Lactococci

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 in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp . lactis ML3 . Using this fragment, we recovered and sequenced the entire lactococcal recA gene . The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.

Appl Environ Microbiol, 1992 Aug, 58(8), 2479 - 84
Characterization of Lactococcus lactis phage antigens; Schouler C et al.; Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail) . In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector . Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli . Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined . Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope . This epitope was very immunogenic . Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter . Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope . Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.

J Ind Microbiol, 1992 Aug, 10(2), 71 - 8
Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures; Kim JH et al.; The strategy and implementation of a unique system for engineering bacteriophage resistant starter cultures of Lactococcus lactis employing antisense RNA is reviewed . As a necessary prerequisite for developing this system, we have cloned and sequenced a number of bacteriophage genes coding for minor and major structural proteins . In addition, we have also identified a series of genes whose function(s) is not known but their sequences appear to be conserved in a vast number of isolates . One of these latter sequences, designated gp51C, codes for a 51-kDa protein which is extremely charged and shares some homology with yeast translation initiation factor . Resistance to a broad class of isometric bacteriophages has been achieved by expression of an antisense RNA targeted against, for example, gp51C . In the best case, expression of the antisense gp51C RNA results is a greater than 99% reduction in the total number of plaque forming units . Additional antisense RNA constructs directed against other bacteriophage genes, including the major capsid protein, also appear effective at inhibiting infection from 40-55% suggesting that this approach may prove useful for engineering a set of truly isogenic strains to be used in a starter culture rotation plan.

FEBS Lett, 1992 Jul 13, 306(1), 9 - 16
Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N; Tan PS et al.; The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined . The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase . The amino acid sequence of the aminopeptidase of L . lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical . Also, a highly conserved area was identified which has similarity with the active site of thermolysin . A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch . Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

Biochim Biophys Acta, 1992 Jul 8, 1108(1), 31 - 9
Effect of the unsaturation of phospholipid acyl chains on leucine transport of Lactococcus lactis and membrane permeability; In 't Veld G et al.; The effect of the degree of unsaturation of the phospholipid acyl chains on the branched-chain amino acid transport system of Lactococcus lactis was investigated by the use of a membrane fusion technique . Transport activity was analyzed in hybrid membranes composed of equimolar mixtures of synthetic unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in which the number of cis double bonds in the 18-carbon acyl chains was varied . The accumulation level and initial rate of both counterflow and protonmotive-force driven transport of leucine decreased with increasing number of double bonds . The reduction in transport activity with increasing number of double bonds correlated with an increase in the passive permeability of the membranes to leucine . The membrane fluidity was hardly affected by the double bond content . It is concluded that the degree of lipid acyl chain unsaturation is a minor determinant of the activity of the branched chain amino acid transport system, but effects strongly the passive permeability of the membrane.

Carbohydr Res, 1992 Jul 2, 231, 273 - 91
Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk; Gruter M et al.; The structure of the exopolysaccharide of Lactococcus lactis subsp . cremoris H414, isolated from a defined medium or skimmed milk, was established by linkage analysis on the native polysaccharide, and by characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, FABMS, EIMS, and 1H-NMR spectroscopy . The polysaccharide has the branched-pentasaccharide repeating unit: {formula: see text}

J Bacteriol, 1992 Jul, 174(14), 4838 - 41
Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp . lactis bv . diacetylactis; Snoep JL et al.; The pyruvate dehydrogenase complex of Lactococcus lactis subsp . lactis bv . diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate . The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively . The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa) . The L . lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate . Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity . The native enzyme measures 172 kDa and consists of 62-kDa monomers . The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.

J Dairy Sci, 1992 Jul, 75(7), 1761 - 7
Bacteriophage resistance in Lactococcus lactis ssp . lactis using antisense ribonucleic acid; Kim SG et al.; Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp . lactis strain renders it resistant to bacteriophage infection . Two open reading frames have been identified in a L . lactis ssp . lactis bacteriophage that are conserved in a majority of isolates . They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages . The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions . Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp . cremoris Wg2 promoter, p59 . These antisense RNA-producing vectors inhibited the efficiency of plaquing of L . lactis ssp . lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape . The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.

Int J Food Microbiol, 1992 Jul, 16(3), 227 - 36
Is thermotolerance correlated to heat-shock protein synthesis in Lactococcus lactis subsp . lactis?
Boutibonnes P, Tranchard C, Hartke A, Thammavongs B, Auffray Y.
Exposure of Lactococcus lactis subsp . lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min) . This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps) . Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis . These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L . lactis subsp . lactis.

J Appl Bacteriol, 1992 Jun, 72(6), 479 - 85
An investigation of dye reduction by food-borne bacteria; Learoyd SA et al.; The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations . Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all . There was also considerable variation in the rates of reduction with any single dye/organism combination . Glucose stimulated the rates of endogenous dye reduction in about half of the organisms . For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli, dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect . In Lactococcus lactis, dye reduction was stimulated by sugars but not by organic acids . Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption . All the organisms showed little cytosolic dye reduction, except L . lactis which showed substantial rates of reduction of some dyes by this fraction . With the membrane fraction of E . coli and Ps . fluorescens, cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.

Appl Environ Microbiol, 1992 Jun, 58(6), 1952 - 61
Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp . lactis biovar diacetylactis WM4; Stoddard GW et al.; The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp . lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis . The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation . DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A . The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA . The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems . lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators . LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus . The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs . On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.

Appl Microbiol Biotechnol, 1992 Jun, 37(3), 364 - 8
Batch cultures of recombinant Lactococcus lactis subsp . lactis in a stirred fermentor . II . Plasmid transfer in mixed cultures; el Alami N et al.; The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp . lactis (donor strains) with L . lactis subsp . lactis CNRZ 268M3 (recipient strain) . Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome) . Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10(-7) to 10(-8) after 12 h of fermentation . These frequencies were 60-400 times lower than in unstirred M17 broth and 100,000 times lower than on agar medium . In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10(-5) - 10(-6).

Appl Microbiol Biotechnol, 1992 Jun, 37(3), 358 - 63
Batch cultures of recombinant Lactococcus lactis subsp . lactis in a stirred fermentor . I . Effect of plasmid content on bacterial growth and on genetic stability in pure cultures; el Alami N et al.; The effect of plasmid introduction into Lactococcus lactis subsp . lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures . The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome) . Growth and acidification of L . lactis subsp . lactis IL2661 were similar to those of the derived recombinant lactococci . The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1 . Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively . Different levels of plasmid stability were detected . Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture . A slight loss (1-2%) of pIL205 was observed in all strains . In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells . This result suggested an incompatibility between pIL205 and pIL252 or pIL253.

J Biol Chem, 1992 May 5, 267(13), 8971 - 6
Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae; van der Rest ME et al.; The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D . (1985) EMBO J . 4, 1599-1603) and encodes a 446-amino acid protein . Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions . The delta pH (pH gradient across the membrane) is the major driving force for uptake . It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K . pneumoniae (van der Rest, M . E., Abee, T., Molenaar, D., and Konings, W . N . (1990) Eur . J . Biochem . 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions . The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments . These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop . The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp . lactis var . diacetylactis (CitP) (David, S., van der Rest, M . E., Driessen, A . J . M., Simons, G., and de Vos, W . M . (1990) J . Bacteriol . 172, 5789-5794) and less similar to CitH of K . pneumoniae . We conclude that the citS gene of K . pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.

J Gen Microbiol, 1992 May, 138 ( Pt 5), 945 - 50
Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A; Powell IB et al.; Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis . Infection of L . lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min . A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored . The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA . Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Appl Environ Microbiol, 1992 May, 58(5), 1772 - 5
Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels; Cancilla MR et al.; Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis . Closely related strains produced almost identical fingerprints . Fingerprints of other strains showed only some similarities.

Appl Environ Microbiol, 1992 May, 58(5), 1670 - 6
Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin; Gireesh T et al.; Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp . lactis with derivatives of L . lactis subsp . lactis LM0230 . The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character . Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process . The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments . ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).

Appl Environ Microbiol, 1992 May, 58(5), 1477 - 83
Characterization of two nisin-producing Lactococcus lactis subsp . lactis strains isolated from a commercial sauerkraut fermentation; Harris LJ et al.; Two Lactococcus lactis subsp . lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin . LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3 . NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946 . NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946 . Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946 . Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C . Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin . A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L . lactis subsp . lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII . The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402 . A different hybridization pattern was observed when the same probe was used against Nip+ L . lactis subsp . lactis ATCC 11454 and ATCC 7962 . These phenotypic and genetic data confirmed that unique Nip+ L . lactis subsp . lactis strains were isolated from fermenting sauerkraut.

Appl Environ Microbiol, 1992 May, 58(5), 1429 - 34
Lactococcus lactis release from calcium alginate beads; Champagne CP et al.; Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined . Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads . Rinsing the beads between fermentations did not influence the numbers of free cells in the milk . Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading . Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations . A double coating of PLL and alginate reduced cell release by a factor of approximately 50 . However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads . Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained . Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol . Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.

Int J Biochem, 1992 May, 24(5), 707 - 18
Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp . lactis NCDO 763; Monnet V et al.; 1 . The specificity of the cell envelope-located proteinase of Lactococcus lactis subsp . lactis NCDO 763 towards caseins has been submitted to a statistical study . Positive and negative relations have been evidenced between several amino acids and positions P6 to P'2 of the cleaved bonds . 2 . Fragment 1-23 of alpha s1 and oxidized B chain of insulin are well cleaved by the proteinase while CMP (fragment 106-169 of kappa-casein) is a poor substrate . 3 . Comparison with other cell envelope-located proteinase has been done . The enzyme of the strain 763 hydrolyses alpha s1-casein and fragment 1-23 of alpha s1-casein as the enzyme of the strain Sk11 and beta-casein as the enzyme of the strain Wg2 . 4 . The specificity of these proteinases and the comparison of their amino acid sequences let us postulate a more complex substrate binding area for these lactococcal proteinases than for the subtilisin.

J Bacteriol, 1992 May, 174(10), 3118 - 24
The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis; Molenaar D et al.; Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5{and -6}-carboxyfluorescein (BCECF) (D . Molenaar, T . Abee, and W . N . Konings, Biochim . Biophys . Acta 1115:75-83, 1991) . This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found . This transport system is most likely driven by ATP or a related compound . The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide . Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated . Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter . The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.

Biosci Biotechnol Biochem, 1992 May, 56(5), 704 - 7
Catalytic properties of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp . cremoris nTR; Yan TR et al.; An X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) was identified to be loosely bound on the inner cell membrane fraction of Lactococcus lactis subsp . cremoris nTR . The biosynthesis of X-PDAP was continuously increased before the late-log growth phase of the bacteria . Both Gly-Pro-pNA and Ala-Ala-pNA were hydrolyzed by X-PDAP; the kcat/Km value of the former was about 10-fold that of the latter . The Ki of X-Pro and Pro-X were more specific to X-PDAP than those of X-Ala . The enzyme splitting a dipeptide sequentially from beta-casomorphin as a model catalytic pattern was identified and some properties of the enzyme were further characterized.

Appl Microbiol Biotechnol, 1992 May, 37(2), 216 - 24
Lysozyme expression in Lactococcus lactis; van de Guchte M et al.; Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp . lactis . Hen egg white lysozyme (HEL) could be detected in L . lactis lysates by Western blotting . No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L . lactis product . The functionally related lysozymes of the E . coli bacteriophages T4 and lambda were produced as biologically active proteins in L . lactis . In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin . Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

J Dairy Res, 1992 May, 59(2), 169 - 75
Antibacterial and antiviral activity of camel milk protective proteins; el Agamy EI et al.; Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk . The activity of these protective proteins was assayed against Lactococcus lactis subsp . cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus . Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented . The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ . Bovine and camel milk LF antibacterial activity spectra were similar . The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures . The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk . The LP system was ineffective against rotavirus.

J Gen Microbiol, 1992 Apr, 138 ( Pt 4), 709 - 18
Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp . lactis UC317; Law J et al.; The proteinase genes from Lactococcus lactis subsp . lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain . The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L . lactis subsp . lactis SK112 . This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively . Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L . lactis strains SK112, Wg2 and NCDO763 . In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2 . These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.

Gene, 1992 Apr 1, 113(1), 107 - 12
Sequence of a gene (lap) encoding a 95.3-kDa aminopeptidase from Lactococcus lactis ssp . cremoris Wg2; Stroman P; A gene (lap) coding for a Lactococcus lactis ssp . cremoris Wg2 aminopeptidase was cloned from genomic libraries of size-fractionated lactococcal DNA . The 5' end of the lap gene was isolated by using a polymerase chain reaction hybridization probe of 77 nucleotides (nt) synthesized from two degenerate primers derived from the N-terminal amino acid (aa) sequence of the lactococcal lysine-aminopeptidase (LAP) . The remaining part(s) of the gene were recovered by a search for overlapping sequences in Southern blots of variably restricted genomic DNA . The complete nt sequence of the lap gene has been determined . A large open reading frame of 2538 nt is predicted to encode a polypeptide of 846 aa (approx . 95.3 kDa; pI, 5.93) . A recombinant plasmid containing the lap gene with its flanking sequences was shown to direct in vivo synthesis of LAP activity in Escherichia coli, indicating that the cloned DNA fragment is the lap gene . Primer extension analysis of lap mRNA and Northern blot hybridization indicated the gene transcript to be approx . 3.0 kb in size with a 5'-untranslated region of 19-22 nt . Comparison of the deduced aa sequence indicates that the LAP has extensive homology with the super family of Zn(2+)-metallohydrolases and shows identity in the core deca-peptide consensus sequence for the Zn(2+)-binding motif of these enzymes.

