Lactococci

Appl Environ Microbiol, 2001 Jun, 67(6), 2677 - 82
Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate; Jensen NB et al.; Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp . cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h(-1) . More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate . Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point . Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate . The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed . Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, alpha-acetolactate synthase, and the NADH oxidases . Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.

J Bacteriol, 2001 Jun, 183(12), 3614 - 22
Transcriptional pattern of genes coding for the proteolytic system of Lactococcus lactis and evidence for coordinated regulation of key enzymes by peptide supply; Guedon E et al.; The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), P(I) and P(III) proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) of Lactococcus lactis MG1363 was analyzed in response to different environmental factors . Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37 degrees C, and peptide supply on the transcription of these genes . Only transcription of the pepP gene is modulated by the source of sugar . The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression . Elevated temperature had no significant effect on the level of transcription of these genes . prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium . Moreover, the transcription of prtP1, prtP3, pepC, pepN, and the opp-pepO1 operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine . The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon . The significance of these results with respect to the functions of different components of the proteolytic system in L . lactis are discussed.

J Mol Biol, 2001 Jun 1, 309(2), 361 - 86
Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference; Singh NN et al.; Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA . The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences . Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches . In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand . Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions . These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing . Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon . Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site . Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site .

Mol Genet Genomics, 2001 Mar, 265(1), 189 - 97
Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage phi LC3; Blatny JM et al.; Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp . cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286 . The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T . A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3 . Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region . Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively . In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L . lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein . These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

Biotechnol Bioeng, 2001 Jul 20, 74(2), 108 - 15
Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism; Garrigues C et al.; Two strains of Lactococcus lactis ssp . cremoris, MG 1820 and MG 1363, which differed by the presence or absence of the lactose plasmid, respectively, were cultivated in batch-mode fermentation on lactose as carbon substrate . A correlation between the rate of sugar consumption, the growth rate, and the type of metabolism was observed . The MG 1820 strain grew rapidly on lactose and homolactic fermentation occurred . The major regulating factor was the NADH/NAD(+) ratio proportional to the catabolic flux, which inhibited glyceraldehyde-3-phosphate dehydrogenase activity . This control led to an increase in metabolite concentration upstream of this enzyme, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, and inhibition of pyruvate formate lyase activity, while lactate dehydrogenase was strongly activated by the high coenzyme ratio . The contrary was observed during growth of the MG 1363 strain . Further investigation during growth of L . lactis ssp . lactis NCDO 2118 on galactose as carbon substrate and on various culture media enabling the growth rate to proceed at various rates demonstrated that the relative flux between catabolism and anabolism was the critical regulating parameter rather than the rate of glycolysis itself . In a minimal medium, where anabolism was strongly limited, the rate of sugar consumption was reduced to a low value to avoid carbon and energy waste . Despite this low sugar consumption rate, the catabolic flux was in excess relative to the anabolic capability and the NADH/NAD+ ratio was high, typical of a situation of nonlimiting catabolism leading to a homolactic metabolism .

Prikl Biokhim Mikrobiol, 2001 Mar-Apr, 37(2), 227 - 31
{Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis}; Kisrieva IuS et al.; Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp . lactis biovar . diacetylactis 4 and purified . Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM . The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape . The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate . Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively . The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5 . The molecular weight of acetolactate synthase was 150 kDa . In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

J Bacteriol, 2001 Jun, 183(11), 3458 - 67
Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux; Andersen HW et al.; Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed . These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level . In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels . In complex medium growth was even further reduced . Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions . One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates . Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites . The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.

J Bacteriol, 2001 Jun, 183(11), 3391 - 8
Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis; Monedero V et al.; In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46 . This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase . We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose . In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect . In vitro experiments carried out with L . lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG) . In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion . Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant . It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

Genome Res, 2001 May, 11(5), 731 - 53
The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp . lactis IL1403; Bolotin A et al.; Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter . It is also the best-characterized lactic acid bacterium . We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step . The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements . Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes . A complete set of late competence genes is present, indicating the ability of L . lactis to undergo DNA transformation . Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration . It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group.

FEBS Lett, 2001 Apr 27, 495(3), 178 - 83
Evolution and function of the neisserial dam-replacing gene; Cantalupo G et al.; Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis . In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam . We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3' . drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter . This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 20 - 3
{Fusion expression of a peptide antibiotic-apidaecin gene in Lactococcus lactis}; Sun C et al.; The ubiquitin fusion of apidaecin was expressed in Lactococcus lactis, using a novel nisin-inducible expression system . After induction, a specific band could be detected in the extracts of the host strain by Tricine-SDS-PAGE and Western blotting . Production of the fusion was up to 7.2% of the total soluble protein of the host strain . While the fusion was cut by ubiquitin specific protease-UBP1, the product had distinct antibacterial activity.

Clin Diagn Lab Immunol, 2001 May, 8(3), 545 - 51
Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin; Chatel JM et al.; The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen . Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle . The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L . lactis . Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture . To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations . Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG . Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form . The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined . Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain . Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG . No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice . These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.

Lett Appl Microbiol, 2001 May, 32(5), 326 - 30
Rapid, high-throughput extraction of bacterial genomic DNA from selective-enrichment culture media; Wilson T et al.; AIMS: To create a fast, sensitive and inexpensive high-throughput method for the extraction of bacterial genomic DNA from selective-enrichment culture media . METHODS AND RESULTS: Lysis of bacteria was achieved using guanidinium isothiocyanate, and DNA was extracted using 96-well glass microfibre filtration plates . Extraction-PCR detected the presence of 1 cfu Yersinia ruckeri and 16 cfu Lactococcus garvieae 200 microl(-1) sample of selective-enrichment medium . CONCLUSION: An efficient method for high-throughput extraction of bacterial genomic DNA from selective-enrichment culture media was achieved . SIGNIFICANCE AND IMPACT OF THE STUDY: This method enables detection of covert bacterial infections in fish . The simultaneous extraction of large numbers of samples allows for its use in bacterial monitoring programmes and quarantine.

Microbiology, 2001 May, 147(Pt 5), 1223 - 33
Identity elements in tRNA-mediated transcription antitermination: implication of tRNA D- and T-arms in mRNA recognition; van de Guchte M et al.; tRNA-mediated transcription antitermination has been shown to control the expression of several amino acid biosynthesis operons and aminoacyl-tRNA-synthetase-encoding genes in Gram-positive bacteria . A model originally put forward by Grundy & Henkin describes the conserved structural features of the leader sequences of these operons and genes . Two sequences of 3 and 4 nt, respectively, take a central position in this model and are thought to be responsible for the binding of the system-specific uncharged tRNA, an interaction which would stabilize the antiterminator conformation of the leader . Here a further evolution of this model is presented based on an analysis of trp regulation in Lactococcus lactis in which a function is assigned to hitherto unexplained conserved structures in the leader sequence . It is postulated that the mRNA-tRNA interaction involves various parts of the tRNA in addition to the anticodon and the acceptor in the original model and that these additional interactions contribute to the recognition of a specific tRNA, and hence to the specificity and efficacy of the regulatory response.

Res Microbiol, 2001 Mar, 152(2), 131 - 9
Distinctive features of homologous recombination in an 'old' microorganism, Lactococcus lactis; Quiberoni A et al.; Homologous recombination is needed to assure faithful inheritance of DNA material, especially under stress conditions . The same enzymes that repair broken chromosomes via recombination also generate biodiversity . Their activities may result in intrachromosomal rearrangements, assimilation of foreign DNA, or a combination of these events . It is generally supposed that homologous recombination systems are conserved, and function the same way everywhere as they do in Escherichia coli, the accepted paradigm . Studies in an 'older' microorganism, the gram-positive bacterium of the low GC branch Lactococcus lactis, confirm that many enzymes are conserved across species lines . However, the main components of the double strand break (DSB) repair system, an exonuclease/helicase (Exo/hel) and a short DNA modulator sequence Chi, differ markedly between bacteria, especially when compared to the gram-negative analogues . Based on our studies, a model is proposed for the functioning of the two-subunit Exo/hel of L . lactis and other gram-positive bacteria, which differs from that of the three-subunit E . coli enzyme . The differences between bacterial DSB repair systems may underlie a selection for diversity when dealing with DSB . These and other features of homologous recombination in L . lactis are discussed.

Virology, 2001 Apr 25, 283(1), 93 - 109
Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages; Brondsted L et al.; A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned . This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation . Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination . Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution . Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L . lactis . This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool .

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 330 - 7
Growth of nisin-producing lactococci in cooked rice supplemented with soybean extract and its application to inhibition of Bacillus subtilis in rice miso; Kato T et al.; Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated . By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration . Lactococcus lactis subsp . lactis IFO12007 (Lc . lactis, a producer of the bacteriocin nisin) proliferated at 10(8 to approximately 9) cells/g after 24 h of incubation and produced high activity of nisin . The fermented rice with Lc . lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains . While some strains of LAB markedly inhibited the growth of Asp . oryzae, resulting in failure of koji fermentation, Lc . lactis did not affect the growth of these molds . When Lc . lactis was used for rice miso fermentation as a lactic acid starter culture, Lc . lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B . subtilis, which had been inoculated at the beginning of the koji fermentation . The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color . Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.

J Bacteriol, 2001 May, 183(9), 2785 - 94
The pyrimidine operon pyrRPB-carA from Lactococcus lactis; Martinussen J et al.; The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp . lactis MG1363 . The functions of the different genes were established by mutational analysis . The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation . The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil . The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate . The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis . The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.

Appl Environ Microbiol, 2001 Apr, 67(4), 1700 - 9
Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis; Emond E et al.; pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system . Sequence analysis revealed five open reading frames and two putative cis-acting regions . None appears to code for undesirable phenotypes with regard to food applications . Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis . A two-component food-grade cloning system was derived from the pCD4 replicon . The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L . lactis DNA and has no selection marker . The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker . The pCOM1 construct can only replicate in L . lactis if trans complemented by the RepB initiator provided by pVEC1 . Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1 . Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step . The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L . lactis and two industrial strains . The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains . The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

Appl Environ Microbiol, 2001 Apr, 67(4), 1423 - 8
Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic; Enouf V et al.; Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice . To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary . We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable . The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system . Production of recombinant NSP4 (rNSP4) was observed in L . lactis for both locations . In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4 . Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited . The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus . We showed that L . lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.

