kDa, which is in accordance with the theoretical protein size of 117.5 kDa as calculated from the nisB open reading frame . Several amphipathic transmembrane alpha-helices indicated that NisB is associated with the membrane . This was confirmed by preparing L . lactis vesicles . The NisB protein was tightly associated with the vesicle fraction and was released by sodium dodecyl sulfate treatment only . These results suggest that NisB is membrane associated and that nisin biosynthesis occurs at the cell membrane. Appl Environ Microbiol, 1992 Nov, 58(11), 3683 - 93 A lactococcal expression system for engineered nisins; Dodd HM et al.; The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants . Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity . However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity . Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions. Mol Gen Genet, 1992 Nov, 235(2-3), 359 - 64 Isolation of purine auxotrophic mutants of Lactococcus lactis and characterization of the gene hpt encoding hypoxanthine guanine phosphoribosyltransferase; Nilsson D et al.; Five purine auxotrophic mutants of Lactococcus lactis were isolated . L . lactis was capable of converting adenine, guanine and hypoxanthine to AMP, GMP and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activities . A 1.3 kb DNA fragment from L . lactis was cloned by complementation of the hpt mutation in Escherichia coli . Introduction of this fragment into L . lactis resulted in an increase in HGPRT activity . In vitro transcription and translation analysis showed that the fragment coded for a polypeptide with M(r) of 22,000 . The nucleotide sequence of this hpt gene was determined. J Dairy Sci, 1992 Nov, 75(11), 2946 - 51 B-cell mitogenic activity of slime products produced from slime-forming, encapsulated Lactococcus lactis ssp . cremoris; Kitazawa H et al.; The mitogenic activities of whole cell lyophylized preparations, cell-wall components, and slime products obtained from Lactococcus lactis ssp . cremoris KVS20 were examined on murine spleen cells . Whole cell lyophylized preparations and slime products significantly (P < .05) stimulated mitogenic responses of the cells . The highest activity was induced by slime products in which the optimal concentration was 116 microg/ml . The significant (P < .05) increase of mitogenic activity induced by slime products occurred at 24 h, and the peak response was obtained 48 h after the stimulation . The activity was much higher in the fraction enriched with B cells than in the fraction enriched with T cells . In addition, slime products induced mitogenic activity to spleen cells of athymic nu/nu mice . The chemical analysis of lipopolysaccharide and the minimal concentration for mitogenic response eliminated the possibility that the activity of slime products may be due to the contamination of lipopolysaccharide . The data demonstrate that slime products are a potent B-cell-dependent mitogen. Mol Microbiol, 1992 Nov, 6(21), 3213 - 23 Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712; Gasson MJ et al.; High-frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor . Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size . This suggested that more than simple cointegration with a sex factor plasmid was involved . Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA . Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids. J Bacteriol, 1992 Nov, 174(22), 7463 - 9 Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp . lactis ME2; Durmaz E et al.; The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp . lactis ME2 has been characterized and sequenced . The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system . Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L . lactis subsp . lactis LM0230 . However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host . Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die . Thus, phage infections in Prf+ cells are aborted . Prf is effective in both L . lactis subsp . lactis and L . lactis subsp . cremoris strains against several small isometric-headed phages but not against prolate-headed phages . The Prf determinant was localized by Tn5 mutagenesis and subcloning . DNA sequencing identified a 1,056-nucleotide structural gene designated abiC . Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e . abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L . lactis subsp . lactis ME2) and abi416 (Abi+, from L . lactis subsp . lactis IL416) . Unlike abiA, the action of abiC does not appear to affect DNA replication . Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle. J Bacteriol, 1992 Nov, 174(21), 6956 - 64 Cloning, nucleotide sequence, expression, and chromosomal location of ldh, the gene encoding L-(+)-lactate dehydrogenase, from Lactococcus lactis; Llanos RM et al.; A gene (designated ldh) that encodes fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase was cloned from Lactococcus lactis subsp . lactis . Plasmids containing ldh conferred fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase activity on Escherichia coli cells . This activity was conferred only when a promoter had been introduced into the plasmid to express the cloned ldh . The nucleotide sequence of ldh predicted a chain length of 324 amino acids and a subunit molecular weight of 34,910 for the enzyme, after removal of the N-terminal methionine residue . Northern analyses of L . lactis subsp . lactis RNA showed that a 4.1-kb transcript hybridized strongly with ldh and that 1.2- and 1.1-kb transcripts hybridized to much lesser extents . Promoter- and terminator-cloning studies in which we used the vectors pGKV210 and pGKV259 in L . lactis subsp . lactis revealed that the 5' flanking DNA of ldh is devoid of transcription initiation signals and that transcription entering the 3' flanking DNA from either direction is efficiently terminated . These data and the data from Northern analyses led to the conclusion that ldh is expressed as the 3' gene of the 4.1-kb transcript and suggested that posttranscriptional processing yielded the shorter transcripts . We determined that ldh is located on the L . lactis subsp . lactis chromosome between coordinates 1.619 and 1.669 of the previously reported physical map (D . L . Tulloch, L . R . Finch, A . J . Hillier, and B . E . Davidson, J . Bacteriol . 173:2768-2775, 1991). J Bacteriol, 1992 Nov, 174(21), 6752 - 62 Physical and genetic map of the chromosome of Lactococcus lactis subsp . lactis IL1403; Le Bourgeois P et al.; A combined physical and genetic map of the chromosome of Lactococcus lactis subsp . lactis IL1403 was determined . We constructed a restriction map for the NotI, ApaI, and SmaI enzymes . The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments . The strain IL1403 chromosome was found to be circular and 2,420 kb in size . A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L . lactis and various other bacteria . Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome . Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped . At least seven copies of IS1076 were present and were located on 50% of the chromosome . In contrast, no copy of ISS1RS was detected . Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction . A comparison of the physical maps of L . lactis subsp . lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters . In contrast, despite major restriction pattern dissimilarities between L . lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains. Res Microbiol, 1992 Nov-Dec, 143(9), 879 - 90 Regulation of nisin biosynthesis by continuous cultures and by resting cells of Lactococcus lactis subsp . lactis; Meghrous J et al.; Nisin production by Lactococcus lactis subsp . lactis has been investigated using lactose as carbon source . Whether or not continuous cultures were lactose-limited, maximum nisin titre was observed at an intermediate mu value with a sharp peak of activity between 0.2 and 0.3/h . The maximum specific growth rate obtained in the medium used was 0.6/h and the maximum titre of nisin at mu = 0.25/h (160 AU/ml) was about nine-fold higher as compared with activity obtained at a dilution rate of 0.05/h or 0.4/h . With a constant dilution rate of 0.25/h and varying initial lactose concentrations from 3 to 40 g/l, there is an increase in nisin biosynthesis with increasing lactose concentration correlated with higher rates of sugar consumption . A Ymax value of 0.2 g bacterial dry weight and a maintenance coefficient of 124 mg lactose/g bacterial dry weight/h were determined . Lactose consumption increased from 1 to 3.28 g of lactose/g (dry wt) of cell mass/h and the nisin titre from 12.5 to 164.2 AU/ml . At higher values, nisin production declined . This implies that biosynthesis of nisin is regulated by a system of repression and derepression . Addition of lanthionine and beta-methyllanthionine precursors to the medium decreased the nisin titre when either threonine, threonine-cysteine, or cysteine-serine-threonine was added at the optimal dilution rate of 0.25/h; however, simultaneous addition of serine and cysteine elicited a slight increase in nisin activity . Studies with resting cells confirm that the biosynthesis of nisin is tightly regulated, since the production rate can be 5.6-fold higher than in cells grown in continuous culture . In addition, cell-adhered nisin appears to play a role in the production of the enzyme: low levels of cell-adhered nisin elicited high production rates, whereas high levels were not associated with nisin biosynthesis . In addition to pH, magnesium sulphate and lactose concentrations, nitrogen sources were also able to interfere in cell-adherence nisin. Biochimie, 1992 Nov, 74(11), 1007 - 17 Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins of Lactococci, Streptococci, and Pediococci; Limas Nzouzi N et al.; Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins are equally effective procedures for detecting differences and similarities in the Lactococci, Streptococci and Pediococci examined . Electrophoretic distribution patterns of ribosomal