J Bacteriol, 1992 Apr, 174(7), 2273 - 80
Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity; van Rooijen RJ et al.; We determined the location, activity, and regulation of the promoter of the Lactococcus lactis 8-kb lactose operon (lacABCDFEGX), which encodes the enzymes of the lactose phosphotransferase system and the tagatose 6-phosphate pathway . The lac promoter sequence corresponds closely to the consensus promoter described for gram-positive bacteria and is located in a back-to-back configuration with the promoter of the divergently transcribed lacR gene, which encodes the LacR repressor . The transcription start sites used under induced (lactose) and noninduced (glucose) conditions were determined . The minimal promoter region that could be isolated on a single restriction fragment included sequences ranging from -75 to +42 . The effect of the presence of flanking sequences and the lacR gene on promoter activity and regulation was studied in Escherichia coli and L . lactis strains by using transcriptional fusions with promoterless chloramphenicol acetyltransferase reporter genes . The results showed that transcriptional regulation of the lac operon is mediated by the interaction between the LacR repressor, the lac promoter, and sequences in the noncoding region between the lacR and lacA genes . Sequences flanking the minimal promoter region appeared to enhance lac promoter activity much more in L . lactis (5- to 38-fold) than in E . coli (1.3- to 5-fold).

Appl Microbiol Biotechnol, 1992 Apr, 37(1), 79 - 83
Antisense RNA directed against the major capsid protein of Lactococcus lactis subsp . cremoris bacteriophage 4-1 confers partial resistance to the host; Chung DK et al.; Antisense RNA targeted against the major capsid protein (MCP) of Lactococcus lactis subsp . cremoris bacteriophage F4-1 reduced bacteriophage replication by up to 50% . The region containing the mcp gene was oriented to transcribe the antisense strand using a L . lactis subsp . cremoris Wg2 promoter . The size of the mcp insert transcribed affected the level of bacteriophage inhibition and the greatest level of inhibition was achieved using a 301-bp fragment from the 5' end of the mcp . Antisense mcp RNA constructs were stable and did not alter the endogenous plasmid profile in the host, L . lactis subsp . cremoris F4-1 . There were, however, some adverse effects on the host during the stationary phase as exhibited by a decline in cell density.

Biochemistry, 1992 Mar 31, 31(12), 3038 - 43
Zinc, a novel structural element found in the family of bacterial adenylate kinases; Glaser P et al.; The adk gene from Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli under the control of the lac promoter . The primary structure of B . stearothermophilus adenylate kinase exhibited 76% identity with the enzyme from Bacillus subtilis, 60% identity with the enzyme from Lactococcus lactis, and 42% identity with the enzyme from E . coli . The most striking property of the adenylate kinase from B . stearothermophilus is the presence of a structural zinc atom bound to four cysteines in a zinc finger-like fashion . The ability to coordinate zinc is predicted also for a number of other isoforms of bacterial adenylate kinases . Furthermore, the tightly bound metal ion contributes to the high thermodynamic stability of adenylate kinase from B . stearothermophilus.

Biochim Biophys Acta, 1992 Mar 2, 1104(2), 250 - 6
Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis; In 't Veld G et al.; The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl beta-D-gluco-pyranoside and reconstituted into proteoliposomes . Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids . Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step . Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L . lactis . Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes (Driessen, A.J.M., Zheng, T., In't Veld, G., Op den Kamp, J.A.F . and Konings, W.N . (1988) Biochemistry 27, 865-872) . We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation.

J Gen Microbiol, 1992 Mar, 138 ( Pt 3), 571 - 8
Influence of the carbon source on nisin production in Lactococcus lactis subsp . lactis batch fermentations; De Vuyst L et al.; Nisin production by Lactococcus lactis subsp . lactis NIZO 22186 was studied in batch fermentation using a complex medium . Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase . A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide . Moreover, it seemed likely that nisin had a growth control function . A physiological link is proposed between sucrose fermentation capacity and nisin production ability . Carbon source regulation appears to be a major control mechanism for nisin production.

Eur J Biochem, 1992 Mar 1, 204(2), 815 - 20
Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis; Laan H et al.; Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished . Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified . By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components . The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule . In a L . lactis proteinase which was inactivated by the substitution Asp30----Asn under native conditions, only one epitope could be detected . This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.

Carbohydr Res, 1992 Feb 7, 224, 245 - 53
Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp . cremoris SBT 0495; Nakajima H et al.; The extracellular polysaccharide obtained from slime-forming Lactococcus lactis subsp . cremoris SBT 0495 is composed of D-glucose, D-galactose, L-rhamnose, and phosphate . Methylation analysis of the native and dephosphorylated polysaccharides provided information on the linkage of the sugar residues and the location of the phosphate group . N.m.r . spectroscopy confirmed the structure of the polysaccharide, which is assigned the following repeating-unit: {formula: see text}

Appl Environ Microbiol, 1992 Feb, 58(2), 750 - 3
The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp . cremoris BC101; Nissen-Meyer J et al.; The plasmid-free strain Lactococcus lactis subsp . cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains . The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located . The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L . lactis subsp . cremoris Wg2 . The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101 . By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found . DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid . The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA . These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.

Appl Environ Microbiol, 1992 Feb, 58(2), 572 - 7
Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6; van Belkum MJ et al.; On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp . cremoris 9B4, a third bacteriocin determinant was identified . The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M . J . van Belkum, B . J . Hayema, R . E . Jeeninga, J . Kok, and G . Venema, Appl . Environ . Microbiol . 57:492-498, 1991) . The fragment was sequenced and analyzed by deletion and mutation analyses . The bacteriocin determinant consisted of two genes which were transcribed as an operon . The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity . The second gene (lciB) contained 91 codons and was responsible for immunity . The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6 . Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6 . This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene . When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.

FEMS Microbiol Rev, 1992 Feb, 8(2), 73 - 92
Gene expression in Lactococcus lactis; van de Guchte M et al.; Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods . A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible . Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired . An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L . lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L . lactis-derived genes in the latter bacterial species . Several studies have indicated, however, that in spite of the similarities, the expression signals from E . coli, B . subtilis and L . lactis are not equally efficient in these three organisms.

Lett Appl Microbiol, 1992 Feb, 14(2), 17 - 21
Genus- and species-specific oligonucleotide probes derived from 16S rRNA for the identification of vagococci; Williams AM et al.; Synthetic oligonucleotide probes specific for vagococci were designed from 16S rRNA sequence data . Molecular hybridizations with PCR-amplified rDNA targets provided an unequivocal means of differentiating vagococci from related lactic acid bacteria (eg . Carnobacterium, Enterococcus, Lactococcus) and identification at the generic and species levels.

Appl Environ Microbiol, 1992 Feb, 58(2), 699 - 702
IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp . lactis; Romero DA et al.; The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp . lactis . Electroporation of L . lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA . pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid . Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.

Appl Environ Microbiol, 1992 Feb, 58(2), 476 - 84
Development of a lactococcal integration vector by using IS981 and a temperature-sensitive lactococcal replication region; Polzin KM et al.; A vector (pKMP10) capable of Campbell-like integration into the Lactococcus lactis subsp . lactis LM0230 chromosome via homologous recombination with chromosomal IS981 sequences was constructed from the replication region of lactococcal plasmid pSK11L, an internal fragment of IS981, and the erythromycin resistance gene and Escherichia coli replication origin of pVA891 . The pSK11L replication region is temperature sensitive for maintenance in L . lactis subsp . lactis LM0230, resulting in loss of unintegrated pKMP10 during growth at greater than 37 degrees C . pKMP10 integrants made up 8 to 75% of LM0230(pKMP10) erythromycin-resistant cells following successive growth at 25 degrees C with selection, 39 degrees C without selection, and 39 degrees C with selection . pKMP10 integrants were also isolated from L . lactis subsp . lactis MG1363(pKMP10) but at a 10-fold-lower frequency (4%) . No integrants were isolated form L . lactis subsp . lactis MMS368(pKMP10) (a Rec-deficient strain) or LM0230(pKMP1-E) (the corresponding plasmid lacking the IS981 fragment) . Examination of 17 LM0230 integrants by Southern hybridization revealed pKMP10 integration into five different chromosomal sites . Four of the integration sites appeared to be chromosomal IS981 sequences, while one was an uncharacterized chromosomal sequence . The four IS981 integrants seemed to have pKMP10 integrated in a tandem repeat structure of undetermined length . Integrated pKMP10 was more stable (0 to 2% plasmid loss) than unintegrated pKMP10 (100% plasmid loss) when grown for 100 generations at 32 degrees C without selection.

Zentralbl Hyg Umweltmed, 1992 Feb, 192(5), 419 - 31
{Virucidal effectiveness of some commercial products for chemothermal disinfection methods for temperature resistant viruses and bacteriophages--evaluation of a test model}; von Rheinbaben F et al.; In the laboratory assay of chemothermal virus disinfection procedures a test system is required which allows to differentiate between the physical effects of the temperature and the chemical effects by the disinfectant used . From the four test viruses recommended by the German Association for the Control of Virus Diseases (Deutsche Vereinigung zur Bekampfung der Viruskrankheiten) only SV 40 tumor virus showed a sufficient thermal stability at 55 degrees C to take it in consideration as test virus for chemothermal disinfection procedures . Bacteriophage Phi x 174 and lactococci phages P001, P008 and P109 are suggested for the evaluation of chemothermal disinfection procedures in food processing industries because of their thermal stability at 55 degrees C, for short exposition times even at 60 degrees C . For procedures which work at 60 degrees C, e.g . laundry disinfection, especially bovine parvovirus can be recommended as test virus . In case of a binary product consisting of two different compounds for mechanically cleaning and disinfection of surgical instruments we were able to evaluate the virucidal effectivity at 55 degrees C for both parts separately . Generally our results showed that products with insufficient virucidal activity against naked viruses even at 60 degrees C may not be assumed as sure virucidal disinfectants.

Gene, 1992 Feb 1, 111(1), 109 - 14
New tools for the physical and genetic mapping of Lactococcus strains; Le Bourgeois P et al.; Tools for the genetic and physical analysis of the Lactococcus lactis subsp . lactis genome were developed . Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L . lactis subsp . lactis . When a chromosomal L . lactis subsp . lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner . The integration lead to the generation of new rare restriction sites near to the host fragment . This procedure allows precise mapping of cloned genes onto the chromosomal restriction map . The mapping of the his operon of L . lactis subsp . lactis provides an illustration . The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome . The L . lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1 . Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L . lactis subsp . lactis chromosome, via transposition . Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.

J Bacteriol, 1992 Feb, 174(4), 1280 - 7
Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis; Rauch PJ et al.; A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized . It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system . Conjugal transfer of Tn5276 was demonstrated from L . lactis NIZO R5 to different L . lactis strains and a recombination-deficient mutant . The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA) . The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1) . By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located . The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614 . The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.

J Dairy Sci, 1992 Jan, 75(1), 43 - 50
Antagonism between Listeria monocytogenes and lactococci during fermentation of products from ultrafiltered skim milk; el-Gazzar FE et al.; Tyndallized samples of unfiltered skim milk and retentate (concentrated fivefold or twofold by volume) and permeate from UF skim milk were inoculated with 5.5 x 10(3) to 1.5 x 10(5) cfu/ml of Listeria monocytogenes strains California or V7 together with 4 x 10(7) to 2.3 x 10(8) cfu/ml of mesophilic lactic acid bacteria . Numbers of L . monocytogenes (McBride Listeria agar) and lactic acid bacteria (all purpose Tween agar) were determined after 0, 6, 12, 24, 30, and 36 h of incubation at 30 degrees C . Lactic acid bacteria significantly inhibited or inactivated L . monocytogenes in all three products . Inactivation was greater in permeate (6.77 orders of magnitude) than in unfiltered skim milk (3.67 orders of magnitude) or in retentate (4.21 orders of magnitude) . Degree of inactivation in retentate was related to the extent of concentration . Inactivation was not complete, and L . monocytogenes survived in these products during fermentation for up to 36 h . When fermented products were refrigerated (4 degrees C), L . monocytogenes survived for 4 to 6 wk in skim milk, 3 to 5 wk in retentate, and 1 wk in permeate . At refrigeration temperature, length of survival was dependent on type of product and strain of the pathogen.

Appl Environ Microbiol, 1992 Jan, 58(1), 78 - 84
Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk; Bruinenberg PG et al.; Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp . cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains . The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media . In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L . lactis subsp . cremoris SK112 . In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L . lactis subsp . lactis SH4109 . In all strains proteinase production appeared to be regulated by the medium composition . Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium . Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk . Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109 . These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.

Appl Environ Microbiol, 1992 Jan, 58(1), 125 - 31
Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp . cremoris SK11 into the chromosome of L . lactis subsp . lactis; Petzel JP et al.; When Lactococcus lactis subsp . lactis LM0230 is transformed by the lactose plasmid (pSK11L) from Lactococcus lactis subsp . cremoris SK11, variants with pSK11L in the integrated state can be derived (J . M . Feirtag, J . P . Petzel, E . Pasalodos, K . A . Baldwin, and L . L . McKay, Appl . Environ . Microbiol . 57:539-548, 1991) . In the present study, a 1.65-kb XbaI-XhoI fragment of pSK11L was subcloned for use as a probe in Southern hybridization analyses of the mechanism of integration, which was shown to proceed via a Campbell-like, single-crossover event . Furthermore, the presence of the XbaI-XhoI fragment in a nonreplicating vector facilitated the stable, Rec-dependent integration of the vector into the chromosome of L . lactis subsp . lactis LM0230 and other lactococci . DNA sequence analysis of the fragment revealed an open reading frame of 885 bp with lactococcal expression sequences . The putative gene did not have significant homology with other genes in computer data bases . The XbaI-XhoI fragment is a naturally occurring piece of lactococcal DNA that can be used as a recombinogenic cassette in the construction of integration vectors for the industrially important lactococci.