Yi Chuan Xue Bao, 2001, 28(3), 285 - 90
{Cloning and expression of nisZ gene in Lactococcus lactis}; Chen XZ et al.; The gene encoding the precursor of nisin was amplified by PCR using the lambda HJ-3 DNA as the template, which contained the entire nisin biosynthesis gene cluster from Lactococcus lactis AL2 with high yield of nisin, and was cloned into pMG36e . The recombinant plasmid pHJ201 was introduced into Lactococcus lactis NZ9800 by electroporation . pHJ201 is very stable in L . lactis NZ9800 . Antimicrobial activity test and Tricine-SDS-PAGE analysis revealed that L . lactis NZ9800 harbouring pHJ201 restored ability of nisin production, but the production level was markedly lower than L . lactis AL2 . The result of DNA sequence analysis indicated that Nisin Z is produced by L . lactis AL2.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 71 - 80
Physiological function of exopolysaccharides produced by Lactococcus lactis; Looijesteijn PJ et al.; The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L . lactis ssp . cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors . There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin . A model system showed that EPS production did not affect the survival of L . lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage . The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin . Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme . However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 183 - 8
Determination of peroxy radical-scavenging of lactic acid bacteria; Stecchini ML et al.; Responses of lactic acid bacteria (LAB) to peroxy radicals generated via thermal (40 degrees C) decomposition of the diazocompound 2,2,-azo-bis (2-amidinopropane) dihydrochloride (ABAP), were studied . In general, LAB displayed survival curves with shoulders and tails indicative of 'multihit' killing by exposure to peroxy radicals . One strain, Lactococcus lactis subsp . lactis DIP15, producing a slope of 0.0105 in the kinetic analysis when exposed to 4 mM ABAP, exhibited a measurable antioxidant capacity . The other LAB failed to show any significant antioxidant capacity . The antioxidant capacity of strain DIP15 remained constant after cells have been heat-treated, suggesting that compounds bearing free radical scavenging capacity are rather stable.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 151 - 9
Pre-inoculation enrichment procedure enhances the performance of bacteriocinogenic Lactococcus lactis meat starter culture; Scannell AG et al.; Sodium nitrite and sodium chloride may inhibit growth and bacteriocinogenesis of protective starter cultures . To reduce sensitivity of a lacticin 3147-producing starter culture to nitrite, prior to production of salami, Lactococcus lactis DPC 4275 was placed in a number of pre-inoculation treatments, containing (a) 1% glucose, (b) 2.5 ppm manganese (Mn), (c) 250 ppm magnesium (Mg), (d) 2.5 ppm manganese + 250 ppm magnesium (Mn + Mg), and held at ambient temperature for 30 min and 4 degrees C for 2 h . The growth, pH reduction, and bacteriocin production was monitored in beaker sausage over a period of 10 days at 28 degrees C, corresponding to typical salami production time, and compared to untreated starter culture . The effect of 1% tryptone and inoculum level on growth and bacteriocin production was also determined . Challenge tests were performed using Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 as target strains . All treatments gave a significantly higher (P < 0.05) initial starter level than the untreated starter . Beaker sausage inoculated with either low (10(7)) or high (10(9)) levels of starter culture, treated with Mn + Mg reached significantly (P < 0.05) higher levels by day 10 than other treatments . Trends indicate that Mn + Mg also gave best pH reduction in sausage containing the low-level starter culture, sausage and significantly lower (P < 0.05) values for sausage produced with higher inoculum . Bacteriocin production was also higher in starter culture treated with Mn, or glucose . Pre-treatment with Mg gave a 2-fold increase in bacteriocin, the addition of Mn augmenting this increase further . The incorporation of tryptone gave no additional effect . In beaker sausage, both L . innocua and S . aureus populations showed significant reductions (P < 0.05) in the presence of the bacteriocinogenic strain compared to a non-bacteriocinogenic control strain.

Mol Microbiol, 2001 Feb, 39(4), 982 - 93
Regulation of immunity to the two-component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR; McAuliffe O et al.; Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp . lactis DPC3147 . In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively . Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream beta-galactosidase (beta-gal) fusion beyond an intragenic stem-loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery . The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR . A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147 . Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the beta-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR . Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

Structure Fold Des, 2000 Dec 15, 8(12), 1227 - 38
Structure of dihydroorotate dehydrogenase B: electron transfer between two flavin groups bridged by an iron-sulphur cluster; Rowland P et al.; BACKGROUND: The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD) . Based on their sequences, DHODs are grouped into two major families . Lactococcus lactis is one of the few organisms with two DHODs, A and B, belonging to each of the two subgroups of family 1 . The B enzyme (DHODB) is a prototype for DHODs in Gram-positive bacteria that use NAD+ as the second substrate . DHODB is a heterotetramer composed of two different proteins (PyrDB and PyrK) and three different cofactors: FMN, FAD, and a {2Fe-2S} cluster . RESULTS: Crystal structures have been determined for DHODB and its product complex . The DHODB heterotetramer is composed of two closely interacting PyrDB-PyrK dimers with the {2Fe-2S} cluster in their interface centered between the FMN and FAD groups . Conformational changes are observed between the complexed and uncomplexed state of the enzyme for the loop carrying the catalytic cysteine residue and one of the lysines interacting with FMN, which is important for substrate binding . CONCLUSIONS: A dimer of two PyrDB subunits resembling the family 1A enzymes forms the central core of DHODB . PyrK belongs to the NADPH ferredoxin reductase superfamily . The binding site for NAD+ has been deduced from the similarity to these proteins . The orotate binding in DHODB is similar to that in the family 1A enzymes . The close proximity of the three redox centers makes it possible to propose a possible electron transfer pathway involving residues conserved among the family 1B DHODs.

Lett Appl Microbiol, 2001 Jan, 32(1), 36 - 41
Characterization of lactic acid bacteria in the larval midgut of the keratinophagous lepidopteran, Hofmannophila pseudospretella; Shannon AL et al.; In two of eight Hofmannophila pseudospretella specimens studied by microscopy, the larval midgut contained an unidentified micro-organism . Although not seen microscopically in midgut sections, bacteria were isolated from dissected midgut . Microscopy, carbohydrate utilization and ribosomal sequence data all separated the isolates into the same three classes . These were identified as Lactococcus lactis, Carnobacterium piscicola and, tentatively, Bacillus subtilis, the first two being facultative anaerobes and the latter, an aerobe . The bacteria were therefore not specifically adapted to the reducing conditions of the midgut.

Lett Appl Microbiol, 2001 Jan, 32(1), 20 - 5
Peptide utilization by Lactococcus lactis and Leuconostoc mesenteroides; Foucaud C et al.; To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin . Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases . Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc . mesenteroides assimilated only shorter peptides containing up to seven amino acids . Growth was limited by the transport of peptides and not by their hydrolysis . The nutritional value of peptides varied with the strains and the composition of the peptides, L . lactis being advantaged over Leuc . mesenteroides.

Microbiology, 2001 Jan, 147(Pt 1), 215 - 24
Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk; Dudley E et al.; Amino acid aminotransferases (ATases), which catalyse the last biosynthetic step of many amino acids, may have important physiological functions in Lactococcus lactis during growth in milk . In this study, the aspartate ATase gene (aspC) from L . lactis LM0230 was cloned by complementation into Escherichia coli DL39 . One chromosomal fragment putatively encoding aspC was partially sequenced . A 1179 bp ORF was identified which could encode for a 393 aa, 43.2 kDa protein . The deduced amino acid sequence had high identity to other AspC sequences in GenBank and is a member of the Igamma family of ATases . Substrate-specificity studies suggested that the lactococcal AspC has ATase activity only with aspartic acid (Asp) . An internal deletion was introduced into the L . lactis chromosomal copy of aspC by homologous recombination . The wild-type and mutant strain grew similarly in defined media containing all 20 amino acids and did not grow in minimal media unless supplemented with asparagine (Asn) . The mutant strain was also unable to grow in or significantly acidify milk unless supplemented with Asp or Asn . These results suggest that only one lactococcal ATase is involved in the conversion of oxaloacetate to Asp, and Asp biosynthesis is required for the growth of L . lactis LM0230 in milk.

Appl Environ Microbiol, 2001 Feb, 67(2), 929 - 37
Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker; O' Sullivan D et al.; The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D . O'Sullivan, D . P . Twomey, A . Coffey, C . Hill, G . F . Fitzgerald, and R . P . Ross, Mol . Microbiol . 36:866-876; 2000) . In this communication we provide evidence that the recombination event is favored by phage infection . The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization . The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation . In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria . The functionality of this intron was evident by demonstrating splicing in vivo . Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.

Appl Environ Microbiol, 2001 Feb, 67(2), 858 - 64
Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum; Cibik R et al.; A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach . The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide . In contrast to most of the other known peptidoglycan hydrolases, L . citreum Mur protein does not contain amino acid repeats involved in cell wall binding . The purified L . citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis . An active chimeric protein was constructed by fusion of L . citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis . Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L . lactis; normal cell separation after cell division was restored by Mur-cA expression.

Appl Environ Microbiol, 2001 Feb, 67(2), 774 - 81
Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1; Ostergaard S et al.; DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene . Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13) . The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans . Site-specific mutations were introduced into the replication protein and into the repeats . The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon . Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification . In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo . These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.

Appl Environ Microbiol, 2001 Feb, 67(2), 608 - 16
Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis; McGrath S et al.; Homologous replication module genes were identified for four P335 type phages . DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17 . One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages . DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector . Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred . A number of antisense plasmids targeting specific genes of the replication module were constructed . Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication . These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication . Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.

J Biol Chem, 2000 Apr 14, 275(15), 10962 - 7
Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis . A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis; Vigano C et al.; LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis . A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes . These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle . The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released . (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process . Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place . After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.

J Appl Microbiol, 2001 Jan, 90(1), 59 - 67
Enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway; Ayad EH et al.; Combinations of lactococcal strains from various origins with divers properties were developed as new starters for new dairy products . Flavour formation by such tailor-made cultures was studied . In some cases, a strongly enhanced flavour was observed . For instance, the combination of B1157 and SK110 strains in milk resulted in a very strong chocolate-like flavour . B1157 produces only a moderate chocolate-like flavour, whereas SK110 alone fails to produce this flavour . Headspace gas chromatography results corroborate the organoleptic evaluations . High levels of branched-chain aldehydes were found when B1157 and SK110 were grown together . The enzyme activities involved in this pathway were studied; both strains contain transaminase activity . Although B1157 had a very high amino acid decarboxylating activity, its release of amino acids from milk protein was limited . SK110 was strongly limited in decarboxylating activity, although this strain is very active in proteolysis . By combining these strains, the substrates released by SK110 can directly be used by the other strain, resulting in the completion of the whole flavour-formation pathway . This opens new avenues for the preparation of tailor-made cultures.

J Bacteriol, 2001 Jan, 183(2), 500 - 11
Evidence for horizontal transfer of SsuDAT1I restriction-modification genes to the Streptococcus suis genome; Sekizaki T et al.; Different strains of Streptococcus suis serotypes 1 and 2 isolated from pigs either contained a restriction-modification (R-M) system or lacked it . The R-M system was an isoschizomer of Streptococcus pneumoniae DpnII, which recognizes nucleotide sequence 5'-GATC-3' . The nucleotide sequencing of the genes encoding the R-M system in S . suis DAT1, designated SsuDAT1I, showed that the SsuDAT1I gene region contained two methyltransferase genes, designated ssuMA and ssuMB, as does the DpnII system . The deduced amino acid sequences of M . SsuMA and M.SsuMB showed 70 and 90% identity to M.DpnII and M.DpnA, respectively . However, the SsuDAT1I system contained two isoschizomeric restriction endonuclease genes, designated ssuRA and ssuRB . The deduced amino acid sequence of R.SsuRA was 49% identical to that of R.DpnII, and R.SsuRB was 72% identical to R.LlaDCHI of Lactococcus lactis subsp . cremoris DCH-4 . The four SsuDAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order: purF-purM-purN-purH-ssuMA-ssuMB-ssuRA++ +-ssuRB-purD-purE . The G+C content of the SsuDAT1I gene region (34.1%) was lower than that of the pur region (48.9%), suggesting horizontal transfer of the SsuDAT1I system . No transposable element or long-repeat sequence was found in the flanking regions . The SsuDAT1I genes were functional by themselves, as they were individually expressed in Escherichia coli . Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of ssuMA to 5 bp downstream of ssuRB was inserted in the intergenic sequence between purH and purD and that the insertion target site was not the recognition site of SsuDAT1I . No notable substitutions or insertions could be found, and the structures were conserved among all the strains . These results suggest that the SsuDAT1I system could have been integrated into the S . suis chromosome by an illegitimate recombination mechanism.