subunit proteins may be a useful tool in taxonomic studies. J Clin Microbiol, 1992 Oct, 30(10), 2657 - 61 Description and evaluation of the semiautomated 4-hour rapid ID 32 Strep method for identification of streptococci and members of related genera; Freney J et al.; The rapid ID 32 Strep system (bioMerieux, La Balme les Grottes, France) is a new system which allows the identification in 4 h of most streptococci and members of related genera encountered in medical and veterinary bacteriology . Four hundred thirty-three isolates first identified by conventional methods and belonging to the genera Streptococcus, Lactococcus, Enterococcus, Aerococcus, Gemella, Leuconostoc, Erysipelothrix, Gardnerella, and Listeria were tested . Overall, rapid ID 32 Strep correctly identified 413 (95.3%) of the strains, with 109 (25.1%) requiring extra tests for complete identification . Sixteen strains (3.7%) were not identified, and 4 (1.0%) were misidentified . The rapid ID 32 Strep system is a suitable alternative for rapid identification of members of the genus Streptococcus and of related genera. J Bacteriol, 1992 Oct, 174(20), 6699 - 702 Determination of the sequence of spaE and identification of a promoter in the subtilin (spa) operon in Bacillus subtilis; Chung YJ et al.; An 851-residue open reading frame (ORF) called SpaE has been discovered in the subtilin (spa) operon . Interruption of this ORF with a chloramphenicol acetyltransferase gene destroys the ability of Bacillus subtilis LH45 delta c (a derivative of B . subtilis 168) to produce subtilin, which is an antimicrobial peptide belonging to the class of ribosomally synthesized peptide antibiotics called lantibiotics . SpaE shows strong homology to NisB, which is in the nisin (nis) operon in Lactococcus lactis ATCC 11454 . Despite the strong sequence homology between SpaE and NisB, the spaE and nisB genes occupy very different locations in their respective operons, indicating that they have been evolving separately for a long time . Primer extension analysis was employed to identify a promoter upstream from the spaE gene, which appears to define the 5' end of the spa operon, which contains four other ORFs (Y . J . Chung, M . T . Steen, and J . N . Hansen, J . Bacteriol . 174:1417-1422, 1992). J Bacteriol, 1992 Oct, 174(20), 6580 - 9 Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp . lactis; Godon JJ et al.; The genes for biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine in Lactococcus lactis subsp . lactis NCDO2118 were characterized by cloning, complementation in Escherichia coli and Bacillus subtilis, and nucleotide sequence analysis . Nine structural genes are clustered on a 12-kb DNA fragment in the order leuABCD ilvDBNCA . Upstream of these genes, the nucleotide sequence suggests the existence of regulation by transcriptional attenuation . Between the leuD and ilvD genes is an unexpected gene, encoding a protein which belongs to the ATP-binding cassette protein superfamily. J Bacteriol, 1992 Oct, 174(20), 6571 - 9 Histidine biosynthesis genes in Lactococcus lactis subsp . lactis; Delorme C et al.; The genes of Lactococcus lactis subsp . lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing . Complementation of E . coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B . subtilis hisH gene (the E . coli hisC equivalent) allowed localization of the corresponding lactococcal genes . Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon . The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis . The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance . The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases. J Bacteriol, 1992 Oct, 174(20), 6563 - 70 Tryptophan biosynthesis genes in Lactococcus lactis subsp . lactis; Bardowski J et al.; The Lactococcus lactis chromosomal region containing the seven structural genes required for tryptophan biosynthesis was characterized by cloning and sequencing . All of the trp genes were identified by the homology of their products with known Trp proteins from other organisms . The identification was confirmed for five genes by their ability to complement trp mutations in Escherichia coli . The seven structural genes are present in the order trpEGDCFBA and span a 7,968-bp segment . Each gene is preceded by a putative ribosome binding site complementary to the 3' end of the L . lactis 16S rRNA . Three pairs of genes (trpG-trpD, trpC-trpF, and trpB-trpA) overlap, and there is intercistronic spacing of 124, 46, and 585 bp between the trpE-trpG, trpD-trpC, and trpF-trpB gene pairs, respectively . No gene fusion was found . Upstream of the trp genes, a 457-bp noncoding DNA segment contains several regions fitting the consensus for gram-positive promoters and one region strongly resembling a transcription terminator . However, it seems unlikely that an attenuation mechanism similar to the one found in E . coli regulates tryptophan biosynthesis in L . lactis, since no potential leader peptide was detected . We propose that a mechanisms resembling that described in Bacillus spp . can regulate trp genes expression in L . lactis. J Bacteriol, 1992 Oct, 174(19), 6152 - 8 Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster; Jagusztyn-Krynicka EK et al.; DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase . The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment . Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products . Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes . Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits . The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively . These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis. Gene, 1992 Sep 21, 119(1), 145 - 6 Sequence encoding ribosomal protein L33 of Lactococcus lactis; Koivula T et al.; A cloned fragment from Lactococcus lactis chromosome encoding the L33 ribosomal protein was sequenced . Two incomplete open reading frames (ORFs) were also found: the upstream ORF shows similarity to the tetracycline-resistance protein (Tet) of Bacillus stearothermophilus, and the downstream ORF shows homology to a protein of Bacillus subtilis participating in sporulation (SpoVE), and to proteins of Escherichia coli involved in cell division (FtsW) and the maintenance of cell shape (RodA). FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 135 - 41 Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis; Harrington A et al.; Lactococcus lactis subsp . lactis biovar . diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1 . Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721 . The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33 . The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain . Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system . The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study. J Bacteriol, 1992 Sep, 174(17), 5686 - 92 A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides; Nissen-Meyer J et al.; A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL-4B, and reverse-phase chromatography . The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained . The bacteriocin activity was associated with three peptides, termed alpha 1, alpha 2, and beta, which were separated by reverse-phase chromatography . Judging from their amino acid sequences, alpha 1 and alpha 2 were the same gene product . Differences in their configurations presumably resulted in alpha 2 having a slightly lower affinity for the reverse-phase column than alpha 1 and a reduced bacteriocin activity when combined with beta . Bacteriocin activity required the complementary action of both the alpha and the beta peptides . When neither alpha 1 nor beta was in excess, about 0.3 nM alpha 1 and 0.04 nM beta induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio . As judged by the amino acid sequence, alpha 1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long) . Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long) . Molecular weights of 4,376 and 4,109 for alpha 1 and beta, respectively, were obtained by mass spectrometry . The N-terminal halves of both the alpha and beta peptides may form amphiphilic alpha-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism . The C-terminal halves of both peptides consist largely of polar amino acids. Infect Immun, 1992 Sep, 60(9), 3739 - 46 Genetic analysis of scrA and scrB from Streptococcus sobrinus 6715; Chen YY et al.; A DNA fragment containing scrA and scrB, which encode enzyme II of the phosphoenolpyruvate-dependent sucrose phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, was isolated from a lambda gt10 genomic DNA library of Streptococcus sobrinus 6715 . Both genes were located on a 4.2-kb DNA fragment which was maintained stably in Escherchia coli on low-copy-number vector pGB2 . The recombinant E . coli clone expressed sucrose-hydrolytic activity on MacConkey agar base supplemented with raffinose or sucrose . Results from deletion analysis showed that the sucrose-metabolic activity was contained within a 3.5-kb region . The lactic acid bacterium Lactococcus lactis subsp . lactis LM0230, which is devoid of sucrose-metabolic activity, was used to study the enzyme activities encoded by scrA and scrB from S . sobrinus 6715 . L . lactis transformants carrying the 4.2-kb S . sobrinus-derived DNA fragment on E . coli-Streptococcus shuttle vector pDL278 were able to grow at the expense of sucrose and exhibited enzyme II and sucrose-6-phosphate hydrolase activities . Results from hybridization studies and a comparison of the restriction endonuclease maps of the scrA- and scrB-containing chromosomal regions from S . mutans GS5 and S . sobrinus 6715 suggested considerable divergence. J Dairy Sci, 1992 Sep, 75(9), 2344 - 52 Growth and activities of Lactococcus lactis in milk enriched with low mineral retentate powders; St-Gelais D et al.; The growth and activities of three strains of Lactococcus lactis ssp . cremoris (Wg2, E8, and HP) and their proteinase-negative variants were studied in skim milk enriched with three types of retentate powder . The performance of these strains in enriched milks