J Bacteriol, 1991 Dec, 173(23), 7573 - 81
Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp . cremoris; Horng JS et al.; The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp . cremoris (L . cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L . lactis subsp . lactis (L . lactis) LM0230 . Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment . DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep . The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times . The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism . Like pSK11L, pKMP1 was unstable in L . lactis LM0230 . Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L . lactis LM0230 . pKMP1 was significantly more stable in L . cremoris EB5 than in L . lactis LM0230 at all of the temperatures compared . This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L . lactis LM0230 . Other regions affecting stability in L . cremoris EB5 but not in L . lactis LM0230 were also identified . Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L . lactis LM0230 and L . cremoris EB5, although the plasmid was more unstable in L . lactis LM0230 . The region responsible for the temperature sensitivity phenotype in L . lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L . Our results suggest that the closely related L . lactis and L . cremoris subspecies behave differently regarding maintenance of plasmids.

J Bacteriol, 1991 Dec, 173(23), 7491 - 500
Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum; Hastings JW et al.; Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat . The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity . Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin . It is inactivated by a range of proteolytic enzymes . The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4 . Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3 . A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain . The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19 . DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator . The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension . No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.

J Clin Microbiol, 1991 Dec, 29(12), 2731 - 4
Differentiation of Lactococcus lactis and Lactococcus garvieae from humans by comparison of whole-cell protein patterns; Elliott JA et al.; We tested 12 reference and 24 clinical strains of lactococci for physiologic characteristics using a conventional test system, the Gen-Probe Enterococcus 2 chemiluminescence assay (Gen-Probe Inc., San Diego, Calif.), the Rapid Strep identification system (Analytab Products, Plainview, N.Y.), and whole-cell protein analysis . The Gen-Probe Enterococcus 2 chemiluminescence assay for Enterococcus identification was negative with all strains . Neither the conventional test nor the Rapid Strep identification system could differentiate between the two Lactococcus spp . most commonly isolated from humans . A simple procedure, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was developed for comparing the whole-cell protein patterns of Lactococcus spp . L . lactis and L . garvieae were differentiated by unique protein patterns.

FEMS Microbiol Lett, 1991 Dec 1, 68(3), 307 - 12
Differentiation of lactococci by rRNA gene restriction analysis; Kohler G et al.; Strains of the subspecies of Lactococcus lactis could be differentiated by rRNA gene restriction fragment length polymorphisms (RFLP) . 16S rRNA-specific oligonucleotide as well as polynucleotide DNA probes were used for the detection of restriction fragments . In addition, a site-specific probe was designed for the intergenic spacer region of 23S and 5S rRNA genes . For all lactococcal strains the putative presence of six rRNA operons was confirmed . A non-radioactive hybridization assay was used based on hybrid detection by chemiluminescence . Specific patterns were found for any of the strains investigated . Subspecies-specific restriction fragments could be identified in addition to the strain-specific patterns.

J Appl Bacteriol, 1991 Dec, 71(6), 509 - 16
Specific and intraspecific molecular typing of lactococci based on polymorphism of DNA encoding rRNA; Rodrigues UM et al.; The rRNA gene restriction patterns or species of the genus Lactococcus were determined . Chromosomal DNA was digested with endonucleases and probed with radiolabelled DNA complementary to rRNA synthesized by random oligonucleotide priming using reverse transcriptase . Highly discriminatory restriction patterns were obtained which served to distinguish the five currently recognized lactococcal species . In addition the observed variations in the patterns at intra-specific level indicate that rRNA gene restriction fingerprinting may be of value in distinguishing the individual strains for epidemiological studies, and monitoring and checking authenticity of starter strains.

Appl Environ Microbiol, 1991 Dec, 57(12), 3547 - 51
Phage abortive infection mechanism from Lactococcus lactis subsp . lactis, expression of which is mediated by an Iso-ISS1 element; Cluzel PJ et al.; A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp . lactis IL416 . The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISS1 element and an open reading frame of 753 bp designated ORFX . Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi-416 gene . The expression of abi-416 was shown to be mediated by the Iso-ISS1 element, which contains a sequence fitting the consensus sequence for gram-positive promoters.

J Bacteriol, 1991 Dec, 173(23), 7599 - 606
Construction of an IS946-based composite transposon in Lactococcus lactis subsp . lactis; Romero DA et al.; An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946 . A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+ . When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA) . pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01 . An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L . lactis LM2345 . Transfer of either marker alone occurred at frequencies of ca . 2 x 10(-4) per input donor . Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker . No Cmr Ems transconjugants were recovered (n = 550) . Plasmid analysis showed that the Cms Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element . A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids . DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA . This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms.

Appl Microbiol Biotechnol, 1991 Dec, 36(3), 344 - 51
Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp . cremoris {corrected}; Reid JR et al.; The action of the cell-wall-associated proteinases from Lactococcus lactis subsp . cremoris strains H2 and SK112 on bovine beta-casein was compared . The proteinase from the H2 strain was characterised as a PI-type proteinase since it did not hydrolyse alpha s1-casein and the initial trifluoroacetic acid-soluble products of beta-casein hydrolysis were identical to those previously identified as hydrolysis products of PI-type lactococcal proteinase action . The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr193-Gln194 and Gln182-Arg183 were the first to be hydrolysed . Cleavage of the bonds Gln175-Lys176, Ser168-Lys169, Ser166-Gln167 and Leu163-Ser164 was also very rapid . Four of the five bonds in beta-casein most susceptible to hydrolysis by the PIII-type proteinase from strain SK112 were different from those cleaved by the PI-type proteinase, initial hydrolysis being at the sites Tyr193-Gln194, Leu192-Tyr193, Asp43-Glu44, Gln46-Asp47 and Phe52-Ala53 . Early hydrolysis at the three sites in the N-terminal region of beta-casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of PI-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly.

J Biol Chem, 1991 Nov 25, 266(33), 22626 - 33
Purification and properties of fructokinase I from Lactococcus lactis . Localization of scrK on the sucrose-nisin transposon Tn5306; Thompson J et al.; Two electrophoretically distinct proteins with fructokinase (ATP:fructose-6-phosphotransferase) activity were detected in Lactococcus lactis subsp . lactis K1 . Whereas fructokinase I was induced specifically by growth of the organism on sucrose, fructokinase II was derepressed during growth on ribose, galactose, maltose, and lactulose . Fructokinase I was purified about 1000-fold to electrophoretic homogeneity (specific activity 112 units/mg) . The amino acid composition, N-terminal sequence, nucleoside triphosphate, and metal requirement(s) of the enzyme are reported . Ultracentrifugal analysis showed that the enzyme was primarily dimeric with subunits of 33.5 kDa (+/- 5%) . When completely reduced, fructokinase I migrated as a single protein (Mr = 32,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in the absence of reducing agent two polypeptides (apparent Mr = 29,000 and 31,000) were detected . Isoelectric focusing also revealed two polypeptides (pI 5.6 and 5.8), and both species catalyzed the phosphorylation of fructose and mannose . Hybridization studies showed that: (i) a sucrose-negative mutant lacking the fructokinase I gene (scrK) retained fructokinase II activity and (ii) scrK is closely linked to scrA and scrB which encode Enzyme IIScr and sucrose-6-phosphate hydrolase, respectively . In L . lactis K1, these genes and the N5-(1-carboxyethyl)-L-ornithine synthase gene (ceo) are encoded on the sucrose-nisin transposon Tn5306 in the order ceo-scrKAB.

Biochim Biophys Acta, 1991 Nov 14, 1115(1), 75 - 83
Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator; Molenaar D et al.; The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF) . A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe . This results in a pH gradient, which drives accumulation of the probe in the cytoplasm . After neutralization the probe was well retained in cells stored on ice . BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization . The probe leaks out slowly at elevated temperatures . Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system . It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH . This allowed a correction of the fluorescence signal for efflux of the probe . After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.

Eur J Biochem, 1991 Nov 1, 201(3), 581 - 4
Identification and characterization of the lantibiotic nisin Z, a natural nisin variant; Mulders JW et al.; Lactococcus lactis strain NIZO 22186 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity . Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin . The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide . NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.

Appl Environ Microbiol, 1991 Nov, 57(11), 3206 - 11
Characterization of phiLC3, a Lactococcus lactis subsp . cremoris temperature bacteriophage with cohesive single-stranded DNA ends; Lillehaug D et al.; The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp . cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins . Infection of a permissive L . lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min . A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes . The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5% . Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C . Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA . This result indicates that lysogeny involves integration of the phage into the host chromosome . A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.

J Gen Microbiol, 1991 Nov, 137 ( Pt 11), 2595 - 600
Nucleotide sequence of a Lactococcus lactis gene cluster encoding adenylate kinase, initiation factor 1 and ribosomal proteins; Koivula T et al.; We have previously isolated a putative promoter from the Lactococcus lactis subsp . lactis chromosome . We now report the sequence of the promoter fragment and its extension in the 5'-direction . The region contains several open-reading frames which correspond to ribosomal protein L15, SecY, adenylate kinase, initiation factor 1 and ribosomal proteins B and S13 . The order of the genes, rplO (L15), secY, adk, infA, rpmJ (B) and rpsM (S13), is similar to that in the spc and alpha operon region of Bacillus subtilis, with the exception of the map gene, coding for methionine amino peptidase, which is located between adk and infA in B . subtilis . The putative promoter is located between adk and infA.

Appl Environ Microbiol, 1991 Nov, 57(11), 3390 - 3
Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes; Klijn N et al.; Specific DNA probes based on variable regions V1 and V3 of 16S rRNA of lactic acid bacteria were designed . These probes were used in hybridization experiments with variable regions amplified by using the polymerase chain reaction . In this way, a rapid and sensitive method was developed for the identification and classification of Lactococcus and Leuconostoc species.

J Bacteriol, 1991 Oct, 173(19), 6095 - 100
A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp . lactis C2; Valyasevi R et al.; Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp . lactis C2 infected with phage c2, did not form plaques but bound phage normally . The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller . Binding to phage sk1 was reduced about 10% . Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage . Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells . However, a difference was observed in the interactions of the phage with the cytoplasmic membranes . Membranes from the wild-type cells, but not mutant cells, inactivated phage c2 . Phage sk1 was not inactivated by membrane from either strain . Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect . On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography . Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000 . The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components . It is proposed that this protein is required for phage c2 infection.

J Bacteriol, 1991 Oct, 173(19), 6030 - 7
Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy; Poolman B et al.; The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated . In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate . Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide . Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate . The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate . Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L . lactis mutant deficient in malolactic enzyme . In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction . In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate . A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton) . The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate . The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.

J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2423 - 9
Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide; Dufour A et al.; Lactococcin, a bacteriocin produced by Lactococcus lactis subsp . lactis ADRIA 85LO30, was purified as a 2.3-2.4 kDa peptide . Six non-bacteriocin-producing (Bac-) and non-immune (Imm-) strains were isolated after curing experiments . These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb) . By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.

Appl Environ Microbiol, 1991 Oct, 57(10), 3046 - 8
Development of an improved chemically defined minimal medium for Listeria monocytogenes; Premaratne RJ et al.; A chemically defined minimal medium for Listeria monocytogenes has been developed by modification of Welshimer's medium . The growth factors required by L . monocytogenes Scott A are leucine, isoleucine, arginine, methionine, valine, cysteine (each at 100 mg/liter), riboflavin and biotin (each at 0.5 micrograms/ml), thiamine (1.0 micrograms/ml), and thioctic acid (0.005 micrograms/ml) . Growth was stimulated by 20 micrograms of Fe3+ per ml as ferric citrate . Glucose (1%) and glutamine (600 mg/liter) are required as primary sources of carbon and nitrogen . Glucose could not be replaced by various organic acids or amino acids . Of several sugars tested, fructose, mannose, cellobiose, trehalose, maltose (weak), glycerol (weak), and the amino sugars glucosamine, N-acetylglucosamine, and N-acetylmuramic acid supported growth in the absence of glucose . Evidence was found that chitin and cell walls of starter bacteria (Lactococcus lactis) supported survival of L . monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods, such as cheeses, by assimilating bacteria or molds that are present.

Int J Food Microbiol, 1991 Oct, 14(1), 1 - 9
Heat shock induces thermotolerance and inhibition of lysis in a lysogenic strain of Lactococcus lactis; Boutibonnes P et al.; In this preliminary work, the heat shock response of lactic acid bacteria was investigated and characterized . Log-phase Lactococcus lactis cells pre-incubated at 40 degrees C before heat challenge at 52 degrees C for 30 min demonstrated increased thermotolerance as compared with cells pre-incubated at 30 degrees C . The response persisted for at least 60 min . Additionally, we demonstrated that: (i) the physiological expression of the heat shock response is temperature dependent; (ii) ethanol 4.0% (v/v) caused, to a lesser extent, a response similar to the heat shock; and (iii) hydrogen peroxide failed to induce a detectable response . Furthermore, we suggest that the induction of the heat shock response increases the resistance of a lysogenic strain of L . lactis, treated by mitomycin C (1.25 micrograms/ml), to lysis by the bacteriophage.

J Bacteriol, 1991 Oct, 173(19), 5992 - 8
Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway; Rosey EL et al.; The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented . The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively . The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli . A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described . Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity . LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E . coli . Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD . The LacD protein is highly homologous with LacD of L . lactis . Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S . aureus.