Appl Environ Microbiol, 2001 Jan, 67(1), 251 - 9
Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways; Walker SA et al.; A novel system that leaks beta-galactosidase (beta-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes . The late promoter of the lytic lactococcal bacteriophage phi31 is an 888-bp fragment (P(15A10)) encoding the transcriptional activator . When a high-copy-number P(15A10)::lacZ.st fusion was introduced into L . lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant beta-gal activity were detected in the supernatant (approximately 85% of the total beta-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t) . Studies showed that the phenotype resulted from expression of Tac31A from the P(15A10) fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains . Despite the high levels of beta-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy . Integration of the holin-lysin cassette of phage r1t, under the control of the phage phi31 late promoter, into the host genome of MG1363 yielded a similar "leaky" phenotype, indicating that holin and lysin might play a critical role in the release of beta-gal into the medium . In addition to beta-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system . Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell . These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.

Appl Environ Microbiol, 2001 Jan, 67(1), 33 - 41
Relationship between glycolysis and exopolysaccharide biosynthesis in Lactococcus lactis; Ramos A et al.; The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps(+)) and in nonproducer strain MG5267 (Eps(-)) were characterized . The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by (31)P nuclear magnetic resonance . The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps(+) strain than in the Eps(-) strain . The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps(+) strain, indicating that there is competition between EPS synthesis and cell growth . An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating . Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate . The activities of relevant enzymes, such as phosphoglucose isomerase, alpha-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains . (13)C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool . This study provided clues for ways to enhance EPS production by genetic manipulation.

J Appl Microbiol, 2000 Dec, 89(6), 960 - 8
Isolation of a bacteriocin-producing Lactococcus lactis subsp . lactis and application to control Listeria monocytogenes in Moroccan jben; Benkerroum N et al.; AIM: Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben . METHODS AND RESULTS: A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin . Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1 . Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures . Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used . For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h . Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days . CONCLUSIONS: In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L . monocytogenes in jben and of allowing shelf-life extension . SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

Enzyme Microb Technol, 2000 Dec, 27(10), 761 - 765
Secretion of biologically active murine interleukin-10 by Lactococcus lactis; Schotte L et al.; We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10) . mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein . The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L . lactis can efficiently secrete biologically active, murine IL-10 . Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase . The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH . Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.

Curr Microbiol, 2001 Jan, 42(1), 45 - 8
Molecular differentiation of Lactococcus lactis subspecies lactis and cremoris strains by ribotyping and site specific-PCR; Basaran P et al.; Twenty-five strains of Lactococcus lactis subspecies lactis and subspecies cremoris obtained from dairy industry and environmental collections were examined by 16S RNA automated ribotyping profiles and site-specific PCR (S-PCR) . By automated ribotyping, the majority of strains were classified in accordance with phenotypic characterization, with the exception of one lactis (220) and two cremoris (BO32 and 140) strains . A complete differentiation of subspecies lactis and cremoris in agreement with conventional phenotypic methods was achieved by S-PCR with a set of site-specific primer pairs (PR1, RM4, and F3) designed particularly from a deletion region found in subspecies cremoris, but not in lactis . Therefore, S-PCR with primers (PR1, RM4, and F3) is a rapid and very sensitive method for the distinction of lactis and cremoris subspecies in dairy production.

Appl Environ Microbiol, 2000 Dec, 66(12), 5518 - 20
Diacetyl and alpha-acetolactate overproduction by Lactococcus lactis subsp . lactis biovar diacetylactis mutants that are deficient in alpha-acetolactate decarboxylase and have a low lactate dehydrogenase activity; Monnet C et al.; Lactococcus lactis subsp . lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor alpha-acetolactate . Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in alpha-acetolactate decarboxylase and had low lactate dehydrogenase activity . The mutants produced large amounts of alpha-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.

Appl Environ Microbiol, 2000 Dec, 66(12), 5134 - 40
The autoproteolysis of Lactococcus lactis lactocepin III affects its specificity towards beta-casein; Flambard B et al.; The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards beta-casein was investigated . beta-Casein degradation was performed by using either an autolysin-defective derivative of L . lactis MG1363 carrying the proteinase genes of L . lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L . lactis SK11 . Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences . To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry . More than 100 oligopeptides were released from beta-casein by the cell-anchored proteinase . Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.

DNA Seq, 2000, 11(3-4), 239 - 45
LldI, a plasmid-encoded type I restriction and modification system in Lactococcus lactis; Deng YM et al.; A plasmid-encoded type I restriction and modification (R-M) system, designated LldI, was identified in Lactococcus lactis biovar diacetylactis LD10-1 . LldI consists of three genes encoding endonuclease, methylase and specificity subunits, respectively . RT-PCR analysis revealed that the three genes are co-transcribed as a polycistronic mRNA in L . lactis . The specificity subunit of LldI differs significantly in the target recognition domains from those of other type I R-M systems, suggesting that LldI confers a novel specificity in L . lactis.

Carbohydr Res, 2000 Oct 20, 329(1), 75 - 85
Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp . cremoris B39 and B891; van Casteren WH et al.; Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp . cremoris B39 and B891 . The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C . Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases . The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate . The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures . The enzyme degraded O-deacetylated EPS B891 faster than EPS B39 . Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.

Infect Immun, 2000 Dec, 68(12), 6871 - 8
Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells; Sinha B et al.; Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S . aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B . Sinha et al., Cell . Microbiol . 1:101-117, 1999) . However, it is unknown whether this mechanism is sufficient for S . aureus invasion . To address this question, various S . aureus adhesins (FnBPA, FnBPB, and clumping factor {ClfA}) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp . cremoris . Both noninvasive gram-positive microorganisms are genetically distinct from S . aureus, lack any known S . aureus surface protein, and do not bind fibronectin . Transformants of S . carnosus and L . lactis harboring plasmids coding for various S . aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin) . FnBPA or FnBPB but not of ClfA conferred invasiveness to S . carnosus and L . lactis . Invasion of 293 cells by transformants was comparable to that of strongly invasive S . aureus strain Cowan 1 . Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting . Thus, S . aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci . Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S . aureus-specific) coreceptors.

Lett Appl Microbiol, 2000 Nov, 31(5), 343 - 8
Conditions for conjugative transposon transfer in Lactococcus lactis; Blaiotta G et al.; Three different techniques for bacterial mating were applied to wild type and culture collection strains of Lactococcus lactis harbouring transposons: direct plate conjugation, filter mating and mating on milk agar . Efficiencies and frequencies of transfer were compared . Transconjugants were characterized by marker properties and molecular assays . Transposon-coded Suc+ Nis+ phenotype as well as Suc+ Bac+ Nis- phenotype were transferred with frequencies ranging between 10-9 and 10-6 . Milk agar plate mating was the best technique for obtaining gene transfer events involving wild type lactococci.

Fiziol Zh, 2000, 46(3), 78 - 83
{The production of antihypertensive peptides in beta-casein proteolysis}; Iukalo VH et al.; Antihypertensive peptides were obtained after the proteolysis of beta-casein by starter cells Lactococcus lactis ssp . lactis and lactococci with pepsin or fromase . The peptides have shown the effect as inhibitors of angiotensin-converting enzyme . The strongest action the peptide obtained after the proteolysis of beta-casein by synergic action of lactococci with pepsin has shown . It demonstrates a capability of formation of such peptides directly in milk products during their making and maturation under the action of proteolytic system of lactic acid bacteria and milk clothing proteases.

J Appl Microbiol, 2000 Oct, 89(4), 573 - 9
Continuous production of lacticin 3147 and nisin using cells immobilized in calcium alginate; Scannell AG et al.; Bacteriocinogenic strains, Lactococcus lactis subsp . lactis DPC 3147 and L . lactis DPC 496, producing lacticin 3147 and nisin, respectively, were immobilized in double-layered calcium alginate beads . These beads were inoculated into MRS broth at a ratio of 1:4 and continuously fermented for 180 h . Free cells were used to compare the effect of immobilization on bacteriocin production . After equilibrium was reached, a flow rate of 580 ml h(-1) was used in the immobilized cell (IC), and 240 ml h(-1) in free-cell (FC) bioreactors . Outgrowth from beads was observed after 18 h . Bacteriocin production peaked at 5120 AU ml(-1) in both IC and FC bioreactors . However, FC production declined after 80 h to 160 AU ml(-1) at the end of the fermentation . Results of this study indicate that immobilization offers the possibility of a more stable and long-term means of producing lacticin 3147 in laboratory media than with free cells.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12487 - 92
Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis; Detmers FJ et al.; The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues . The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA . To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA . The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity . The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA . This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.

Mol Cell Biol, 2000 Nov, 20(22), 8432 - 46
Multiple homing pathways used by yeast mitochondrial group II introns; Eskes R et al.; The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing . Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent . All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein . The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences . This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele . The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing . Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences . This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron . Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site . This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Biochemistry, 2000 Oct 24, 39(42), 13059 - 67
The conserved C-terminus of the citrate (CitP) and malate (MleP) transporters of lactic acid bacteria is involved in substrate recognition; Bandell M et al.; The membrane potential-generating transporters CitP of Leuconostoc mesenteroides and MleP of Lactococcus lactis are homologous proteins with 48% identical residues that catalyze citrate-lactate and malate-lactate exchange, respectively . The two transporters are highly specific for substrates containing a 2-hydroxycarboxylate motif (HO-CR(2)-COO(-)) in which substitutions of the R groups are tolerated well . Differences in substrate specificity between MleP and CitP are based on subtle changes in the interaction of the protein with the R groups affecting both binding and translocation properties . The conserved, 46-residue long C-terminal region of the transporters containing the C-terminal putative transmembrane segment XI was investigated for its role in substrate recognition by constructing chimeric transporters . Replacement of the C-terminal region of MleP with that of CitP and vice versa did not alter the exchange kinetics with the substrates malate and citrate, indicating that the main interactions between the proteins and di- and tricarboxylate substrates were not altered . In contrast, the interaction of the proteins with the monocarboxylate substrates mandelate and 2-hydroxyisovalerate changed in a complementary manner . The affinity of CitP for the S-enantiomers of these substrates was at least 1 order of magnitude lower than observed for MleP . Introduction of the C-terminal residues of MleP in CitP resulted in a higher affinity and vice versa . Interchanging the C-termini had a more complicated effect on the R-enantiomers, affecting different kinetic parameters with different substrates, indicating multiple interactions of the R groups at this side of the binding pocket . It is suggested that the binding pocket is located between transmembrane segment XI and the other transmembrane segments of the transporters.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 119 - 24
The life cycles of the temperate lactococcal bacteriophage phiLC3 monitored by a quantitative PCR method; Lunde M et al.; We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome . The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e . the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers . By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage phiLC3 after infection of its bacterial host Lactococcus lactis ssp . cremoris IMN-C18.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 85 - 9
Zinc uptake, oxidative stress and the FNR-like proteins of Lactococcus lactis; Scott C et al.; Lactococcus lactis ssp . cremoris MG1363 contains two FNR homologues, FlpA and FlpB, encoded by the distal genes of two paralogous operons (orfX(A/B)-orfY(A/B)-flpA/B) . An flpA flpB double mutant strain is hypersensitive to hydrogen peroxide and has a depleted intracellular Zn(II) pool . The phenotypes of the flp mutant strains suggest that FlpA and FlpB control the expression of high and low affinity ATP-dependent Zn(II) uptake systems, respectively . Plate tests revealed that expression from a orfX(B)::lac reporter was activated by Cd(II), consistent with other Zn(II)-regulated systems . The link between a failure to acquire Zn(II) and hypersensitivity to oxidative stress suggests that Zn(II) may be required to protect vulnerable protein thiols from oxidation.