was compared with that determined in reconstituted skim milk . Proteinase-positive strains of L . lactis ssp . cremoris exhibited higher maximum specific growth rates than protease-negative variants . Moreover, maximum specific growth rates of lactococci were lower in skim milk than in enriched milk with a high buffering capacity . The performance of proteinase-positive strains was better than that of proteinase-negative variants . Growth of proteinase-positive lactococci in milk media increased alpha-amino groups as determined by the increase of equivalent glutamic acid concentration . Available alpha-amino groups decreased with proteinase-negative variants . Proteinase-positive strain Wg2 exhibited the most proteolytic activity but showed the least specific overall productivity of lactic acid despite high biomass concentration in milk . Among proteinase-positive lactococci, strain E8 produced more lactic acid than other strains, and, among proteinase-negative variants, strain HP had the best specific overall productivity of lactic acid. Appl Environ Microbiol, 1992 Sep, 58(9), 3142 - 9 Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L . lactis; van der Vossen JM et al.; The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp . cremoris Wg2 were mapped by primer extension . The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2 . In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259 . The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L . lactis . Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription . The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L . lactis Wg2. Mol Gen Genet, 1992 Sep, 234(3), 401 - 11 Protein export elements from Lactococcus lactis; Perez-Martinez G et al.; Broad-host-range plasmids carrying alpha-amylase or beta-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences . Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli . Several of the selected export elements were also active in Bacillus subtilis and L . lactis, although the efficiencies depended strongly on the host organism and reporter gene used . The export elements AL9 and BL1 were highly efficient in L . lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis alpha-amylase and E . coli TEM-beta-lactamase) . AL9 even permitted growth of this organism on starch as the sole carbon source . Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides . The putative expression signals had a limited similarity to previously described expression signals for E . coli, B . subtilis and L . lactis . Differences in both expression and export efficiency are likely to underlie the host-specific effects. Gene, 1992 Sep 1, 118(1), 115 - 20 Location, characterization and expression of lytic enzyme-encoding gene, lytA, of Lactococcus lactis bacteriophage phi US3; Platteeuw C et al.; Gene lytA, which encodes lytic enzyme (LytA), of the isometric Lactococcus lactis bacteriophage phi US3, was cloned and expressed in Escherichia coli . The lytA gene was located on the physical map of the phi US3 32-kb DNA that contains cohesive ends . Initial expression of lytA was detected by lysis of an overlay of cells of the phage-sensitive strain, L . lactis SK112 . However, LytA appeared to have a broad spectrum and induced lysis in more than 30 different lactococcal strains . The nucleotide sequence of lytA showed a single open reading frame (ORF) of 774 bp encoding a protein of 258 amino acids (aa) with a calculated M(r) of 28,977 . This is in agreement with the size of 29 kDa as determined for LytA produced in E . coli using a T7 expression system . The lytA gene is preceded by an ORF that may code for a hydrophobic peptide of 66 aa containing a putative secretion signal, and two putative transmembrane helices . The deduced aa sequence of the phage phi US3 LytA shows similarities to that of the autolysin of Streptococcus pneumoniae which is known to be an amidase. J Bacteriol, 1992 Sep, 174(18), 5840 - 7 Transfer of Tn916 between Lactococcus lactis subsp . lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363; Bringel F et al.; Lactococcus lactis subsp . lactis MG1363 can act as a conjugative donor of chromosomal markers . This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff) . Using inter- and intrastrain crosses, we identified other L . lactis strains (LMO230 and MMS373) that appear to lack Laff . The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome . The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L . lactis cannot act as a donor in Tn916-promoted conjugation (F . Bringel, G . L . Van Alstine, and J . R . Scott, Mol . Microbiol . 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host . In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor . In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L . lactis . Thus, it is possible that Clu and Laff are identical. Appl Environ Microbiol, 1992 Aug, 58(8), 2674 - 8 Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp . lactis recA gene; Duwat P et al.; Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used