Gene, 1991 Sep 30, 106(1), 115 - 9
Isolation, sequence and expression in Escherichia coli, Bacillus subtilis and Lactococcus lactis of the DNase (streptodornase)-encoding gene from Streptococcus equisimilis H46A; Wolinowska R et al.; A partial library of BclI-generated chromosomal DNA fragments from Streptococcus equisimilis H64A (Lancefield Group C) was constructed in Escherichia coli . Clones displaying either streptokinase or deoxyribonuclease (streptodornase; SDC) activities were isolated . The gene (sdc) expressing the SDC activity was allocated on the 1.1-kb AccI DNA subfragment . Sequence analysis of this DNA fragment revealed the presence of one open reading frame, which could encode a protein of 36.8 kDa . The N-terminal portion of the deduced protein exhibited features characteristic of prokaryotic signal peptides . The sdc gene was expressed in E . coli, Bacillus subtilis and Lactococcus lactis . As observed for S . equisimilis, in the heterologous Gram + hosts, at least part of the SDC protein was secreted into the medium.

J Dairy Sci, 1991 Sep, 74(9), 2820 - 30
The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk; Eckner KF et al.; Whole milk was pasteurized and concentrated two times by ultrafiltration . Starter cultures, Lactococcus lactis ssp . cremoris and Lactococcus lactis ssp . lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture . The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var . Hillfarm . Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures . The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels . The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels . Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses . During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased . No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.

Appl Environ Microbiol, 1991 Sep, 57(9), 2568 - 75
Chromosomal stabilization of the proteinase genes in Lactococcus lactis; Leenhouts KJ et al.; The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp . cremoris Wg2 were integrated by a Campbell-like mechanism into the L . lactis subsp . lactis MG1363 chromosome by using the insertion vector pKLG610 . Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies . The amplifications were stably maintained during growth in milk in the absence of antibiotics . The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5 . All three strains showed rapid growth in milk with concomitant rapid production of acid . The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.

Appl Environ Microbiol, 1991 Sep, 57(9), 2562 - 7
Lactococcal plasmid pWV01 as an integration vector for lactococci; Leenhouts KJ et al.; A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome . This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene . The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid . Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome . The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.

Appl Environ Microbiol, 1991 Sep, 57(9), 2555 - 61
Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N; van Alen-Boerrigter IJ et al.; The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction . The pepN gene was localized and subcloned in E . coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P . S . T . Tan and W . N . Konings, Appl . Environ . Microbiol . 56:526-532, 1990) . The L . lactis pepN gene appeared to complement an E . coli strain carrying a mutation in its pepN gene . High-level expression of the pepN gene in E . coli was obtained by using the T7 system . The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots . Cloning of the pepN gene on a multicopy plasmid in L . lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein . Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N . The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein . The intracellular location of aminopeptidase N in L . lactis was confirmed by immunogold labeling of lactococcal cells.

FEBS Lett, 1991 Aug 19, 288(1-2), 114 - 8
Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins; Koivula T et al.; Sec Y is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane . We have cloned the sec Y gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined Sec Y proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum . Comparison of the L . lactis Sec Y to the 3 other Sec Y proteins revealed 90 conserved amino acid residues (21%) . Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions . Some of the conserved regions are apparently responsible for the interactions of Sec Y with signal sequences, and the proteins SecE and SecA.

J Biol Chem, 1991 Aug 5, 266(22), 14573 - 9
Transposon-encoded sucrose metabolism in Lactococcus lactis . Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1; Thompson J et al.; Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp . lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity . Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography . The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively) . Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200 . The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000) . However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate . The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined . Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment . This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904 . In L . lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Int J Food Microbiol, 1991 Aug, 13(4), 309 - 14
Effect of lactic cultures on Escherichia coli in ewes' milk stored at low temperatures; Chavarri FJ et al.; The behaviour of Escherichia coli in pasteurized ewes' milk inoculated with different lactic starter cultures and incubated at temperatures in the range 4-16 degrees C for 96 h was investigated . Growth temperature of lactic starter cultures before inoculation had a significant effect on inhibition of E . coli . The growth temperature of lactic starter inoculum which resulted in the highest inhibitory activity was 24 degrees C . Size of lactic starter inoculum also significantly influenced growth of E . coli, with a higher inhibition for 1% inoculum than for 0.1% or 0.3% inocula . Single cultures of Lactococcus lactis showed a stronger inhibitory activity than single cultures of Leuconostoc cremoris or Leuconostoc dextranicum . A lactic starter culture comprising Lactococcus lactis, Leuconostoc cremoris and Leuconostoc dextranicum resulted in the strongest inhibition . Stimulation of E . coli by the lactic starter cultures was frequently recorded at 4 degrees C and 8 degrees C . However, none or a very limited growth of E . coli was seen at these temperatures.

Int J Food Microbiol, 1991 Aug, 13(4), 285 - 93
Comparison of lactococcal bacteriophage isolated in the United States and Argentina; de Fabrizio SV et al.; Bacteriophage of Lactococcus lactis ssp . lactis and ssp . cremoris, isolated in the United States and Argentina, were compared with respect to host range, adsorption, latent period, burst size and immunological cross-reactivity . Only 1 out of 13 U.S . culture isolates was sensitive to Argentinian phage . Argentinian L . lactis ssp . lactis C2 mutants were resistant to 13 U.S . phage isolates (4 prolate and 9 isometric) . While Argentinian phage Stl-3 multiplied on U.S . culture isolate 59-1, low adsorption (38%) and insignificant burst size and latent period data were evident . Antisera prepared against U.S . phage D59-1 (prolate) and F4-1 (isometric) neutralized the lytic activities of all Argentinian prolate phage although the F4-1 antiserum was less effective . The data suggest homology especially between U.S . phage D59-1 and the Argentinian phage.

J Bacteriol, 1991 Aug, 173(15), 4794 - 8
Replacement recombination in Lactococcus lactis; Leenhouts KJ et al.; In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP) . The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence . Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis . In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L . lactis chromosome . The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid . Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.

Biochem Soc Trans, 1991 Aug, 19(3), 670 - 4
Proteinase genes of cheese starter cultures; Kok J; The proteolytic enzymes of lactococci are of eminent importance for milk fermentations . By the combined action of proteinases and peptidases milk protein is degraded to peptides and amino acids which are required for cell growth and contribute to the organoleptic properties of the foods . The importance of the proteolytic system for dairy product quality has resulted in an increased fundamental research of the enzymes and genes involved . Proteinase plasmids have been identified and plasmid stability problems offered an explanation for the apparent instability of proteolysis in certain strains of lactococci . Chromosomal integration has recently been used to stably anchor the proteinase genes in the chromosome of Lactococcus lactis . The structural proteinase genes of a number of strains have been cloned and sequenced, and some of the properties of the enzymes they specify will be discussed . The product of a second gene is necessary for the activation of the proteinase, a proteinase maturation process that is unique in the bacterial world.

Mol Gen Genet, 1991 Aug, 228(1-2), 129 - 35
Nisin biosynthesis genes are encoded by a novel conjugative transposon; Horn N et al.; Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301 . The element is 70 kb in size and lacks inverted repeats at its termini . Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process . The integrated element is flanked by the directly repeated sequence 5'-TTTTTG-3' . Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology . The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties . Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.

Appl Environ Microbiol, 1991 Aug, 57(8), 2324 - 31
Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys; Baloga AO et al.; Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI . Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA . The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L . monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes . A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII . The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L . monocytogenes into 6 ribotype groups . DNA fingerprinting and ribotyping differentiated L . monocytogenes from other Listeria spp., including L . ivanovii, L . welshimeri, and L . innocua as well as the lactic acid bacteria Lactococcus lactis subsp . lactis and subsp . cremoris . L . monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods . Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis . DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates . This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L . monocytogenes implicated in food-borne illness.

J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1611 - 8
Purification and characterization of the free form of the lactococcal extracellular proteinase and its autoproteolytic cleavage products; Nissen-Meyer J et al.; In addition to the cell wall proteinase, Lactococcus lactis subsp . cremoris produced significant amounts of a free extracellular proteinase . The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase . Both proteinase forms had a pH optimum between 4-6 and 5-8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related . Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50,000-80,000-fold increase in the specific activity of the free proteinase . It contained two major protein species (termed pro150 and pro115) with proteinase activity . As judged by SDS-PAGE, the Mr values of pro150 and pro115 were 150,000 and 115,000, respectively, and by chromatofocusing the isoelectric points were 4.3 and 4.1, respectively . Upon gel filtration, pro150 and pro115 had Mr values of 300,000 and 125,000, respectively, indicating that pro150 was a dimer and pro115 a monomer . Pro115 was an autodegradation product of pro150 . Other distinct autodegradation products had Mr values of 90,000 (p90), 53,000 (p53), 37,000 (p37) and 30,000 (p30) . These had little if any proteinase activity . Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase . Judging from its N-terminal sequence and Mr, p30 was derived from the C-terminal half of p53 . Cleavage of pro150 to pro115 generated p37.

Appl Environ Microbiol, 1991 Jul, 57(7), 1899 - 904
Processing of the lactococcal extracellular serine proteinase; Haandrikman AJ et al.; Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membrane-associated lipoprotein PrtM . To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed . This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme . The secreted form of this inactivated proteinase was the same size as the inactive secreted form of the proteinase produced in the absence of PrtM . Both inactive proteinases are larger than the active proteinase . Isolation of proteinase by washing lactococcal cells carrying the complete proteinase gene in a Ca(2+)-free buffer was prevented by the absence of prtM or the absence of a functional active site . We propose that PrtM, during or after membrane translocation of the proteinase, effects the autoproteolytic removal of the N-terminal pro region of the proteinase . Subsequent C-terminal autodigestion results in the release of the enzyme from the lactococcal cells.

J Bacteriol, 1991 Jul, 173(14), 4517 - 25
Lactococcal proteinase maturation protein PrtM is a lipoprotein; Haandrikman AJ et al.; The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM . A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter was purified and used to obtain PrtM-specific antibodies . With these antibodies, immunogold labeling of lactococcal cells revealed that PrtM was associated with the lactococcal cell envelope . Western blot (immunoblot) analysis of whole lactococcal cells and isolated membrane vesicles indicated that PrtM was a membrane-associated protein . Radiolabeling of Lactococcus lactis with {3H}palmitic acid showed that PrtM was a lipoprotein . Partial secretion of PrtM into the culture medium was observed after Cys-24, the target residue for lipid modification, was replaced by an Ala residue by means of site-directed mutagenesis . This mutation did not affect proteinase activity.

J Bacteriol, 1991 Jul, 173(14), 4363 - 70
In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage; Hill C et al.; The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+) . pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures . One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid . Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030 . A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced . A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes . Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI . The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI . In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome . The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans . This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage . Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.

Plasmid, 1991 Jul, 26(1), 55 - 66
Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1; Leenhouts KJ et al.; The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined . This analysis revealed four open reading frames (ORFs) . ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication . The ORF C product was assumed to play a regulatory role in replication . Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1 . In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1 . Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L . lactis, suggesting that this plasmid uses the rolling-circle mode of replication . In B . subtilis, but not in L . lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA) . Apparently, in L . lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

J Dairy Sci, 1991 Jul, 74(7), 2082 - 8
Functional alteration of macrophages by a slime-forming Lactococcus lactis ssp . cremoris; Kitazawa H et al.; The effect of a slime-forming, encapsulated Lactococcus lactis ssp . cremoris KVS20 on macrophage function has been examined in vivo and in vitro in short-term studies . Peritoneal macrophages in which 21 to 34% of macrophage was presenting Fc gamma-receptor positive macrophages were elicited by intraperitoneal injection of 10 to 50 mg/kg of L . lactis ssp . cremoris KVS20 . The peritoneal macrophage exhibited cytotoxic activity against Sarcoma-180 cells in which the maximum activity was obtained in macrophage from mice injected with 10 mg/kg on d 5 . However, L . lactis ssp . cremoris KVS20 rendered the elicited macrophage cytotoxic in vitro . The cytotoxicity was significantly augmented by 6- and 24-h treatment at the concentration of 50 to 500 micrograms/ml . These results obtained in the short-term studies demonstrated that the antitumor activity of L . lactis ssp . cremoris KVS20 may be mediated through the enhanced cytotoxic activity of macrophage.

Appl Microbiol Biotechnol, 1991 Jul, 35(4), 477 - 83
Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp . cremoris AM1 in its action on bovine beta-casein; Visser S et al.; The action of the cell-envelope proteinase (PIII-type) from Lactococcus lactis ssp . cremoris AM1 on bovine beta-casein was studied . The results were compared with those obtained earlier with (PI-type) proteinases from the cell envelope of other L . lactis strains . From a 4-h digest (pH 6.2; 15 degrees C) of beta-casein made with the PIII-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC . Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in beta-casein by the PIII-type proteinase . In nine cases the primary cleavage site (P1-P'1) was a Glx-X or X-Glx peptide bond . In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond . The P2-P3 and/or P'2-P'3 regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds . Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by PI-type proteinases, although the kinetics may be different . The PIII-type proteinase shows a broader specificity in its initial cleavage of beta-casein than does the PI-type.

FEMS Microbiol Lett, 1991 Jun 15, 65(2), 201 - 8
A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis; van de Guchte M et al.; Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences . Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression . Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed . Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L . lactis . The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L . lactis.

J Gen Microbiol, 1991 Jun, 137 ( Pt 6), 1355 - 62
Cloning and characterization of the determinant for abortive infection of bacteriophage from lactococcal plasmid pCI829; Coffey AG et al.; The genetic determinant for abortive infection of bacteriophage (Abi) from the lactococcal plasmid pCI829 was cloned on a 6.2 kb StuI fragment in Escherichia coli using the shuttle vector pSA3 . In Lactococcus lactis subsp . lactis MG1363Sm the resulting recombinant plasmid pCI816 conferred complete insensitivity to the small isometric-headed phage 712 and a reduced plaque size in the case of the prolate-headed phage c2 . The determinant was further localized by subcloning and nuclease Bal31 deletion analysis; approximately 2.0 kb of DNA was essential for the expression of the Abi+ phenotype . Nucleotide sequence analysis of this region revealed a putative open reading frame of 1887 base pairs preceded by a putative promotor sequence and ribosome-binding site which exhibited similarity to consensus E . coli and Bacillus subtilis transcription/translation signals . Hybridization experiments indicated that this region was not homologous to the abi determinant from the phenotypically similar lactococcal plasmid pCI750.