Virology, 2000 Oct 25, 276(2), 315 - 28
Mutational analysis of two structural genes of the temperate lactococcal bacteriophage TP901-1 involved in tail length determination and baseplate assembly; Pedersen M et al.; Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp . cremoris 901-1 . The adsorption efficiencies of the mutated phages to the indicator strain L . lactic ssp . cremoris 3107 were determined and electron micrographs were obtained . Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages . Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively . The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination . Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure . The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate . Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented .

Diagn Microbiol Infect Dis, 2000 Oct, 38(2), 115 - 8
In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics; Giacometti A et al.; The in-vitro activity of nisin, a 34-residue peptide produced by several Lactococcus lactis strains, and ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana, alone and in combination with amoxycillin, amoxycillin-clavulanate, imipenem, clarithromycin, ciprofloxacin, rifampin and vancomycin was investigated against 40 nosocomial isolates of methicillin-resistant Staphylococcus aureus (MRSA) . All isolates were inhibited at concentrations of 1 to 32 microg/ml . Synergy was observed when the peptides were combined with other agents, with the exception of the beta-lactams . Finally, the consecutive exposures to each peptide did not result in selection of stable mutants with decreased susceptibility . Our finding show that nisin and ranalexin are active against MRSA, and that their activity is enhanced when they are combined with several antimicrobial agents.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 237 - 40
Novel sucrose transposons from plant strains of Lactococcus lactis; Kelly WJ et al.; Lactococcus lactis strains isolated from vegetable products transferred the ability to ferment sucrose in conjugation experiments with the recipient strain L . lactis MG1614 . Nisin production and sucrose fermentation were transferred together from two strains, but transfer also occurred from several other strains which did not produce nisin . Pulsed-field gel electrophoresis analysis showed that all transconjugants had acquired large chromosomal insertions at two main sites . Nisin sucrose transconjugants had gained inserts of 70 kb, while those that fermented sucrose without nisin production contained inserts of between 50 and 110 kb . Transconjugants from one donor had acquired a separate insertion of 55 kb which correlated with enhanced bacteriophage resistance, but contained neither nisin nor sucrose fermentation genes.

Int J Food Microbiol, 2000 Sep 10, 59(3), 141 - 56
Effects of mixed starter composition on nisin Z production by lactococcus lactis subsp . lactis biovar . diacetylactis UL 719 during production and ripening of Gouda cheese; Bouksaim M et al.; A starter culture system that produced both acid and nisin at acceptable rates in milk for manufacture of Gouda cheese was developed using nisin Z-producing L . lactis subsp . lactis biovar . diacetylactis UL 719 (UL 719) and a commercial Flora Danica (FD) starter culture . Different compositions of mixed cultures (0, 0.2, 0.4, 0.6 or 0.8% UL 719 with 1.4% FD) were tested for acidification and nisin Z production in milk after 12 h incubation at 30 degrees C . The 0.6/1.4% combination, selected as the optimal mixture of starter cultures, acidified milk to a suitable pH and produced nisin Z at a high concentration of 512 IU/ml . With this optimal combination, FD numbers of citrate-fermenting and non-fermenting bacteria did not change compared with the control (1.4% FD) . However, with 0.8% of L . lactis strain UL 719 and 1.4% of the FD starter culture, the numbers of citrate-fermenting and non-fermenting bacteria in fermented milk decreased compared with those obtained when milk was inoculated with 0.2, 0.4 or 0.6% of UL 719 added to 1.4% FD or control cultures (1.4% FD) . Mixed starter culture ratios 0.6/1.4%, 0.4/1.4% and 0.5/1.4% (UL 719/FD) were used to manufacture nisin Z containing Gouda cheese which was ripened up to 45 weeks . The composition of control cheeses made with 1.4% FD, and nisin Z-containing Gouda cheeses were similar with respect to percent moisture, fat, salt and protein . During the ripening period, the cell counts observed were approximately two logs higher in cheese made with the 0.6/1.4% mixed starter culture than in control cheese . In experimental cheese produced with 0.6/1.4% (UL 719/FD) mixed starter culture, nisin activity increased from 256 IU/g at the end of manufacture to a maximum of 512 IU/g after 6 weeks of ripening; the levels then decreased to 128 and 32 IU/g after 27 and 45 weeks of ripening, respectively . In contrast, nisin Z was not detected in experimental cheeses made with 0.4/1.4% or 0.5/1.4% (UL 719/FD) mixed starters . Using an affinity purified anti-nisin polyclonal antibody, anti-rabbit gold-conjugate and transmission electron microscopy, nisin Z was found to be localized in the cheese matrix, in fat globules, in the casein phase and concentrated at the fat-casein interface . After 27 weeks of ripening, nisin Z was detected preferentially in the fat globules of the experimental cheese.

J Microbiol Methods, 2000 Oct, 42(2), 139 - 47
A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material; Millar BC et al.; This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material . The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/alkali/heat lysis . The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method . This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material . Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert FAN aerobic blood culture material examined.

Int J Food Microbiol, 2000 Sep 25, 60(2-3), 241 - 9
Development of bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin; Scannell AG et al.; Immobilisation of the bacteriocins nisin and lacticin 3147 to packaging materials was investigated . Stability of both cellulose-based bioactive inserts and anti-microbial polyethylene/polyamide pouches was examined over time . Anti-microbial activity against the indicator strain Lactococcus lactis subsp . lactis HP, in addition to Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 was observed for all bacteriocin-adsorbed materials . Activity retention of the inserts showed an initial decrease in the first week of storage but remained stable for the remaining 3 months of the trial . However, adsorption of lacticin 3147 to plastic film was unsuccessful, nisin bound well and the resulting film maintained its activity for 3-month period, both at room temperature and under refrigeration . When applied to food systems, the anti-microbial packaging reduced the population of lactic acid bacteria in sliced cheese and ham stored in modified atmosphere packaging (MAP) at refrigeration temperatures, thus extending the shelf life . Nisin-adsorbed bioactive inserts reduced levels of Listeria innocua by > or = 2 log units in both products, and Staphylococcus aureus by approximately 1.5 log units in cheese, and approximately 2.8 log units in ham . Similar reductions were observed in cheese vacuum-packaged in nisin-adsorbed pouches.

J Clin Microbiol, 2000 Oct, 38(10), 3791 - 5
Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks and comparison with isolates of other countries and sources; Vela AI et al.; The phenotypic and genetic analysis results for 84 isolates of Lactococcus garvieae (including 62 strains from trout with lactococcosis from four different countries, 7 strains from cows and water buffalos with subclinical mastitis, 3 from water, and 10 from human clinical samples) are presented . There was great phenotypic heterogeneity (13 different biotypes) based on the acidification of saccharose, tagatose, mannitol, and cyclodextrin and the presence of the enzymes pyroglutamic acid arylamidase and N-acetyl-beta-glucosaminidase . L . garvieae also exhibited high genetic diversity by pulsed-field gel electrophoresis (PFGE), with 19 different pulsotypes among the isolates of L . garvieae studied . Only epidemiologically related strains, like the Spanish and Italian fish isolates and the cow and water buffalo isolates, displayed a close genetic relationship by PFGE, while the strains isolated from sporadic clinical cases, like the human isolates, were genetically unrelated . Overall, a general correlation between phenotypic and genetic data was observed . Epidemiological analysis of biotype and PFGE results indicated that the trout lactococcosis outbreaks in Spain and Portugal and those in France and Italy were produced by genetically unrelated clones . In Spain, two different clones were detected; the outbreaks diagnosed from 1995 onward were produced by a clone (biotype 2, pulsotype A1) which, although genetically related, was different from the one that was responsible for the outbreaks studied between 1991 and 1994 (biotype 1, pulsotype B) . The Portuguese isolate had a biochemical profile identical to that of the Spanish strain isolated from 1995 onward and is also genetically closely related to this strain (pulsotype A2) . There was a close relationship between the two pulsotypes (E and F) found in the Italian isolates . The French isolate (biotype 3, pulsotype D) was not genetically related to any other L . garvieae fish isolate . These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.

J Bacteriol, 2000 Oct, 182(20), 5823 - 31
The N-terminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells; Sao-Jose C et al.; The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated . We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner . Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site . The lethal effect of Lys44 expression observed in E . coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product . We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing . An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O . oeni-infected cells . The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation . Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin . Database searches and alignment of protein sequences support the prediction that other known O . oeni and Lactococcus lactis phages also encode secretory lysins . The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.

J Dairy Sci, 2000 Sep, 83(9), 1912 - 8
Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp . lactis C2; Wang H et al.; We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library . Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified . However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together . The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein . The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein . Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp . lactis C2 . Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Carbohydr Res, 2000 Aug 7, 327(4), 411 - 22
Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp . cremoris B891; van Casteren WH et al.; Lactococcus lactis subsp . cremoris B891 grown on whey permeate produced an exopolysaccharide containing D-Gal and D-Glc in a molar ratio of 2:3 . The polysaccharide was partially O-acetylated . By means of HF solvolysis, O-deacetylation, enzymic modification, sugar linkage analysis and ID/2D NMR studies the exopolysaccharide was shown to be composed of repeating units with the following structure: {structure: see text}.

J Bacteriol, 2000 Oct, 182(19), 5600 - 5
IS1675, a novel lactococcal insertion element, forms a transposon-like structure including the lacticin 481 lantibiotic operon; Dufour A et al.; Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon . The whole structure could be a composite transposon designated Tn5721 . This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant . We identified five other IS1675 copies not associated with the lacticin 481 operon . The conservation of IS1675 flanking sequences suggested a 24-bp target site.

J Bacteriol, 2000 Oct, 182(19), 5399 - 408
Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp . lactis; Goupil-Feuillerat N et al.; The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis . Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis . It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3) . In this paper we show that the production of ALDC is limited by two mechanisms . First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response . Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts . The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure . The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity . The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation . The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation . Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity . Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability . The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate . Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.

J Food Prot, 2000 Sep, 63(9), 1189 - 96
Efficacy of nisin-coated polymer films to inactivate Salmonella Typhimurium on fresh broiler skin; Natrajan N et al.; Nisin is an antimicrobial peptide produced by the food-grade microorganism Lactococcus lactis subsp . lactis . This peptide inhibits many gram-positive bacteria, and when combined with chelating agents it inhibits gram-negative bacteria such as Salmonella sp . The efficacy of packaging films treated with nisin-containing formulations to reduce Salmonella contamination of fresh broiler drumstick skin and increase the refrigerated shelf life was investigated . Three films (5.1 cm2) of varying hydrophobicities (polyvinyl chloride {PVC}, linear low density polyethylene, nylon) were coated with one of three liquid formulations (pH 3.5 to 3.8) composed of 100 microg/ml nisin and varying concentrations of citric acid, EDTA, and Tween 80 . The treated films were applied either wet or dry to 5.1-cm2 broiler drumstick skin samples inoculated with a nalidixic acid-resistant (NAr) strain of Salmonella Typhimurium . After incubation at 4 degrees C for 24 h the populations of surviving Salmonella TyphimuriumNAr organisms were recovered from the skin and film samples using a rinse procedure and enumerated on brain heart infusion agar containing 800 ppm NA . Log reductions (untreated versus treated skin) in Salmonella TyphimuriumNAr populations ranged from 0.4 to 2.1 . Treatment formulation compositions and wet versus dry treatment application also influenced the extent of kill . The shelf life of refrigerated broiler drumsticks was extended by 0.6 to 2.2 days following a 3-min immersion in a nisin-containing treatment solution and subsequent storage in a foam tray pack containing a nisin-treated PVC overwrap and a nisin-treated absorbent tray pad . These findings demonstrated that Salmonella Typhimurium and spoilage microorganism populations on the surface of fresh broiler skin and drumsticks can be significantly reduced using immersion treatments, absorbent tray pads, and packaging films treated with nisin-containing formulations.

Mol Microbiol, 2000 Aug, 37(4), 717 - 26
Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC; Charpentier E et al.; The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis . By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC . The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis . Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein . Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases . The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin . The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells . Finally, the clpC disruption resulted in decreased genetic transformation . Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ . These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

J Appl Microbiol, 2000 Aug, 89(2), 249 - 60
Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105; Martinez-Cuesta MC et al.; The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105 . The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography . Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively . The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L . lactis DPC3147 . LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type . The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine . Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference . Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters . In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition . The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

J Appl Microbiol, 2000 Aug, 89(2), 191 - 7
Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese; Mannu L et al.; Twenty-nine Lactococcus lactis isolates from one traditional 24 h-old Pecorino Sardo cheese were characterized phenotypically, technologically and genotypically in order to assess the biodiversity within this wild microbial population . Two DNA-based techniques, plasmid profiling and PFGE, were used for the genetic typing of the isolates . All 29 isolates were characterized at strain level and eight different genotypes were recognized . In addition, by combining the results from plasmid profile analysis and PFGE, it was possible to identify closely related isolates probably belonging to the same clonal lineage . The dominant biotype was identified in the 24 h-old cheese, as were the strains believed to act as starters for the curd . Atypical lactococci, able to grow in 6.5% NaCl, were isolated . The results suggest that wild bacterial populations should be preserved in order to protect the traditional raw milk cheeses, and to select new starter strains for the dairy industry.

Appl Environ Microbiol, 2000 Sep, 66(9), 4112 - 4
Lactococcus lactis as a cell factory for high-level diacetyl production; Hugenholtz J et al.; We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and alpha-acetolactate decarboxylase inactivation . Eighty percent of the carbon flux was found to be rerouted via alpha-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.

Appl Environ Microbiol, 2000 Sep, 66(9), 3756 - 63
Physiological and regulatory effects of controlled overproduction of five cold shock proteins of Lactococcus lactis MG1363; Wouters JA et al.; The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied . CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained . Northern blot analysis revealed low abundance of the cspC transcript, indicating that the stability of cspC mRNA is low . The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased . Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of cold-induced proteins of L . lactis, were induced . Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed . Overproduction of CspB and overproduction of CspE result in increased survival when L . lactis is frozen (maximum increases, 10- and 5-fold, respectively, after 4 freeze-thaw cycles) . It is concluded that in L . lactis CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both.

Appl Environ Microbiol, 2000 Sep, 66(9), 3686 - 91
Changes in glycolytic activity of Lactococcus lactis induced by low temperature; Wouters JA et al.; The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied . The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis . Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures . In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain . This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr {HPr(Ser-P)} probably is involved . Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures . The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed . Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock . This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures . Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

Plasmid, 2000 Sep, 44(2), 196 - 200
Characterization of a novel plasmid-encoded HsdS subunit, S.LlaW12I, from Lactococcus lactis W12; Madsen A et al.; A novel type I restriction-modification specificity subunit, S . LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp . cremoris W12 . Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits . Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC . Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit .

J Bacteriol, 2000 Sep, 182(18), 5196 - 201
Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter; Yokota A et al.; The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L . lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux . However, L . lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L . lactis MG1363 . The cholate efflux activity in L . lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force . In contrast, cholate efflux in L . lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters . Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L . lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG) . Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L . lactis C41-2 . Further experiments with L . lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other . These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L . lactis.

Science, 2000 Aug 25, 289(5483), 1352 - 5
Treatment of murine colitis by Lactococcus lactis secreting interleukin-10; Steidler L et al.; The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD) . Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine . Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice . This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

J Appl Microbiol, 2000 Jul, 89(1), 116 - 22
Influence of different substrate limitations on the yield, composition and molecular mass of exopolysaccharides produced by Lactococcus lactis subsp . cremoris in continuous cultures; Looijesteijn PJ et al.; The type of substrate limitation had a remarkable influence on the molecular mass of exopolysaccharides (EPS) produced by Lactococcus lactis subsp . cremoris NIZO B40 and NIZO B891 . Under glucose/energy limitation, the molecular mass was much smaller than under leucine or phosphate limitation, resulting in a marked decrease of the intrinsic viscosity of this EPS . The sugar composition of EPS produced by both strains, and the phosphate content of EPS produced by NIZO B40, were not affected by the type of nutrient limitation . Both strains produced comparable amounts of EPS under leucine and glucose limitation, but the efficiency of EPS production was highest under glucose limitation . The EPS yields of the phosphorylated B40 EPS as well as the unphosphorylated B891 EPS were reduced, with about 40% under conditions of phosphate limitation.

J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 337 - 46
The role of Escherichia coli RNase E and RNase III in the processing of the citQRP operon mRNA from Lactococcus lactis biovar diacetylactis; Drider D et al.; Citrate transport in Lactococcus lactis biovar diacetylactis (L . diacetylactis) is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene . Two partial overlapping open reading frames citQ and citR are located upstream of citP . These two genes, together with citP, constitute the citQRPoperon . In this report it was shown that in L . diacetylactis and Escherichia coli, cit mRNA is subject to the same specific cleavages at a complex secondary structure which includes the central region of citQ and the 5'-end of citR . The role of ribonucleases in the fate of the cit mRNA processing was investigated in E . coli RNase mutant strains . The results obtained indicate that both endoribonucleases RNase E and RNase III are involved in the generation of mRNA processed species . RNase E is responsible for the major cleavages detected within citQ and upstream of citR, whereas RNase III cleaves citR within its ribosomal binding site . Preliminary results indicate the existence of a RNaselll-like enzyme in L . diacetylactis . Based on these results, a model for the role of cit mRNA processing in the expression of citP is presented.

J Bacteriol, 2000 Sep, 182(17), 4783 - 8
Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis; Melchiorsen CR et al.; The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L . lactis PFL . It was demonstrated that activated L . lactis PFL was sensitive to oxygen, as in E . coli, resulting in the cleavage of the PFL polypeptide . The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions . The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose . This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively . During anaerobic growth in defined medium, complete activation of PFL was observed . Strikingly, although no formate was produced during aerobic growth of L . lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen . In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium . This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation . In E . coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE . It was shown that in L . lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed . Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E . coli and L . lactis.

J Bacteriol, 2000 Sep, 182(17), 4738 - 43
The membrane-bound H(+)-ATPase complex is essential for growth of Lactococcus lactis; Koebmann BJ et al.; The eight genes which encode the (F(1)F(o)) H(+)-ATPase in Lactococcus lactis subsp . cremoris MG1363 were cloned and sequenced . The genes were organized in an operon with the gene order atpEBFHAGDC; i.e., the order of atpE and atpB is reversed with respect to the more typical bacterial organization . The deduced amino acid sequences of the corresponding H(+)-ATPase subunits showed significant homology with the subunits from other organisms . Results of Northern blot analysis showed a transcript at approximately 7 kb, which corresponds to the size of the atp operon . The transcription initiation site was mapped by primer extension and coincided with a standard promoter sequence . In order to analyze the importance of the H(+)-ATPase for L . lactis physiology, a mutant strain was constructed in which the original atp promoter on the chromosome was replaced with an inducible nisin promoter . When grown on GM17 plates the resulting strain was completely dependent on the presence of nisin for growth . These data demonstrate that the H(+)-ATPase is essential for growth of L . lactis under these conditions.

Electrophoresis, 2000 Jul, 21(12), 2546 - 9
Towards a proteomic map of Lactococcus lactis NCDO 763; Anglade P et al.; Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening . Numerous lactococcal enzymes and proteins are involved in this process . Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two-dimensional (2-D) gels . However, an annotated reference map is necessary prior to analyzing protein variations . We have begun to construct such a map in easily reproducible conditions and identify proteins.

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 199 - 205
Characterization of OpuA, a glycine-betaine uptake system of Lactococcus lactis; Bouvier J et al.; A Lactococcus lactis glycine-betaine transport system was identified by functional complementation of an Escherichia coli proP proU mutant with a gene library from L . lactis sbsp . cremoris . The cloned locus forms an operon highly homologous to opuA, encoding a glycine-betaine uptake system of Bacillus subtilis . Disruption of opuA in L . lactis abolished protection by glycine-betaine against elevated osmolarity . OpuA belongs to the so-called "ABC transporters" family, which comprise an extracellularly localized substrate-binding protein . In B . subtilis OpuA system, this binding protein is a lipoprotein, attached to the external face of the cytoplasmic membrane by its lipidic moiety . In contrast, in the L . lactis opuA operon, and in other gram-positive homologues as well, a fusion between the gene encoding the integral membrane protein and the substrate-binding protein components gave rise to a hybrid protein presumably attaching the substrate-binding protein to the surface of the cell via its covalent link to the integral membrane component . Mapping of L . lactis opuA transcription start identified one mRNA, more abundant in cells grown at elevated osmolarity . Construction of an opuA-gusA fusion confirmed that opuA transcription is directed by a promoter osmotically inducible in L . lactis . When recombined upstream from a lac transcriptional fusion in the chromosome of E . coli, the opuA promoter appeared as very strong, and only poorly stimulated by elevated osmotic pressure, suggesting the existence of a specific machinery involved in the osmotic signal transduction in L . lactis.

Metab Eng, 1999 Jul, 1(3), 198 - 205
Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity; Even S et al.; Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium . As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate . This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio . The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined . Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio . Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Metab Eng, 1999 Oct, 1(4), 291 - 8
Glucose/citrate cometabolism in Lactococcus lactis subsp . lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase; Curic M et al.; The pyruvate metabolism of a Lactococcus lactis subsp . lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations . A chemically defined medium was used containing glucose as carbon- and energy-source . The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate . Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions . The product formation was monitored throughout the cultivations . The carbon- and redox-balances were within the accuracy of the experimental data . When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites . By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions . The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.