Can J Microbiol, 1991 Jun, 37(6), 488 - 90
Stability of plasmids in lactococci during extended incubation in growth media; Sinha RP; The stability of plasmids in Lactococcus lactis ssp . lactis strains C2 and ML3, and L . lactis ssp . cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions . Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h) . Lac- variants were obtained from each strain in milk and (M17-) broth . The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands . None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa) . Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.

FEMS Microbiol Immunol, 1991 Jun, 3(3), 159 - 64
In Lactococcus lactis subsp . cremoris SK110 protein, instead of lipoteichoic acid, reacts with the group-N-specific antiserum; Sijtsma L et al.; The reaction between cell-surface components, isolated from two Lactococcus lactis subsp . cremoris strains, with their Group-specific antiserum were studied . No reaction between purified lipoteichoic acid and the antiserum was observed . Both strains, however, did belong to the lactococci (Group-N streptococci), as was demonstrated by the positive reaction between the antiserum and an acid- (Lancefield) or alkaline-extract . Experiments with proteolytic enzymes demonstrated the involvement of protein in the antigenic material in the latter reaction.

J Bacteriol, 1991 Jun, 173(12), 3879 - 87
Lactococcin A, a new bacteriocin from Lactococcus lactis subsp . cremoris: isolation and characterization of the protein and its gene; Holo H et al.; A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp . cremoris LMG 2130 was purified and sequenced . The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins . Only lactococci were killed by the bacteriocin . Of more than 120 L . lactis strains tested, only 1 was found resistant to LCN-A . The most sensitive strain tested, L . lactis subsp . cremoris NCDO 1198, was inhibited by 7 pM LCN-A . By use of a synthetic DNA probe, lcnA was found to be located on a 55-kb plasmid . The lcnA gene was cloned and sequenced . The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension . An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as lcnA . We propose that this open reading frame encodes an immunity function for LCN-A . In Escherichia coli lcnA did not cause an LCN-A+ phenotype . L . lactis subsp . lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying lcnA . The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.

Gene, 1991 May 15, 101(1), 121 - 5
Cloning and nucleotide sequence of the major capsid protein from Lactococcus lactis ssp . cremoris bacteriophage F4-1; Chung DK et al.; The gene (mcp) coding for the major capsid protein (MCP) of the Lactococcus lactis ssp . cremoris bacteriophage F4-1 has been cloned and its nucleotide sequence determined . The mcp gene was localized, by Western blotting with rabbit antiserum against intact bacteriophage, within a 3.3-kb HindIII-Spe I fragment and the sequence of the entire region determined . The 35-kDa MCP is coded for by a 905-bp open reading frame preceded by a putative ribosome-binding site . Deletion analysis and N-terminal sequencing of the MCP confirmed the identification of the gene coding for this bacteriophage MCP.

FEMS Microbiol Lett, 1991 May 15, 64(2-3), 253 - 8
Identification, cloning and sequencing of the replication region of Lactococcus lactis ssp . lactis biovar . diacetylactis Bu2 citrate plasmid pSL2; Jahns A et al.; The replication region of the 7.8 kilobase (kb) citrate plasmid pSL2 from Lactococcus lactis ssp . lactis biovar . diacetylactis Bu2 was identified . Deletion derivatives of pSL2 were introduced into plasmid-free strain Bu2-60 and tested for their ability to replicate autonomously . The region necessary for replication was identified by comparison of the pSL2 derivatives, cloned and sequenced . No homologies were detected by comparing the putative Rep protein of pSL2 with replicons of other plasmids of Gram-positive bacteria . A part of an IS-element flanking the replication region was found.

FEMS Microbiol Lett, 1991 May 15, 64(2-3), 311 - 7
Distribution of the IS elements ISS1 and IS904 in lactococci; Schafer A et al.; A broad distribution of the lactococcal IS elements ISS1 {1} and IS904 {2} in several lactococcal plasmids and chromosomal DNA was observed . Hybridization of the ISS1 and IS904 oligonucleotide gene probes with DNA of lactococcal phages showed that none of these tested bacteriophages contained one of the IS elements . On the transductionally shortened lactose plasmid pTD1 an insertion sequence homologous to ISS1 was identified closely downstream to the P-beta-galactosidase gene . Sequence analysis of ISS1/pTD1 showed 82% homology in the deduced amino acid sequence to the putative transposase of ISS1, ISS1W, ISS1N, and IS946.

Mol Microbiol, 1991 May, 5(5), 1273 - 83
A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export; Kontinen VP et al.; A gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM-11 expression library . The sites of three closely linked prs mutations (prs-3, prs-29 and prs-40) were found to reside in a 5.3 kb DNA fragment, which also complemented the secretion defect in prs-3 and prs-29 mutants . Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prsA) encoding a putative protein of 292 amino acids (33 kDa) . Sequence analysis indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane . Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase . The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export.

Mol Gen Genet, 1991 May, 227(1), 65 - 71
Distance-dependent translational coupling and interference in Lactococcus lactis; van de Guchte M et al.; The possibility of raising the expression level of a heterologous gene in Lactococcus lactis by exploiting the principle of translational coupling was investigated . For this purpose, the Escherichia coli lacZ gene was transcriptionally fused to a short open reading frame (ORF) of lactococcal origin . A Shine-Dalgarno (SD) sequence was introduced at the boundary of the two ORFs . In a series of otherwise identical plasmids, the relative positions of the translational stop codon of the upstream ORF and the translational start codon of the downstream ORF (lacZ) were varied . The expression of lacZ gradually increased as the stop and start codons were placed in closer proximity . A concomitant switch from translational interference to translational coupling was observed . Best results were obtained with partially overlapping stop and start codons . It is concluded that the principle of translational coupling offers good possibilities to increase the level of heterologous gene expression in L . lactis.

Mol Gen Genet, 1991 May, 227(1), 33 - 9
Genetic analysis of a lactococcal plasmid replicon; Xu FF et al.; The sequence and genetic organization was determined of the 2508 bp lactococcal portion of pFX2, which was derived from a cryptic Lactococcus lactis subsp . lactis plasmid and used as the basis for construction of a series of lactococcal vectors . A lactococcal plasmid plus origin and two replication protein-coding regions (repA and repB) were located . RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins . RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria and Mycoplasma . The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication . Up-stream of the ori site, and in a region which was non-essential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified . The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.

Appl Environ Microbiol, 1991 May, 57(5), 1313 - 8
Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp . cremoris; Salama M et al.; Lactococcus lactis subsp . cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors . The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits . Our purpose was to identify unique rRNA sequences that could be used to discriminate L . lactis subsp . cremoris from related subspecies . The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L . lactis subsp . cremoris . All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus . In this region, all L . lactis subsp . lactis biovar diacetylactis strains examined had a sequence identical to that of L . lactis subsp . lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J . Brosius, J . L . Palmer, J . P . Kennedy, and H . F . Noller, Proc . Natl . Acad . Sci . USA 75:4801-4805, 1978) . Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells . Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested . No hybridization was seen with the control species . All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.

J Bacteriol, 1991 May, 173(9), 2768 - 75
Physical map of the chromosome of Lactococcus lactis subsp . lactis DL11 and localization of six putative rRNA operons; Tulloch DL et al.; A physical map of the chromosome of Lactococcus lactis subsp . lactis DL11 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two-dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA . The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L . lactis subsp . lactis DL11 chromosome . By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed-and fixed-field digestion patterns, six putative rRNA operons were identified in L . lactis subsp . lactis DL11 and placed on the map of the chromosome . Five of these loci are clustered in a region representing only 20% of the chromosome . The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced.

Appl Microbiol Biotechnol, 1991 May, 35(2), 222 - 7
Action of a cell wall proteinase from Lactococcus lactis subsp . cremoris SK11 on bovine alpha s1-casein; Reid JR et al.; The cell wall-associated proteinase from Lactococcus lactis subsp . cremoris SK11 was partially purified and incubated with alpha s1-casein for various times up to 48 h . Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence . Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of alpha s1-casein and were present among the products after the first 60 min of digestion . Three oligopeptides from the N-terminal region and two others from the central region of the alpha s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region . No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.

J Biol Chem, 1991 Apr 15, 266(11), 7176 - 81
Molecular cloning, characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene cluster of the lactose operon of Lactococcus lactis; van Rooijen RJ et al.; The tagatose 6-phosphate pathway gene cluster (lacABCD) encoding galactose-6-phosphate isomerase, tagatose-6-phosphate kinase, and tagatose-1,6-diphosphate aldolase of Lactococcus lactis subsp . lactis MG1820 has been characterized by cloning, nucleotide sequence analysis, and enzyme assays . Transcription studies showed that the four tagatose 6-phosphate pathway genes are the first genes of the lactose-inducible lactose-phosphotransferase operon consisting of the lacABCDFEGX genes . Using a T7 expression system, it could be shown that the lacA, lacB, lacC, and lacD genes code for proteins with apparent molecular masses of 15, 19, 33, and 36 kDa, respectively . Cell-free extracts of induced and noninduced Escherichia coli cells expressing the lacABCD genes were used to determine the functions of the encoded proteins . Expression of both lacA and lacB was required to obtain galactose-6-phosphate isomerase activity . The lacC gene codes for tagatose-6-phosphate kinase, the deduced amino sequence of which is similar to that of E . coli Pfk-2 phosphofructokinase, and Staphylococcus aureus LacC protein . The tagatose-1,6-diphosphate aldolase is encoded by the lacD gene, and its deduced primary sequence, which is homologous to that of the S . aureus LacD protein, predicts an amino acid composition which is virtually identical to that of the previously purified L . lactis E8 tagatose-1,6-diphosphate aldolase.

Appl Environ Microbiol, 1991 Apr, 57(4), 1181 - 8
Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis ATCC 11454; Steen MT et al.; The location and organization of the nisin locus in Lactococcus lactis ATCC 11454 were studied . Primer extension of in vivo mRNA transcripts of the gene that encodes the nisin prepropeptide sequence indicated the presence of a promoter at least 4 kb upstream from the nisin gene and that the mRNA has several processing sites . Restriction fragment patterns using rare-cutting enzymes, orthogonal pulsed-field clamped homogeneous electric field (CHEF) agarose gel electrophoresis, and hybridization with nisin gene probes showed that the nisin prepropeptide gene was located on a megabase-size restriction fragment, which was taken as proof of a chromosomal location . This is contrary to earlier reports, which had indicated that genes for nisin production were located on plasmids . There was no evidence of more than one chromosomal location or more than one copy of the nisin gene . The restriction patterns indicated that the size of the L . lactis genome is about 2,500 kb . The previously observed (G . W . Buchman, S . Banerjee, and J . N . Hansen, J . Biol . Chem . 263: 16260-16266, 1988) downstream open reading frame (ORF) was fully sequenced to reveal an 851-amino-acid coding region, an upstream putative mRNA processing site, and a putative rho-independent terminator . The ORF was analyzed for secondary structural features, and the sequence data bases were searched for homologies . The ORF contained many amphipathic helices, a C-terminal transmembrane helix, and homologies to some membrane-associated proteins . It lacked an N-terminal membrane insertion sequence and accordingly appears to be associated with, and anchored to, the cytoplasmic side of the membrane . An additional ORF that possessed a ribosome-binding sequence and tandem promoters, indicating the beginning of a new operon, was identified still farther downstream . The results were consistent with the nisin gene being part of a polycistronic operon with a size greater than 8.5 kb.

Protein Eng, 1991 Apr, 4(4), 479 - 84
Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes; Vos P et al.; Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11 . These plasmids were introduced into the plasmid-free strain L . lactis MG1363 . The serine proteinases produced by these L . lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated . The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes . As a result, various L . lactis strains were constructed having new proteolytic properties that differ from those of the parental strains . Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity . The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins . This suggests that the 200 kd L . lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.

Appl Environ Microbiol, 1991 Mar, 57(3), 804 - 11
Molecular characterization of the nisin resistance region of Lactococcus lactis subsp . lactis biovar diacetylactis DRC3; Froseth BR et al.; The nisin resistance determinant of Lactococcus lactis subsp . lactis biovar diacetylactis DRC3 was localized onto a 1.3-kb EcoRI-NdeI fragment by subcloning and interrupting the NdeI site by cloning random NdeI fragments into it; the nisin resistance determinant was then sequenced . The nucleotide sequence revealed a large open reading frame containing 318 codons . Putative transcription and translation signal sequences were located directly upstream from the initiation codon . Immediately downstream of the termination codon was a palindromic region resembling a rho-independent termination sequence . This 957-nucleotide open reading frame and its associated transcription and translation signal sequences were cloned into plasmid-free L . lactis subsp . lactis LM0230 and conferred an MIC of 160 IU of nisin per ml . This level of nisin resistance is equivalent to that of the initial nisin-resistant subclone, pFM011, used for further subcloning in this study . The inferred amino acid sequence would result in a protein with a molecular mass of 35,035 Da . This value was in agreement with the molecular mass of a protein detected after in vitro transcription and translation of DNA encoding the nisin resistance gene, nsr . This protein contained a hydrophobic region at the N terminus that was predicted to be membrane associated but did not contain a typical signal sequence cleavage site . No significant homology was detected when the DNA sequence of the nsr gene and the amino acid sequence of its putative product were compared with other available sequences . When subjected to Southern hybridization, a 1.2-kb DraI fragment encoding the nsr gene did not hybridize with the genomic DNA of the nisin-producing strain L . lactis subsp . lactis 11454.