Mol Microbiol, 2000 Aug, 37(3), 639 - 51
Regulation of intron function: efficient splicing in vivo of a bacterial group II intron requires a functional promoter within the intron; Zhou L et al.; Conjugative transfer of the Lactococcus lactis plasmid pRS01 requires splicing of a group II intron, Ll.ltrB, for accurate translation of the mRNA for the exon gene ltrB . The protein product of ltrB is a conjugative relaxase, essential for pRS01 transfer . Using a molecular technique for the identification of transcription initiation sites in bacteria, a functional promoter within Ll.ltrB was identified upstream from the gene for the intron-encoded protein (IEP) LtrA . LtrA is required for efficient splicing of Ll.ltrB in vivo . Mutation of the ltrA promoter dramatically reduced the steady-state level of ltrA mRNA, LtrA, intron splicing and conjugation in L . lactis . These effects could be relieved by expression in trans of the ltrA gene cloned under the control of an inducible promoter . These results suggest that the ltrA mRNAs are translated inefficiently . We hypothesize that this bacterial intron, in contrast to previously studied group II introns in eukaryotes, requires a promoter within the intron to regulate ltrA expression and to produce an adequate level of the protein in the cell for efficient splicing.

Appl Environ Microbiol, 2000 Aug, 66(8), 3543 - 9
Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies; Martinez JM et al.; Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH . Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4 . In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable . Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins . The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403 . Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J . M . Martinez, M . I . Martinez, A . M . Suarez, C . Herranz, P . Casaus, L . M . Cintas, J . M . Rodriguez, and P . E . Hernandez, Appl . Environ . Microbiol . 64:4536-4545, 1998) . In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L . lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.

J Dairy Sci, 2000 Jul, 83(7), 1448 - 50
Short communication: salt extends the upper temperature limit for growth of Lactococcus lactis ssp . cremoris on solid M17 medium; Kilstrup M et al.; We have determined conditions for plating of the Lactococcus lactis ssp . cremoris laboratory strain MG1363 on solid M17 broth at 38 degrees C, which is required for the optimal use of the pGhost plasmids . The addition of 1% NaCl (or KCl, potassium acetate, or sucrose at 170 mM) to M17 agar plates results in extension of the upper temperature limit for growth from 37 to 40 degrees C; no decrease in plating efficiency was detected from 30 to 39 degrees C.

Protein Eng, 2000 Jul, 13(7), 515 - 8
Engineering the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis; Schulte D et al.; Several amino acids in the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis were replaced by the corresponding residues in homologous enzymes of glycosidase family 1 with different specificities . Three mutants, W429A, K435V/Y437F and S428D/ K435V/Y437F, were constructed . W429A was found to have an improved specificity for glucosides compared with the wild-type, consistent with the theory that the amino acid at this position is relevant for the distinction between galactosides and glucosides . The k(cat)/K(m) for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-beta-D-galactose-6-phosphate which is the preferred substrate of the wild-type enzyme . This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group . The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosphorylated substrates . The triple mutant is inactive . The double mutant has a dramatically decreased ability to bind o-nitrophenyl-beta-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-beta-D-galactose is barely altered . This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.

Genes Cells, 2000 Jun, 5(6), 453 - 61
Orientation specificity of the Lactococcus lactis Chi site; El Karoui M et al.; BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase . Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner . ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair . We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis . ChiLl and RexAB are functional analogues of ChiEc and RecBCD . RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner . Analysis of ChiLl distribution on the L . lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication . CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB . The active orientation is preferentially found on the replication leading strand of the L . lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork . We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples . Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.

Science, 2000 Jul 21, 289(5478), 452 - 7
Group II introns designed to insert into therapeutically relevant DNA target sites in human cells; Guo H et al.; Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein . Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions . Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites . Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells . This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

Infect Immun, 2000 Aug, 68(8), 4834 - 7
Identification of saliva-regulated genes of Streptococcus gordonii DL1 by differential display using random arbitrarily primed PCR; Du LD et al.; Attachment of Streptococcus gordonii to the acquired pellicle of the tooth surface involves specific interactions between bacterial adhesins and adsorbed salivary components . To study saliva-regulated gene expression in S . gordonii, we used random arbitrarily primed PCR (RAP-PCR) . Bacteria were incubated in either brain heart infusion medium or saliva . Total RNA from both conditions was purified and RAP fingerprinted and then PCR amplified with an arbitrary primer . The differentially displayed DNA fragments were cloned, sequenced, and analyzed using the BLAST search network service . Three DNA products were up-regulated . One was identified as that of the sspA and -B genes, which encode the salivary agglutinin glycoprotein-binding proteins SspA and SspB of S . gordonii; another had 79% identity with the Lactococcus lactis clpE gene, encoding a member of the Clp protease family; and the third product showed no significant homology to known genes . Five down-regulated genes were identified which encode proteins involved in bacterial metabolism . We have shown, for the first time, direct induction of sspA and -B in S . gordonii by human saliva.

EMBO J, 2000 Jul 17, 19(14), 3649 - 56
On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis; Lanfermeijer FC et al.; Lactococcus lactis degrades exogenous proteins such as beta-casein to peptides of 4-30 amino acids, and uses these as nitrogen sources . The binding protein or receptor (OppA(Ll)) of the oligopeptide transport system (Opp) of L.LACTIS: has the unique capacity to bind peptides from five up to at least 20 residues . To study the binding mechanism of OppA(Ll), nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non-fluorescent or bulky and fluorescent groups . Also, nonameric peptides with a non-natural residue, azatryptophan, at positions 3 or 7 were used . The fluorescence of azatryptophan reports on the polarity of the environment . The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein . The peptide binding mechanism of OppA(Ll) is discussed in relation to known three-dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.

Biomol Eng, 2000 Jun, 16(6), 207 - 9
Human glutathione-S-transferase: cloning and expression in Lactococcus lactis; Xiang H et al.; The glutathione-S-transferase A1 cDNA was amplified from the total RNAs of human liver by RT-PCR, and inserted into plasmid pMG36e . The hGSTA1 was expressed in Lactococcus lactis MG1363 and verified by SDS-PAGE and Western blot, purified by affinity chromatography and showed enzymatic activity.

Biochemistry, 2000 Jul 11, 39(27), 7990 - 7
Role of lys100 in human dihydroorotate dehydrogenase: mutagenesis studies and chemical rescue by external amines; Jiang W et al.; Chemical modification, mutagenesis, chemical rescue, and isotope effect studies are used to identify and probe the roles of several conserved amino acid groups in catalysis by human dihydroorotate dehydrogenase . Time- and pH-dependent inactivation of human dihydroorotate dehydrogenase by trinitrobenzenesulfonate implicates at least one critical lysyl residue in catalysis . Of four highly conserved lysines, only the cognate of Lys255 was previously suggested to have catalytic functionality . We now show that replacement of either Lys184 or Lys186 by mutagenesis does not impact, whereas substitution of Lys100 abolishes, enzymatic activity . However, activity is partially restored to K100C (or K100A) by inclusion of exogenous primary amines in reaction mixtures . This rescued activity saturates with respect to numerous amines and exhibits a steric discrimination reflected in K(d,(amine)) values . For all amines, rescued k(cat) values were only approximately 10% of wild type and independent of amine basicity . K(M) values for dihydroorotate and coenzyme Q(0) were similar to wild type . Thus, exogenous amines (as surrogates for Lys100) apparently complement a chemical, not binding, step(s) of catalysis, which does not entail proton transfer . In support of this postulate, solvent kinetic isotope effect analysis indicates that Lys100 stabilizes developing negative charge on the isoalloxazine ring of flavin mononucleotide during hydride transfer, as has been observed for a number of flavoprotein oxidoreductases . Ser215 of human dihydroarotate dehydrogenase (DHODase) was also studied because of its alignment with the putative active-site base Cys130 of Lactococcus lactisDHODase . Substantial retention of activity by S215C, yet complete loss of activity for S215A, is consistent with Ser215 serving as the active-site base in the human enzyme.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 6 - 9
{Cloning and expression of human Cu/Zn superoxide dismutase gene in Lactococcus lactis}; Xiang H et al.; The cDNA encoding human Cu/Zn SOD was amplified by RT-PCR using the total RNA of human liver as the template, and was cloned into an E . coli expression vector pET23b . After the DNA sequence was determined, the recombinant plasmid pET23bsod was introduced into E . coli BL21(DE3)/pLysS . SDS-PAGE analysis revealed that the recombinant E . coli expressed the predicted 19 kDa human Cu/Zn SOD, and its amount was over 50% of total proteins . The Cu/Zn SOD cDNA was then subcloned into a lactococcal expression vector pMG36e, and resulting pMG36esod was introduced into L . lactis MG1363 by electroporation . The human Cu/Zn-SOD was expressed up to 5% of the soluble proteins, and the enzymatic activity was also observed by SOD activity dying.

Appl Environ Microbiol, 2000 Jul, 66(7), 2859 - 65
Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme; Bruinenberg PG et al.; The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function . Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria . The various deletion mutants were functionally expressed in L . lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates . C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity . Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity . However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity . All truncated enzymes show unaltered proteolytic specificity toward various substrates . This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains) . In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.

J Dairy Sci, 2000 Jun, 83(6), 1196 - 202
Use of hydrolyzed whey peptide to inhibit culture agglutination; Hicks CL et al.; Papain was used to hydrolyze sweet whey to prepare peptides that were harvested with ultrafiltration membranes (molecular weight cutoffs of 10,000, 3000, and 1000) . Insure buffer salts were added to whey peptides (ratio 40:60% solids, respectively) to prepare media that were tested for their ability to inhibit culture agglutination . Commercial Insure medium (75.7 g/l) was used as a control . Skim milk (240 ml) in 250-ml graduated cylinders was inoculated (4%) with Lactococcus lactis spp . lactis B62 or E72 . Culture agglutination was determined by measuring upper center and bottom pH values of the skim milk column during 5 h of incubation . A pH differential was calculated by subtracting the bottom pH from the upper center pH . Cultures grown in media containing whey peptides agglutinated in skim milk to a lesser degree than when grown in the control medium . Culture agglutination was inhibited to a greater degree when cultures were grown in the 1000 molecular weight cutoff peptide medium than when grown in the 10,000 or 3000 molecular weight cutoff peptide medium . When culture E72 was grown in medium containing 1000 molecular weight cutoff peptides, culture agglutination was completely inhibited.

Arch Biochem Biophys, 2000 Jun 1, 378(1), 84 - 92
Catalytic properties of dihydroorotate dehydrogenase from Saccharomyces cerevisiae: studies on pH, alternate substrates, and inhibitors; Jordan DB et al.; Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity from its natural source . Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same . Incubations of the yeast DHOD with dideuterodihydroorotate (deuterated in the positions eliminated in the dehydrogenation) as the donor and {14C}orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the 14C exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 deuteron of the dihydroorotate is sticky on the flavin cofactor . The pH dependencies of the steady-state parameters (k(cat) and k(cat)/Km) are similar, indicating that k(cat)/Km reports the productive binding of substrate, and the parameters are dependent on the donor-acceptor pair . The lower pKa values for k(cat) and k(cat)/Km observed for substrate dihydroorotate (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pKa of the active site base (Cys133) . The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pKa value approximating that assigned to Cys133 . Electron acceptors with dihydroorotate as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Qo (0.28), fumarate (0.15), and O2 (0.035) . Orotate inhibition profiles versus varied concentrations of dihydroorotate with ferricyanide or O2 as acceptors suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme.

Nucleic Acids Res . 2000 Jun 1;28(11):E55.
Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer; Kilstrup M et al.; In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer . The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments . After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5'-end only . Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used . As an example we report the amplification of the era gene from Lactococcus lactis.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 840 - 848
Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering; Kleerebezemab M et al.; Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid . The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering . We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.

Biopolymers, 2000 Aug, 54(2), 143 - 58
Solution properties of viilian, the exopolysaccharide from Lactococcus lactis subsp . cremoris SBT 0495; Higashimura M et al.; The exopolysaccharide (EPS) "viilian" was isolated from a large-batch fermentation of Lactococcus lactis subsp . cremoris SBT 0495 . After applying a newly developed purification procedure, pure viilian with a weight-averaged molar mass of 2.64 x 10(3) kg/mol was obtained in a yield of 0.6 g/L culture broth . The native EPS, as well as lower molar mass fractions obtained by sonication of the native polymer, were studied by capillary viscometry and size-exclusion chromatography (SEC) coupled to multiangle laser light scattering detection (MALLS) . From the viscosity data at various ionic strengths, we extracted a Mark-Houwink-Kuhn-Sakurada exponent a = 0.79, and a Smidsrod B value of 0.03 . By application of the Hearst, Bohdanecky, and Odijk models for stiff polymer coils, in connection to the experimental viscosity data, we established the characteristic ratio to be C(infinity) = 44 and the intrinsic persistence length q(0) = 11.5 nm . The rms radii of gyration predicted from each of the models were in good agreement with the experimental radii (e.g., <S(2)>(1/2)(w) = 162 nm for native viilian in 0.2M NaNO(3)), as determined by SEC-MALLS . In addition, the Odijk model predicts correct ionic strength-linear charge density dependence of the rms radius of gyration . From the combined viscosity and SEC-MALLS experiments we concluded that, in dilute aqueous solutions, viilian behaves as an intermediately stiff, random coil polyelectrolyte system.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7102 - 6
Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane; van der Heide T et al.; An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress . This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain . The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen . A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system . Hypoosmotic medium conditions inhibited the basal activity of the system . The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator . Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane . This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

Infect Immun, 2000 Jul, 68(7), 4245 - 54
Genetic locus for streptolysin S production by group A streptococcus; Nizet V et al.; Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis . Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar . We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S . D . Betschel, S . M . Borgia, N . L . Barg, D . E . Low, and J . C . De Azavedo, Infect . Immun . 66:1671-1679, 1998) . Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project , we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production . The sag locus is conserved among GAS strains regardless of M protein type . Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype . Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon . A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon . Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant . Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis . We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production . Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.

Eur J Biochem, 2000 Jun, 267(12), 3859 - 68
Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR; Neves AR et al.; The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L . lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene {Gasson, M.J., Benson, K., Swindel, S . & Griffin, H . (1996) Lait 76, 33-40} was studied in a noninvasive manner by 13C-NMR . The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of {1-13C}glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s . The metabolism of glucose by the parental wild-type strain was also examined for comparison . A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed . Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted . Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration . Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase . The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration . Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance . The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.

Microbiology, 2000 Jun, 146 ( Pt 6), 1447 - 55
ctsR of Lactococcus lactis encodes a negative regulator of clp gene expression; Varmanen P et al.; Bacteria undergo a complex programme of differential gene expression in response to stress . In Bacillus subtilis, it was recently shown that CtsR, a negative transcriptional regulator, mediates stress-induced expression of components of the Clp protease complex . In this study, a gene was identified in the Gram-positive bacterium Lactococcus lactis that encodes a 17 kDa product with 38% identity to the CtsR protein of B . subtilis . By Northern analyses it was found that in a L . lactis strain carrying a large internal deletion of ctsR, including the region encoding a putative helix-turn-helix motif, the amounts of clpC, clpP, clpB and clpE mRNAs were increased 3-8-fold compared to those present in wild-type L . lactis MG1363 . In another ctsR mutant strain in which only one-third of CtsR was deleted, leaving the putative DNA-binding domain and the C-terminal 29 amino acids intact, only minor derepression of clp gene expression was observed and, furthermore, all the clp genes were still induced by heat . These results indicate that the amino acids of CtsR involved in temperature sensing are located either close to the DNA-binding domain or in the C-terminal part of the protein . Thus, in L . lactis in addition to B . subtilis, CtsR is a key regulator of heat-shock-induced gene expression, suggesting that the presence of CtsR-homologous DNA-binding sites observed in many Gram-positive bacteria reflects functional heat-shock regulatory systems.

Mol Microbiol, 2000 May, 36(4), 866 - 75
Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis; O'Sullivan D et al.; This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82 . The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co-integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain . Analysis of the regions involved in the co-integration revealed that two novel hybrid hsdS genes had been formed during the co-integration event . The HsdS chimeras had interchanged the C- and N-terminal variable domains of the parent subunits, generating two new restriction specificities . Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co-integration event is highly conserved among lactococcal type I hsdS determinants . Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes . This study demonstrates that, similar to previous observations in Gram-negative bacteria, a Gram-positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.

J Dairy Res, 2000 May, 67(2), 233 - 40
Accumulation of casein-derived peptides during growth of proteinase-positive strains of Lactococcus lactis in milk: their contribution to subsequent bacterial growth is impaired by their internal transport; Foucaud C et al.; To explain the limited nutritional value of milk cultured with proteinase-positive (Prt+) strains of Lactococcus lactis for the subsequent growth of dairy lactococci, we investigated further the time courses of modifications in the free amino acid and peptide contents of cultured milk . When growing in milk for up to 24 h, Prt+ strains of Lc . lactis progressively accumulated amino acids and casein-derived peptides . The growth of proteinase-negative (Prt-) wild-type strains and peptide transport mutants of Lc . lactis in cultured milk showed that casein-derived peptides could sustain growth up to 5 x 10(8) cfu/ml, depending on the extent of casein degradation during the preliminary growth of Prt+ strains and the Prt- strains . Of the casein-derived oligopeptides, < 25% were transported into the cell and used for Lc . lactis growth . However, they played a prominent role, contributing 90% to growth . In contrast, di- and tripeptides did not contribute to growth, suggesting that either few were released from caseins or they did not supply essential amino acids.

Appl Environ Microbiol, 2000 Jun, 66(6), 2647 - 51
Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp . lactis KR2; Twomey DP et al.; The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation . The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR . Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes . Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication . These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Appl Environ Microbiol, 2000 Jun, 66(6), 2325 - 9
Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230; Atiles MW et al.; A branched-chain aminotransferase gene (ilvE) from Lactococcus lactis LM0230 was identified on a 9-kb chromosomal insert by complementation in Escherichia coli DL39 . Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340-amino-acid protein . Sequence analysis of the deduced amino acid sequence revealed 62% identity to IlvE of Haemophilus influenzae and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases . Under logarithmic growth in complex medium, ilvE is transcribed monocistronically as a 1.1-kb transcript . Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein . A derivative of LM0230 lacking IlvE activity was constructed by gene replacement . Comparison of the IlvE-deficient strain's ability to grow in defined media lacking an amino acid but containing its alpha-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors . Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively . No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.

Biochemistry, 2000 May 2, 39(17), 4963 - 70
Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus; Kaper T et al.; The substrate specificity of the beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level . Following crystallization and X-ray diffraction of this enzyme, a 3.3 A resolution structural model has been obtained by molecular replacement . CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold . Its active site has been compared to the one of the previously determined 6-phospho-beta-glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis . The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG . To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants . Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0) . Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively . In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold) . The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.

Infect Immun, 2000 Jun, 68(6), 3516 - 22
Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp . cremoris using a new shuttle vector; Que YA et al.; Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection . To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp . cremoris 1363 . This bacterium is devoid of virulence factors and has a known genetic background . A new Escherichia coli-L . lactis shuttle and expression vector was constructed for this purpose . First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E . coli . Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site . Gene expression was assessed by a luciferase reporter system . The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid . Transcription was absent in E . coli . The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system . Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls . This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma . ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S . aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen . These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Infect Immun, 2000 Jun, 68(6), 3251 - 60
Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis; Gilbert C et al.; In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogen Streptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization . Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter(-1)) of an extracellular type 3 CPS in L . lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L . lactis . The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses . Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L . lactis and S . pneumoniae showed that the two CPS structures were identical . When mice were immunized intraperitoneally with 3.5 x 10(6) CFU of live recombinant lactococci expressing a total of approximately 0.5 microgram of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 microgram of type 3 CPS purified from S . pneumoniae . These findings show that L . lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules . Additionally, L . lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

FEMS Microbiol Lett, 2000 May 15, 186(2), 181 - 5
Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density; Franz CM et al.; The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system . The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway . The culture supernatants of C . piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C . piscicola LV17A or E . faecium CTC492 . The cbaX fusion product induced bacteriocin production by Bac(-) C . piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E . faecium CTC492 . This represents a relatively simple method of confirming the role of presumptive IPs . The transformation of C . piscicola LV17A with the CbaX gene under expression of the P32 promoter from L . lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.

Nature, 2000 Apr 27, 404(6781), 1018 - 21
Retrotransposition of a bacterial group II intron; Cousineau B et al.; Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown . To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis . The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymatic activities . Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming . In retrohoming, the intron reverse splices into a cognate intronless DNA site . Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA . Here we report the first experimental demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism . This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets . These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.

J Mol Evol, 2000 Apr, 50(4), 339 - 47
Proteobacterial histidine-biosynthetic pathways are paraphyletic; Bond JP et al.; In Lactococcus lactis there is a protein, HisZ, in the histidine-biosynthetic operon that exhibits significant sequence identity with histidyl-tRNA synthetase (HisRS) but does not aminoacylate tRNA . HisRS homologs that, like HisZ, cannot aminoacylate tRNA are represented in a highly divergent set of bacteria (including an aquificale, cyanobacteria, firmicutes, and proteobacteria), yet are missing from other bacteria, including mycrobacteria and certain proteobacteria . Phylogenetic analysis of the HisRS and HisRS-like family suggests that the HisZ proteins form a monophyletic group that attaches outside the predominant bacterial HisRS clade . These observations are consistent with a model in which the absences of HisZ from bacteria are due to its loss during evolution . It has recently been shown that HisZ from L . lactis binds to the ATP-PRPP transferase (HisG) and that both HisZ and HisG are required for catalyzing the first reaction in histidine biosynthesis . Phylogenetic analysis of HisG sequences shows conclusively that proteobacterial HisG and histidinol dehydrogenase (HisD) sequences are paraphyletic and that the partition of the Proteobacteria associated with the presence/absence of HisZ corresponds to that based on HisG and HisD paraphyly . Our results suggest that horizontal gene transfer played an important role in the evolution of the regulation of histidine biosynthesis.

Lett Appl Microbiol, 2000 May, 30(5), 415 - 8
Citrate can partially replace carbon dioxide required for growth of Lactococcus lactis subsp . lactis biovar diacetylactis; Henriksen CM et al.; Lactococcus lactis subsp . lactis biovar diacetylactis was grown as batch cultures on a chemically defined medium . No growth was observed when the cultures were sparged with pure nitrogen (1.3 l l-1 min-1) whereas the cultures displayed exponential growth in the presence of minute amounts of carbon dioxide (0.035 mol-% of the inlet gas) . However, in the former case, the addition of citrate restored growth . This suggested that oxaloacetate required for aspartate biosynthesis can be formed by the carboxylation of pyruvate or by citrate catabolism . When the cultures were heavily sparged with nitrogen (2.6 l l-1 min-1), no growth was observed even in the presence of citrate . This indicated that growth in these conditions was repressed by the absence of carbon dioxide required in some other biosynthetic reaction than in the carboxylation of pyruvate leading to oxaloacetate/aspartate biosynthesis.