Plasmid, 1991 Mar, 25(2), 105 - 12
The bacteriophage resistance plasmid pTR2030 forms high-molecular-weight multimers in lactococci; Hill C et al.; Lactococcus lactis ME2 can transfer a 46-kb plasmid, pTR2030, which encodes abortive phage infection (Hsp) and restriction/modification (R/M) activities . pTR2030 can be detected as a monomeric plasmid in transconjugants at low copy number, but not in ME2 . pTR2030-specific probes were cloned and used to determine the location of the element in ME2 . No homology was observed between these pTR2030-specific probes and the CsCl-purified plasmid content of ME2 . However, probes specific for pTR2030 hybridized strongly to a high-molecular-weight moiety, and not to chromosomal DNA, in total DNA isolated by a gentle lysis procedure . The absence of junction fragments indicates that pTR2030 forms high-molecular-weight multimers in lactococci . A phage-sensitive derivative of ME2, L . lactis N1, is cured of pTR2030 and no longer possesses the high-molecular-weight species . When pTR2030 was reintroduced to N1 via conjugation, an ME2-like phage-insensitive phenotype was restored . pTR2030 could remain as a detectable monomeric plasmid in the N1 transconjugants or could revert to the high-molecular-weight structure.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 319 - 23
Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp . lactis; Beresford T et al.; A cosmid gene library of the genome of Lactococcus lactis subsp . lactis 712 was probed for the presence of 16S rRNA genes, using 32P 5' end-labelled 16S rRNA fragments . Cosmid DNA from positive clones responsible for hybridisation was subcloned into a high copy number vector and a restriction map was constructed . The location of the 16S, 23S and 5S rRNA genes was determined on this map . Transcriptional promoter activity was identified upstream of the 5' end of the 16S rRNA gene . By probing L . lactis 712 chromosomal DNA cut with a range of restriction endonucleases, with a conserved oligonucleotide to the 5' end of the 16S rRNA gene, 6 copies of rRNA genes were identified.

Appl Environ Microbiol, 1991 Mar, 57(3), 734 - 43
Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci; Polzin KM et al.; An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS) . IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase . IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion . IS981 was present on the chromosome of Lactococcus lactis subsp . lactis LM0230 from where it transposed to pGBK17 during transformation . Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L . lactis subsp . cremoris strains containing the highest number of copies . Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.

Anal Biochem, 1991 Feb 1, 192(2), 362 - 6
Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus; Tortorello ML et al.; Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed . In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production . Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique . In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody . In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface . All three assays were quantifiable photometrically.

Biochimie, 1991 Feb-Mar, 73(2-3), 231 - 3
Identification of a RecA-like protein in Lactococcus lactis; Auffray Y et al.; We have identified in Lactococcus lactis, an analogue of Escherichia coli RecA protein . Physiological responses such as ultraviolet (UV) and chemical mutagenesis and induction of prophage have been characterized and suggest the existence of RecA-like functions in this commercially important species . The putative RecA protein was detected at the position of an apparent molecular weight of 39 kDa by Western blot analysis by using antiserum against E coli RecA protein . In addition, the protein level is significantly increased after UV irradiation in a wild-type strain compared to the recombination deficient mutant strain.

Int J Food Microbiol, 1991 Feb, 12(2-3), 167 - 71
Capsular polysaccharide of a slime-forming Lactococcus lactis ssp . cremoris LAPT 3001 isolated from Swedish fermented milk 'långfil'; Toba T et al.; Slime-forming Lactococcus lactis ssp . cremoris strain LAPT 3001 isolated from Swedish ropy sour milk 'langfil' was investigated for the chemical nature of its capsule . The capsular material purified by gel filtration chromatography and ion-exchange chromatography consisted of rhamnose, glucose, galactose, glycerol and phosphorus . It is most likely a deacylated lipoteichoic acid.

FEMS Microbiol Lett, 1991 Feb, 62(1), 69 - 73
Bacteriophage receptors of Lactococcus lactis subsp . 'diacetylactis' F7/2 and Lactococcus lactis subsp . cremoris Wg2-1; Schafer A et al.; Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L . lactis subsp . 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L . lactis subsp . cremoris Wg2-1 . Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions . However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments . This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.

Appl Environ Microbiol, 1991 Feb, 57(2), 539 - 48
Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp . cremoris lactose plasmids in Lactococcus lactis subsp . lactis; Feirtag JM et al.; Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp . cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids . When the Lac plasmids from these strains were introduced into L . lactis subsp . lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C . The stability of the L . lactis subsp . cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions . Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L . lactis subsp . lactis LM0230 chromosome . In addition, the presence of the L . lactis subsp . cremoris Lac plasmids in L . lactis subsp . lactis resulted in a temperature-sensitive growth response; growth of L . lactis subsp . lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L . lactis subsp . cremoris strains with respect to temperature sensitivity of growth.

Appl Environ Microbiol, 1991 Feb, 57(2), 517 - 24
Genetic construction of nisin-producing Lactococcus lactis subsp . cremoris and analysis of a rapid method for conjugation; Broadbent JR et al.; Conjugation was used to construct nisin-producing Lactococcus lactis subsp . cremoris strains . Recipients were obtained by electroporation of L . lactis subsp . cremoris strains with the drug resistance plasmid pGK13 or pGB301 . A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media . This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L . lactis subsp . lactis 11454, to three L . lactis subsp . cremoris recipient strains . Nip+ Suc+ L . lactis subsp . cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU . DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA . Further investigation indicated that L . lactis subsp . cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk . Results suggested that it would be feasible to construct nisin-producing L . lactis subsp . cremoris strains for application as mixed and multiple starter systems . Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.

Appl Environ Microbiol, 1991 Feb, 57(2), 492 - 8
Organization and nucleotide sequences of two lactococcal bacteriocin operons; van Belkum MJ et al.; Two distinct regions of the Lactococcus lactis subsp . cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses . On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon . The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity . Primer extension analysis indicated the presence of a promoter upstream of the ORFs . Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity . The first ORF, containing 75 codons, specified bacteriocin activity . The second ORF, containing 98 codons, specified immunity . The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.

Appl Environ Microbiol, 1991 Feb, 57(2), 385 - 8
Improved vector for promoter screening in lactococci; Bojovic B et al.; Fragments of Lactococcus lactis subsp . lactis NP45 chromosomal DNA provided promoter activity in Escherichia coli when cloned into the promoter probe vector pGKV210 . Only 13% of these recombinant plasmids promoted detectable cat-86 activity when transferred to L . lactis, i.e., expressed chloramphenicol resistance . In these promoter-containing versions of pGKV210, the cat-86 gene specifies chloramphenicol-inducible chloramphenicol acetyltransferase expression . This could be a limiting factor for cloning of promoters with lower activity in L . lactis . Therefore, we have constructed a new promoter probe vector, pBV5030, with the mutated version of the cat-86 gene, which is constitutively expressed when transcriptionally activated by the insertion of a promoter . We found that in L . lactis IL1403 the constitutively expressed cat-86 gene (on a pBV5030 derivative) has four times higher activity than the inducible version of the same gene (on a pGKV210 derivative) when both have the same promoter inserted upstream of the cat-86 gene . These results suggest that plasmid pBV5030 could be a more efficient vector for the cloning of promoters from lactococci.

Gene, 1991 Feb 1, 98(1), 95 - 100
Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages; Kim SG et al.; A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined . Approximately 95-99% of all L . lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment . Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns . The complete nt sequence for a 1.6-kb region from nine independent L . lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp . This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein . Three out of the five changes occur in a 187-bp region, 5' to this large ORF . The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein . The encoded protein is extremely charged and shares some homology with yeast translation initiation factor . In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.

Appl Environ Microbiol, 1991 Feb, 57(2), 333 - 40
Isolation and characterization of Lactococcus lactis subsp . lactis promoters; Koivula T et al.; DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp . lactis . For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L . lactis . Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements . From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B . subtilis sigma 43 promoters were identified . Another set of promoters, together with a signal sequence, were also isolated from the same organism . These fragments promoted secretion of TEM beta-lactamase from L . lactis . When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA) . By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.

Intervirology, 1991, 32(1), 2 - 9
Species and type phages of lactococcal bacteriophages; Jarvis AW et al.; Lactococcal phages are classified according to morphology and DNA homology . Phages are differentiated into 12 phage species, and type phages of each species are proposed . Members and possible members of each species are named . Available data on type phages are tabulated including morphology, DNA characteristics and phage protein bands.

FEMS Microbiol Lett, 1991 Jan 1, 61(1), 55 - 9
Construction of a family of lactococcal vectors for gene cloning and translational fusion; Xu FF et al.; A family of stable lactococcal vectors have been constructed based on the pFX1 replicon using either the alpha fragment or the complete Escherichia coli lacZ gene as a selective marker . These vectors also incorporate multiple cloning sites and examples are given of their use for gene cloning and translational fusion studies in lactococci.

Biochem J, 1991 Jan 1, 273(Pt 1), 135 - 9
Specificity of two genetically related cell-envelope proteinases of Lactococcus lactis subsp . cremoris towards alpha s1-casein-(1-23)-fragment; Exterkate FA et al.; The specificity of two genetically related cell-envelope serine proteinases (PI-type and PIII-type) of Lactococcus lactis subsp . cremoris towards the alpha s1-casein-(1-23)-fragment, an important intermediate product of primary chymosin-directed proteolysis in cheese, has been established . Both enzymes showed, at pH 6.5 and under relatively low-ionic-strength conditions, a characteristic, mutually different, cleavage pattern that seems, in the first instance, to be determined by the charge N-terminal to the cleaved bond . With Pi, three cleavage sites were found in the N-terminal positively charged part of the peptide and, with PIII, three sites were found in the C-terminal negatively charged part . Comparison of the specific cleavage sites in this peptide and those in beta-casein revealed similarities with respect to the different residues which can occur N-terminally to the cleaved bond . The properties of these substrate residues match with the structural and various interactive features of the respective binding regions of the enzymes predicted on the basis of a close sequence similarity of the lactococcal proteinases with the subtilisin family . A hydrophobic interaction and/or hydrogen-bridge formation seems to govern the binding of the first amino acid residue N-terminal to the scissile bond . The more distantly N-terminally positioned sequence of residues apparently is attracted electrostatically by a negative charge in the binding region of PI and by a positive charge in that of PIII, provided that the opposite charge is is present at the appropriate position in this sequence . Hence a specific electrostatic binding may occur; additionally, hydrophobic interaction and/or hydrogen-bond formation is important.

Plasmid, 1991 Jan, 25(1), 16 - 26
Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305; Hayes F et al.; Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp . lactis UC317 . The nucleotide sequence of the pCI305 replication region was determined . A single open reading frame of 1158 bp was identified in the trans-active domain repB . The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter . In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated . repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences . No significant homology between repA or repB and other gram-positive replication regions was evident . Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date . The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.

FEMS Microbiol Lett, 1991 Jan 1, 61(1), 101 - 6
A transposon-like element on the lactose plasmid of Lactococcus lactis subsp . lactis Z270; Huang DC et al.; An inverted repeat previously called IR was identified on the lactose plasmid of Lactococcus lactis subsp . lactis Z270 by self-annealing; it was now named IS1076 . The two sequences were 3.3 kb apart . Both copies were cloned in E . coli, sequenced and found to be identical, except for an additional 44 bp direct repeat at the 5' end of the right-hand copy; they were thus respectively 1296 bp (IS1076R) and 1252 bp (IS1076L) long . Both elements end in near-perfect 39 bp inverted repeats, similar to the IS904 termini . Promoter consensus sequences and a RBS site precede an ORF1 of 384 amino acids . Subclones of IS1076R and IS1076L produced a new 44 kDa protein corresponding to the size of the ORF1 . The distal part of the ORF1 coding region is very similar to the IS3 ORFI sequence and the IS904 ORF sequence, and the proximal part shows some homologies with IS3 ORFII . A three-base target is present as a direct repeat flanking the 5.9 kb genetic block including IS1076L, IS1076R and the internal region, resulting in a structure similar of that of a transposon.

Appl Environ Microbiol, 1991 Jan, 57(1), 45 - 50
Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp . lactis NCDO 763; Nardi M et al.; Lactococcus lactis subsp . lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) . X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate . A DNA bank from L . lactis subsp . lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay . The X-PDAP gene, designated pepXP, was further subcloned and sequenced . It codes for a protein containing 763 residues . Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide . The pepXP gene from L . lactis subsp . lactis NCDO 763 in more than 99% homologous to the pepXP gene from L . lactis subsp . cremoris P8-2-47 described elsewhere (B . Mayo, J . Kok, K . Venema, W . Bockelmann, M . Teuber, H . Reinke, and G . Venema, Appl . Environ . Microbiol . 57:38-44, 1991) and is also conserved in other lactococcal strains.

Appl Environ Microbiol, 1991 Jan, 57(1), 38 - 44
Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp . cremoris; Mayo B et al.; Lactococcus lactis subsp . cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) . A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli . A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E . coli clones . The fragment was also able to confer X-PDAP activity on Bacillus subtilis . The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned . The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L . lactis . We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals . The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787 . No homology was detected between pepXP and genes that had been previously sequenced . A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.

Int J Food Microbiol, 1990 Dec, 11(3-4), 313 - 20
Scanning electron microscopic and texture studies on characteristic consistency of Nordic ropy sour milk; Toba T et al.; The characteristic consistency of Nordic ropy sour milk was studied . Skim milk, reconstituted from non-fat dry milk, was fermented at 20 degrees C for 24 h by addition of 5% (v/v) inoculum of slime-producing (ropy) strain of Lactococcus lactis ssp . cremoris SBT 0495, isolated from Finnish ropy sour milk 'viili' starter culture, and its non-ropy variant SBT 1275 . Measurements of texture showed that milk gel prepared by the ropy strain exhibited remarkably increased adhesiveness as compared to that by the non-ropy variant . Milk gel prepared by the ropy strain also exhibited decreased syneresis (wheying-off) as compared to that by the non-ropy variant . Scanning electron micrographs of milk gel prepared by the ropy strain showed that slime was in the form of a network attaching the bacterial cells to the protein matrix . A thick network of slime attached the casein micelle clusters to each other to make casein conglomerates, which is likely to result in the characteristic consistency of 'viili'.

J Bacteriol, 1990 Dec, 172(12), 7126 - 30
Isolation and characterization of lipoteichoic acid, a cell envelope component involved in preventing phage adsorption, from Lactococcus lactis subsp . cremoris SK110; Sijtsma L et al.; The cell envelope of the phage-resistant Lactococcus lactis subsp . cremoris SK110 differed from its phage-sensitive variant by the presence of a galactosyl-containing component . This component was present in material obtained from SK110 by a mild alkali treatment . In a similar fraction extracted from SK112, no galactosyl-containing components were detected . With respect to gel permeation chromatography and electrophoretic mobility, identical characteristics of the alkali-extracted material and purified lipoteichoic acid (LTA) were measured . Chemical analysis of the latter component showed the absence of galactose in LTA isolated from SK112, whereas it was present in LTA obtained from SK110 . In this paper, we propose that galactosyl-containing LTA is involved in preventing phage adsorption to L . lactis subsp . cremoris SK110.

Microbiologia, 1990 Dec, 6(2), 51 - 64
Genetics of lactic acid bacteria with special reference to lactococci; Rodriguez A et al.; Lactic acid bacteria play an important role in the manufacture of fermented foods . Genetic studies have made these microorganisms, particularly lactococci, accessible to genetic manipulation . The instability of key metabolic traits of lactococci has been explained by the presence of plasmid DNA species . Most genetic interest has been focused to solve the quoted instability and the sensitivity to bacteriophage infection . At the same time, gene transfer systems have been developed and specific genes with commercial significance have been identified and cloned . Lactic acid bacteria can be also used as production organisms of heterologous proteins, e.g . chymosin, lysozyme.

FEMS Microbiol Lett, 1990 Nov, 60(3), 309 - 14
Cloning of a chromosomal fragment from Lactococcus lactis subsp . lactis partially complementing Escherichia coli recA functions; Novel M et al.; A recA-like gene was isolated from a gene library of Lactococcus lactis subsp . lactis by intergeneric complementation of an E . coli recA mutant . A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E . coli recA mutant . The cloned DNA fragment also partially complemented the rec mutation in Lc . lactis MMS36 . Hybridization studies showed that there was no detectable sequence homology between the recA gene of E . coli and Lc . lactis subsp . lactis chromosomal DNA.

J Bacteriol, 1990 Nov, 172(11), 6419 - 26
Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis; Hill C et al.; A number of host-encoded phage resistance mechanisms have been described in lactococci . However, the phage genome has not been exploited as a source of additional resistance determinants . A 4.5-kb BamHI-HindIII fragment of phage nck202.50 (phi 50) was subcloned in streptococcus-Escherichia coli shuttle plasmid pSA3 and introduced into Lactococcus lactis NCK203 and MG1363 by protoplast transformation . This cloned phage fragment directed a bacteriophage resistance phenotype designated Per (phage-encoded resistance) . Both phi 50 and a distantly related phage, nck202.48 (phi 48), formed small plaques on strain NCK213 at a slightly reduced efficiency of plaquing on the Per+ host . The per locus was further reduced to a 1.4-kb fragment through in vitro deletion analysis . The 1.4-kb fragment was sequenced, and the Per phenotype was found to be associated with a ca . 500-bp region rich in direct and inverted repeats . We present evidence that the Per region contains a phage origin of replication which, in trans, may interfere with phage replication by titration of DNA polymerase or other essential replication factors . It was demonstrated that the Per+ phenotype is not a result of reduced adsorption or action of a restriction and modification system . Per+ activity was not detected against six independent phages which were previously shown to be sensitive to the Hsp+ mechanism . The mutually exclusive resistance mechanisms could be combined to confer resistance to both types of phages (Hsp resistant and Per resistant) in a single host . This is the first description in lactococci of a phage resistance phenotype, other than superinfection immunity, originating from a lactococcal phage genome.

Gene, 1990 Oct 30, 95(1), 155 - 60
Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp . lactis MG1363; van Asseldonk M et al.; We have cloned usp45, a gene encoding an extracellular secretory protein of Lactococcus lactis subsp . lactis strain MG1363 . Unidentified secreted 45-kDa protein (Usp45) is secreted by every mesophilic L . lactis strain we tested so far and it is chromosomally encoded . The nucleotide sequence of the usp45 gene revealed an open reading frame of 1383 bp encoding a protein of 461 amino acids (aa), composed of a 27-aa signal peptide and a mature protein initiated at Asp28 . The gene contains a consensus promoter sequence and a weak ribosome-binding site; the latter is rather uncommon for Gram-positive bacteria . Expression studies in Escherichia coli showed efficient synthesis and secretion of the protein . Usp45 has an unusual aa composition and distribution, and it is predicted to be structurally homologous with P54 of Enterococcus faecium . Up to now, no biological activity could be postulated for this secreted protein.

J Biol Chem, 1990 Oct 25, 265(30), 18499 - 503
Molecular cloning, transcriptional analysis, and nucleotide sequence of lacR, a gene encoding the repressor of the lactose phosphotransferase system of Lactococcus lactis; van Rooijen RJ et al.; The repressor gene (lacR) of the lactose phosphotransferase system of Lactococcus lactis subsp . lactis strain MG1820 has been cloned and characterized . Transcription of lacR, into a 1.2-kilobase monocistronic messenger, is repressed approximately 5-fold during growth on lactose . Nucleotide sequence analysis of the lacR gene showed the presence of an open reading frame of 861 base pairs . The deduced amino acid sequence of LacR is homologous to three Escherichia coli regulatory proteins (DeoR, FucR, and GutR) and includes a N-terminal domain (helix-turn-helix) involved in DNA binding and a C-terminal domain that may be responsible for inducer binding . The in vivo function of LacR has been determined by introducing multiple copies of lacR into L . lactis, under control of its own or the unrelated prtP promoter . Growth rates and lactose phosphotransferase system enzyme activities were measured during growth on lactose and glucose . The presence of lacR on a multicopy plasmid resulted in the decrease of lactose phosphotransferase system activity, whereas only on lactose a decrease (25%) of growth rate was observed . No significant difference in growth rate was observed on glucose, indicating that LacR specifically represses the lactose genes of L . lactis.

J Appl Bacteriol, 1990 Oct, 69(4), 512 - 9
Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values; Luchese RH et al.; Aspergillus parasiticus was grown in a modified Lab-Lemco tryptone broth both as a single culture and in association with Lactococcus lactis . Total aflatoxin (B1 + G1) production was higher in the mixed cultures . This stimulation persisted when different batches of media, inoculation procedures and makes of ingredients were used . Aflatoxin yields increased in media with an initial pH of 4.2 compared with a pH close to neutrality . Hydrochloric and/or lactic acid had little effect . The substitution of half the carbon content of the medium by lactate resulted in stimulation or reduction on aflatoxin production when the initial pH was 4.2 or 6.8, respectively.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 209 - 13
Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages; Powell IB et al.; Conjugal matings were performed between Lactococcus lactis DRC1 (a lactose-fermenting (Lac+), bacteriocin-producing (Bac+) strain) and L . lactis HID113 (Lac- and Bac-) . Transconjugant derivatives of HID113 were identified on the basis of lactose fermentation, resistance to the DRC1 bacteriocin (dricin) or reduced sensitivity to phage sk1 . Regardless of how they were identified, all transconjugants gave fewer and smaller plaques with phages c2 and sk1 than did HID113 . All but one of 275 transconjugants tested also produced dricin, suggesting some functional relationship or close genetic linkage between the reduced phage sensitivity and dricin production and resistance . Some transconjugants were also Lac+, but this property was unstable.

J Bacteriol, 1990 Oct, 172(10), 5789 - 94
Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene; David S et al.; The plasmid-encoded citrate determinant of the Lactococcus lactis subsp . lactis var . diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain . From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate . Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate . Energy-dependent {1,5-14C}citrate transport was found with membrane vesicles prepared from E . coli cells harboring the citrate permease-expressing plasmid . The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product . The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.

FEMS Microbiol Rev, 1990 Sep, 7(1-2), 15 - 42
Genetics of the proteolytic system of lactic acid bacteria; Kok J; The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media . The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids . The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane . To that purpose, the cell contains specific transport proteins . The internalized peptides are further degraded to amino acids by intracellular peptidases . The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades . Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated . In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented . As most of the work in this field has been done with lactococci, the emphasis will, inevitably, be on this group of organisms . Where possible, links will be made with other species of lactic acid bacteria.

Plasmid, 1990 Sep, 24(2), 81 - 9
Integration and excision of plasmid DNA in Lactococcus lactis subsp . lactis; Hayes F et al.; The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp . lactis UC317 to recombine with the lactococcal chromosome was examined . Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317 . Excision of integrated sequences was subsequently observed at a low level . Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1 . Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated . The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.

Appl Environ Microbiol, 1990 Sep, 56(9), 2606 - 11
Heterologous gene expression in Lactococcus lactis subsp . lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease; van de Guchte M et al.; The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e . Upon introduction of the recombinant plasmid into Lactococcus lactis subsp . lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates . By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium . This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant . On the basis of these results, it is concluded that the B . subtilis neutral protease gene was expressed in L . lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein . The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.

J Bacteriol, 1990 Sep, 172(9), 5286 - 92
Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis; Smid EJ et al.; Proline, which is the most abundant residue in beta-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp . cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp . lactis ML3) . Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane . On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di- and tripeptide transport system, and these peptides are the major source of proline . Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di- and tripeptides . Growth of L . lactis subsp . lactis ML3 on chemically defined media supplemented with casein does not appear proline limited . Addition of dipeptides (including proline-containing peptides) severely inhibits growth on a casein-containing medium, which indicates that the specific growth rate is determined by the balanced supply of different di- or tripeptides which compete for the same di- and tripeptide transport system.

Appl Microbiol Biotechnol, 1990 Sep, 33(6), 677 - 9
In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp . cremoris plasmid pDI-21; Yu PL et al.; A 4.4-kb EcoR1-EcoR1 DNA fragment from the Lactococcus lactis subsp . cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes . In vitro transcription-translation using Escherichia coli S30 extract showed the synthesis of 41,000-, 23,000- and 12,000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.

J Bacteriol, 1990 Aug, 172(8), 4543 - 8
Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids; Sozhamannan S et al.; Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector . Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA . The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA . Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis) . We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.

J Bacteriol, 1990 Aug, 172(8), 4151 - 60
Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030; Romero DA et al.; The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor . Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor) . Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates . Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030 . Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA . Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12 . A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination . Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca . 96% homology with lactococcal insertion sequence ISS1 . IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase . IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.

Appl Environ Microbiol, 1990 Aug, 56(8), 2551 - 8
Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis; Liu W et al.; Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues . The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans . The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step . Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues . One of the nucleophiles had a pKa of about 9.8 . The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety . After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified . The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred . High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products . The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin . These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.

Lett Appl Microbiol, 1990 Aug, 11(2), 62 - 4
High efficiency electroporation of Lactococcus lactis subsp . lactis LM0230 with plasmid pGB301; Dornan S et al.; Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al . 1981), has been reported by McIntyre & Harlander (1989a) . Improved transformation efficiencies of 10(2)-10(3)/micrograms DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b) . This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc . lactis subsp . lactis.

Appl Environ Microbiol, 1990 Jul, 56(7), 2255 - 8
Nucleotide sequence and distribution of the pTR2030 resistance determinant (hsp) which aborts bacteriophage infection in lactococci; Hill C et al.; The lactococcal plasmid pTR2030 encodes resistance to bacteriophage attack via two mechanisms, an abortive-infection mechanism, designated Hsp, and a restriction and modification system . We present the complete sequence of the hsp structural gene . The gene is 1,887 base pairs in length and encodes a protein with a predicted molecular mass of 73.8 kilodaltons . The upstream region was cloned in a promoter-screening vector and shown to direct the constitutive expression of the cat-86 gene . An internal probe was used to determine the distribution of the hsp sequence in industrially significant lactococcal strains and to evaluate its relatedness to another lactococcal plasmid implicated in an abortive-infection-type mechanism, pNP40 . No homology was detected, suggesting that this gene is not widely distributed in lactococci . Therefore, there are at least two independent abortive-infection genotypes in lactococci.

Appl Environ Microbiol, 1990 Jul, 56(7), 2180 - 5
Taxonomic differentiation of 101 lactococcal bacteriophages and characterization of bacteriophages with unusually large genomes; Prevots F et al.; Sixty-three virulent bacteriophages of Lactococcus lactis were differentiated by DNA-DNA hybridization . The results, including those of a previous classification of 38 phages of the same bacterial species (P . Relano, M . Mata, M . Bonneau, and P . Ritzenthaler, J . Gen . Microbiol . 133:3053-3063, 1987) show that 48% of the phages analyzed belong to a unique DNA homology group (group III) . Phages of this most abundant group had small isometric heads . Group I comprised 29% of the phages analyzed and was characterized by a small phage genome (19 to 22 kilobases) and a particular morphology with a prolate head . Like group III, this group contained representative phages of other classifications . Group II (21%) included virulent and temperate phages with small isometric heads . Two large isometric-headed phages, phi 109 and phi 111, were not related to the three DNA homology groups I, II, and III . The genome of phi 111 was unusually large (134 kilobases) and revealed partial DNA homology with another large isometric phage, 1289, described by Jarvis (type e) (A . W . Jarvis, Appl . Environ . Microbiol . 47:343-349, 1984) . The protein compositions of phi 111 and 1289 were similar (three common major proteins of 21, 28, and 32 kilodaltons).

J Bacteriol, 1990 Jul, 172(7), 4122 - 6
Simultaneous loss of N5-(carboxyethyl)ornithine synthase, nisin production, and sucrose-fermenting ability by Lactococcus lactis K1; Donkersloot JA et al.; A spontaneous derivative of Lactococcus lactis subsp . lactis K1 (formerly Streptococcus lactis K1) lacking N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) was isolated . This mutant had also lost the abilities to ferment sucrose and to produce the antibiotic nisin . Hybridization studies indicate that these linked traits are encoded on the chromosome of L . lactis K1 and that they may be located on a conjugative transposon.

Appl Environ Microbiol, 1990 Jul, 56(7), 2164 - 9
Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes; Ross P et al.; The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp . lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined . The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene . The cloned thyA gene was strongly expressed in L . lactis subsp . lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain . In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria . In transformation experiments with thyA mutants of E . coli and conjugation experiments with thyA mutants of R . meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline . Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon . This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.

Appl Environ Microbiol, 1990 Jul, 56(7), 2156 - 63
Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp . lactis; Ross P et al.; The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp . lactis . The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E . coli thyA mutants . DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L . lactis subsp . lactis . By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment . The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique . The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments . The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms . Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E . coli . An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region . The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.

Appl Environ Microbiol, 1990 Jul, 56(7), 2099 - 103
Cloning of the citrate permease gene of Lactococcus lactis subsp . lactis biovar diacetylactis and expression in Escherichia coli; Sesma F et al.; The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp . lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18 . This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L . lactis biovar diacetylactis . The citrate permease was under the control of the lac promoter of pUC18 . Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Appl Microbiol Biotechnol, 1990 Jul, 33(4), 401 - 6
Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp . cremoris are related to secondary binding specificity; Exterkate FA; Various chromophoric peptides have been tested as substates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp . cremoris . The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength . A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength . This cation binding site most probably resides in the aspartic acid residue 166, which in PIII is substituted by asparagine . Substitution of the threonine residue 138 by lysine in PIII may also play a role . The binding step in the reaction pathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site . In addition, hydrophobic interactions contribute to the binding process . Masking of the cation binding site only increases the Michaelis constant Km; the catalytic constant kcat is not affected . In the absence of the cation binding site (viz . in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively . High activities are measured only if a high ionic strength is introduced . Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1990 Jun, 56(6), 1890 - 6
Insertion elements on lactococcal proteinase plasmids; Haandrikman AJ et al.; DNA segments of 809 and 808 nucleotides, with 18-base-pair terminal inverted repeats, are present on the proteinase plasmids pWV05 from Lactococcus lactis subsp . cremoris Wg2 and pSK111 from L . lactis subsp . cremoris SK11, respectively . These DNA segments are highly similar: 77% identical nucleotides and both contain an open reading frame that can encode a protein of 226 amino acids . Furthermore, both DNA segments are located downstream of the proteinase maturation gene prtM, but they differ individually in their orientation with respect to the prtM gene . On the basis of the striking similarity between ISS1, an 808-base-pair insertion sequence (IS) from L . lactis subsp . lactis ML3 lactose plasmid pSK08, and the DNA segments of pWV05 and pSK111, we propose that these DNA segments comprise IS elements . The IS elements from strains Wg2 and SK11 were named ISS1W and ISS1N, respectively . On pWV05, ISS1W is flanked on one side by only part of a second IS element, indicating that pWV05 evolved as a deletion derivative of a precursor plasmid that carried at least two IS elements.

Appl Environ Microbiol, 1990 Jun, 56(6), 1882 - 9
The bacteriophage kh receptor of Lactococcus lactis subsp . cremoris KH is the rhamnose of the extracellular wall polysaccharide; Valyasevi R et al.; A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp . cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls . The cell wall carbohydrates of L . lactis subsp . cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine . Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages . In addition, rhamnose was required for binding phages kh and ml3 . Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose . Rather, galactose may be an essential structural component located in the vicinity of the receptor . Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages . Inactivation required divalent cations and was irreversible . Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage . Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium . Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.

J Bacteriol, 1990 Jun, 172(6), 3485 - 9
High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs; Hayes F et al.; In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp . lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed . A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L . lactis-Escherichia coli shuttle vector . This latter recombination event was site and orientation specific on both plasmids . Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected . Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.

Appl Environ Microbiol, 1990 Apr, 56(4), 934 - 42
Molecular characterization of promoters of the Lactococcus lactis subsp . cremoris temperate bacteriophage BK5-T and identification of a phage gene implicated in the regulation of promoter activity; Lakshmidevi G et al.; DNA fragments from the temperate lactococcal bacteriophage BK5-T were cloned into the promoter-detecting plasmid pMU1328 . Five DNA fragments conferring promoter activity were selected by transformation of Streptococcus sanguis and were functional in Escherichia coli, S . sanguis, and Lactococcus lactis subspp . lactis and cremoris . The nucleotide sequences of these fragments were determined, and primer extension analysis was used to locate the site of initiation of transcription from each promoter in both E . coli and S . sanguis . Transcription was initiated from the same nucleotide in these two organisms, and the promoters contained -10 and -35 regions similar to the consensus sequence for E . coli promoters . The activities of three of the five promoters were decreased two- to threefold when a compatible plasmid containing a 3.8-kilobase-pair EcoRI fragment (EcoRI-f) of BK5-T was coresident with the promoter-containing plasmid in either L . lactis subsp . cremoris or E . coli . Data from Tn5 mutagenesis, subcloning experiments, and DNA sequence analysis indicate that this decrease in promoter activity requires a region of EcoRI-f that contains a 621-base-pair open reading frame . This region has been designated bpi (for BK5-T promoter inhibitor).

J Clin Microbiol, 1990 Mar, 28(3), 416 - 21
Phenotypic characterization, cellular fatty acid composition, and DNA relatedness of aerococci and comparison to related genera; Bosley GS et al.; Aerococci can be misidentified as streptococci, enterococci, pediococci, lactococci, or leuconostocs . To distinguish the genus and determine if another species is needed in the present taxon, we analyzed 37 aerococci for cellular fatty acids and compared them with 377 strains of gram-positive cocci, including the species type strains from each of the related genera . The cellular fatty acid profile of aerococci was distinguishable from other genera . Two relatively novel fatty acids found in the aerococci were identified as C16:1 omega 9c and C16:1 omega 9t . Eleven strains of aerococci (including a strain originally identified as "Gaffkya" species) were chosen for DNA-DNA reassociation studies with the type strain Aerococcus viridans ATCC 11563; DNAs from eight of these strains were more than 75% related to the type strain and had 1 to 4% divergence in related sequences . The remaining three strains were 60 to 70% related to the type strain, had 7 to 11.5% divergence, and may represent a second species, Aerococcus genospecies 2 . beta-Glucuronidase, alpha-galactosidase, and beta-galactosidase were useful in characterizing the aerococci.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 555 - 66
Analysis of the genetic determinant for production of the peptide antibiotic nisin; Dodd HM et al.; The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized . As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic . The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains . Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule . Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant . This element, termed IS904, is present in multiple copies in the genome of L . lactis . The nisin determinant of L . lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes . Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome . Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 109 - 13
Lactococcus piscium sp . nov . a new Lactococcus species from salmonid fish; Williams AM et al.; Chemical and molecular taxonomic studies were performed on a representative strain of some lactic acid bacteria of unknown taxonomic position isolated from salmonid fish . The results demonstrate that the fish bacterium represents a new species of the genus Lactococcus for which the name Lactococcus piscium sp . nov . is proposed . The type strain of Lactococcus piscium is NCFB 2778.

Arch Microbiol, 1990, 154(6), 560 - 5
Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp . lactis OZS1; Smigielski AJ; A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp . lactis strain OZS1 carrying genes for lactose metabolism was characterised . A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer . Cointegrate formation was found to involve an insertional element located on pOZS550 . The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L . lactis subsp . lactis strain ML3 . Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550 . Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L . lactis subsp . lactis strains and two L . lactis subsp . cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

Arch Microbiol, 1990, 154(1), 99 - 104
Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp . cremoris H2 and construction of a new lactococcal vector pFX1; Xu FF et al.; The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp . cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with lambda NM1149, and also directly into Lactococcus lactis subsp . lactis 4125 using a newly-constructed broad host-range vector pFX1 . Proteinase was expressed in both transformed organisms . The proteinase resembles a PI type since it preferentially degraded beta-casein . The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plasmid proteinase genes . High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.

Plasmid, 1990 Jan, 23(1), 71 - 5
Stacking of three different restriction and modification systems in Lactococcus lactis by cotransformation; Josephsen J et al.; Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2 . The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events . Analysis of cotransformants showed that three different R/M plasmids could be combined in L . lactis MG1363 . The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased . Some plasmid combinations were unstable suggesting replicon incompatibility.

Eur J Biochem, 1989 Dec 22, 186(3), 649 - 55
Distribution analyses of chain substituents of lipoteichoic acids by chemical degradation; Schurek J et al.; The lipoteichoic acid from Lactococcus lactis Kiel 48337 was analyzed . It had 61% of its glycerophosphate residues substituted with alpha-D-galactopyranosyl residues . Non-substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage . The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE-Sephadex into 10 pairs of molecular species with n from 1 to 10 . The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents . A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid . Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern . The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P = 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.

Gene, 1989 Dec 21, 85(1), 169 - 76
Cloning and expression of the Lactococcus lactis subsp . cremoris SK11 gene encoding an extracellular serine proteinase; de Vos WM et al.; The Lactococcus lactis subsp . cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods . The complete prtP gene could not be cloned in E . coli and L . lactis on high-copy-number plasmid vectors . However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L . lactis subsp . lactis 712 and L . lactis subsp . cremoris SK11, respectively . The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene . The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111 . The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products . In this way the expression signals of prtP were localized and overproduction was obtained in L . lactis subsp . lactis . Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci . A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.

J Bacteriol, 1989 Nov, 171(11), 6135 - 40
Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein; Smid EJ et al.; The chlorated dipeptide L-alanyl-beta-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis . Spontaneous mutants resistant to the dipeptide were isolated from plates . The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type . Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant of L . lactis ML3 . Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di- and tripeptide transport . Uptake of di- and tripeptides by L . lactis ML3 was found to be mainly mediated by one peptide transport system . The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids . These results indicate that peptide transport in L . lactis ML3 is an essential component in the process of casein utilization during growth in milk.

Appl Environ Microbiol, 1989 Oct, 55(10), 2621 - 6
Improved electroporation efficiency of intact Lactococcus lactis subsp . lactis cells grown in defined media; McIntyre DA et al.; The impact of growth conditions on electroporation of Lactococcus lactis subsp . lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated . Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) . Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA) . Other parameters influencing electroporation of L . lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions . A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine . A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day . In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample.

FEMS Microbiol Lett, 1989 Oct 1, 52(1-2), 183 - 7
Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene; Ross R et al.; A cosmid gene library of the genome of Lactococcus lactis subsp . lactis 712 has been constructed in the broad host range plasmid pLAFR1 in Escherichia coli LE392 . Three lactococcal genes from the bank were identified by heterologous complementation of specific mutations in strains of E . coli . A cosmid clone encoding a putative lactose transport gene was identified by complementing an E . coli lacY mutant . The complemented clone supported the uptake of 14C lactose in transport assays . The DNA fragment responsible was subcloned and localised to a 1.28 kb fragment of the lactococcal chromosome.

J Appl Bacteriol, 1989 Oct, 67(4), 453 - 60
16S ribosomal ribonucleic acid sequence analyses of lactococci and related taxa . Description of Vagococcus fluvialis gen . nov., sp . nov; Collins MD et al.; The phylogenetic status of members of the genus Lactococcus and some motile strains which react with Lancefield group N antiserum was examined by reverse transcriptase sequencing of 16S ribosomal ribonucleic acid . In agreement with earlier nucleic acid hybridization and immunological studies of superoxide dismutase the 16S sequence data clearly demonstrate that the lactococci represent a distinct phylogenetic group equivalent in rank to the genera Enterococcus and Streptococcus . The motile group N strains from chicken faeces and river water, however, were found to be phylogenetically unrelated to lactococci but displayed a closer, albeit loose, association with members of the genus Enterococcus . On the basis of the present sequence data and earlier chemotaxonomic studies it is proposed that the motile group N strains be classified in a new genus Vagococcus, as Vagococcus fluvialis sp . nov . The type strain of V . fluvialis is NCDO 2497.

Appl Environ Microbiol, 1989 Sep, 55(9), 2416 - 9
The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+); Hill C et al.; pTR2030 is a conjugative plasmid which encodes resistance to bacteriophage in lactococci by a mechanism that aborts the phage infection (Hsp+) . Subcloning and in vivo deletion events showed that two independent mechanisms of resistance are located on a 13.6-kilobase Bg/II fragment cloned in pSA3; one mechanism is responsible for the abortive infection, and the other incodes a restriction modification system . The introduction of pTR2030 or the recombinant plasmid pTK6 resulted in the loss of a resident restriction modification plasmid in Lactococcus lactis NCK202 which was not previously identified.

Appl Environ Microbiol, 1989 Sep, 55(9), 2410 - 3
DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity; Steele JL et al.; DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined . Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant . Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17 . The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.

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