J Appl Microbiol, 2000 Apr, 88(4), 563 - 71
Production of a nisin-like bacteriocin by Lactococcus lactis subsp . lactis A164 isolated from Kimchi; Choi HJ et al.; Lactococcus lactis subsp . lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables) . The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium . The antimicrobial spectrum was nearly identical to that of nisin . Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values . This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase . Cultures of L . lactis subsp . lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin . It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration . Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin . The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced . It showed identical sequences to the nisin gene . These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.

J Dairy Sci, 2000 Apr, 83(4), 633 - 40
Growth associated exopolysaccharide expression in Lactococcus lactis subspecies cremoris Ropy352; Knoshaug EP et al.; A natural lactococcal isolate, Lactococcus lactis ssp . cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid) . This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression . Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media . Ropy352 was examined for inducible lysogenic phages . No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction . The sugar compositions of the two phenotypically distinct exopolysaccharides were determined . The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively . The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively . Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.

J Dairy Sci, 2000 Apr, 83(4), 620 - 7
Characterization of mesophilic mixed starter cultures used for the manufacture of aged cheddar cheese; Bissonnette F et al.; Seventy-one different Lactococcus lactis subsp . cremoris strains were isolated from seven mesophilic mixed starters used in the manufacture of aged Cheddar cheese in Canada . Based on plasmid profiles and growth in milk (with or without glucose, Casamino Acids or both), two mixed starters were highly heterogeneous, containing at least 18 to 24 distinct L . lactis strains . Three mixed starters were comprised of seven to nine strains, whereas two starters were relatively homogeneous, containing two or three strains . Many strains with similar plasmid profiles behaved differently during growth in milk, indicating variability in the phenotypes . Only 20% of the strains could grow in plain milk, whereas 30% could not grow in milk supplemented with glucose and Casamino Acids . Twenty-five lactococcal bacteriophages were also isolated from whey samples with single strains as hosts . Eighteen phages belonged to the 936 species and seven to the c2 species . Thirteen strains were insensitive to all 25 phages . Almost all sensitive strains were phage species-specific . The 936-like phages had a broader host range.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 291 - 4
Fluorescence assessment of Lactococcus lactis viability; Bunthof CJ et al.; The reproduction and activity of lactic acid bacteria (LAB) are essential in their applications in the dairy industry and other fermentations . Traditionally used methods like plate counting and acidification tests require long incubation times and provide limited information . Fluorescence techniques provide possibilities for rapid assessment of cell physiology . We used traditional and fluorescence assays to assess the physiological condition of L . lactis subsp . lactis ML3 cultures that were exposed to various stress conditions . After exposure to some of the stress conditions, carboxyfluorescein (cF) labelling did not agree with plate counts . Therefore, a two-step method was developed in which cF labelling was followed by a lactose-energized efflux assay . The combined assay proved to be a good and rapid indicator for reproduction and acidification capacity of stressed L . lactis . This novel assay has potential for physiological research and dairy applications related to LAB.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 215 - 21
Changes in acid tolerance of Lactococcus lactis during growth at constant pH; Alemayehu D et al.; Cells of Lactococcus lactis MG1363 growing in batch culture in TYG (tryptone, yeast extract, glucose) medium at constant pH 7.0 became gradually more acid sensitive shortly after inoculation until a point of maximum sensitivity was reached in early log-phase . The acid tolerance then gradually increased in the mid- and late-log phase until maximum tolerance was reached at the onset of stationary phase . This pattern has been termed the growth-phase acid tolerance . The variation in acid tolerance seen in pH 7.0 grown cells of L . lactis MG1363 did not result from changes in internal pH or membrane H+ ATPase activity levels . Neither the amount of glucose present during mid-log phase nor the amount of lactate produced by the cells correlated with the pattern of the log-phase acid tolerance . Cells grown in partially spent TYG medium showed a reduced growth rate and increased acid tolerance compared to cells grown in fresh TYG medium . Supplementing the spent medium with tryptone or yeast extract or both restored the growth rate and cells became more sensitive to acid . Fractionation of tryptone yielded a fraction which stimulated the growth of MG1363 in partially spent medium and delayed the acquisition of acid tolerance . The active compound(s) has a putative molecular weight of about 1 kDa and was partially degraded by papain and trypsin.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 161 - 5
Metabolism of Lactococcus lactis subsp . cremoris MG 1363 in acid stress conditions; Mercade M et al.; The metabolism of glucose by Lactococcus lactis subsp . cremoris MG 1363 remains homolactic whatever the pH of the culture medium . The growth rate decreased with the acidification of the medium until a limit pH value of 4.0 for which no growth was observed . In contrast, the specific rate of glucose consumption decreased only for very low pH values, i.e., below 4.5 . The efficiency of biomass synthesis relative to the energy supply decreased when the medium pH diminished, as illustrated by Y(ATP) values . This observation was related to the increase in both components of the proton-motive force when the pH decreased . The growth stopped when the internal pH reached a limit value of 5.4 due to organic acid accumulation.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 127 - 31
Growth of Lactococcus lactis strains at low water activity: correlation with the ability to accumulate glycine betaine; O'Callaghan J et al.; Lactococcus lactis strains were divided into two groups based on their ability to grow in the presence of an upper limit of either 2% w/v NaCl (sensitive) or 4% w/v NaCl (tolerant) . Growth inhibition of NaCl tolerant strains was substantially relieved by glycine betaine which was accumulated in significant amounts when growing at low water activities (a(w)) . Very little accumulation of glycine betaine occurred during growth of the NaCl sensitive strains . The NaCl tolerant strains had substantial levels of glycine betaine transport activity in vitro, whereas the NaCl sensitive strains had little or no such activity . A low a(w) sensitive mutant of L . lactis subsp . cremoris MG1363 (NaCl tolerant) was isolated following ISS1 insertional mutagenesis . This mutant was inhibited at an a(w) of 0.988 produced by addition of 2% w/v NaCl or the equivalent glucose concentration (0.58 M) . The mutant did not accumulate glycine betaine when growing at low a(w), and did not transport glycine betaine when assayed in vitro.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 83 - 6
Lactococcus lactis, a bacterial model for stress responses and survival; Duwat P et al.; The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes . To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis . Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation) . Our results revealed that metabolic flux plays an important role in L . lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors . As previously shown, we also observed an increase of stress resistance during the stationary phase . We have evidence that stationary phase actually initiates very early during growth . Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether . These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L . lactis.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 47 - 51
Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress; Guillot A et al.; Lactococcus lactis subsp . cremoris NCDO763 accumulates glycine-betaine (betaine) when submitted to an osmotic stress with NaCl . Betaine transport activity increases with the extent of the osmotic upshock but also with growth temperature, and supplementation of the medium by Tween-80 . Fatty acid analysis of the lipid fraction of L . lactis NCDO763 reveals significant modifications of the fatty acid composition of the membrane when cells are submitted to osmotic stress, high temperature or Tween-80 medium supplementation . The main modification in L . lactis membrane fatty acid composition in response to high osmolality is the increase of Cyclopropane Fatty Acid (CFA) deltaC19:0, whereas Unsaturated/Saturated ratio remains unchanged.

Appl Environ Microbiol, 2000 May, 66(5), 2235 - 7
Inactivation of the glutamate decarboxylase gene in Lactococcus lactis subsp . cremoris; Nomura M et al.; Lactococcus lactis subsp . lactis strains show glutamate decarboxylase activity, whereas L . lactis subsp . cremoris strains do not . The gadB gene encoding glutamate decarboxylase was detected in the L . lactis subsp . cremoris genome but was poorly expressed . Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.

Appl Environ Microbiol, 2000 May, 66(5), 2192 - 8
Investigation of the relationship between lysogeny and lysis of Lactococcus lactis in cheese using prophage-targeted PCR; O'Sullivan D et al.; The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement in flavor and acceleration of cheese ripening . Using a laboratory-scale cheese manufacturing assay, the autolytic behavior of 31 strains of Lactococcus lactis was assessed . In general, marked variation was observed between strains with a 20-fold difference between the best and worst lysing strains based on the release of the intracellular enzyme lactate dehydrogenase . In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (int) sequences by using conserved primer sequences from known lysogenic phage . Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences . These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful initial screen to assess strains for this important industrial trait.

Appl Environ Microbiol, 2000 May, 66(5), 2021 - 8
Structural changes and interactions involved in the Ca(2+)-triggered stabilization of the cell-bound cell envelope proteinase in Lactococcus lactis subsp . cremoris SK11; Exterkate FA; The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism Lactococcus lactis subsp . cremoris SK11 is protected from rapid thermal inactivation at 25 degrees C by calcium bound to weak binding sites . The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F . A . Exterkate and A . C . Alting, Appl . Env . Microbiol . 65:1390-1396, 1999) . In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called "Ca-free" CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose . The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP . On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species . Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs . It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability . The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.

Microbiology, 2000 Apr, 146 ( Pt 4), 949 - 55
Lactococcin 972, a bacteriocin that inhibits septum formation in lactococci; Martinez B et al.; Addition of lactococcin 972 to exponentially growing sensitive cultures of Lactococcus lactis resulted in cell elongation and widening . Thin sections revealed that septum invagination was blocked . Cell growth progressed until most cells showed equatorial constriction and even initial deposition of material at the septum ring, although cell division did not proceed any further . The increase in the incorporation of labelled precursors into the cell wall shifted from an exponential to a linear mode in treated cultures, subsequently being arrested . Gross degeneration of the cells was observed prior to cell death, followed by slow lysis of the culture . In contrast, stationary-phase cultures remained unaffected.

Microbiology, 2000 Apr, 146 ( Pt 4), 935 - 47
Six putative two-component regulatory systems isolated from Lactococcus lactis subsp . cremoris MG1363; O'Connell-Motherway M et al.; The genetic elements specifying six putative two-component regulatory systems (2CSs) were identified on the chromosome of Lactococcus lactis MG1363 . These 2CSs appear to represent distinct loci, each containing a histidine kinase and response-regulator-encoding gene pair . Transcriptional analysis of the six 2CSs was performed either by generating transcriptional fusions to a reporter gene or by primer extension . Two of the systems appeared to be expressed constitutively at a high level, whilst the remaining four exhibited growth-phase-dependent expression . Insertional mutagenesis established that the two constitutively expressed 2CSs are necessary for normal cell growth and/or survival . Mutational analysis of the remaining four systems revealed that they are implicated in susceptibility to extreme pH, osmotic or oxidative conditions, or the regulation of phosphatase activity in L . lactis.

Prikl Biokhim Mikrobiol, 2000 Mar-Apr, 36(2), 131 - 7
{Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture}; Kisrieva IuS et al.; Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp . lactis biovar . diacetylactis strain 4 . The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield) . The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield) . The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer) . The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM . AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration . In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM) . Leucine, valine, and isoleucine were shown to be activators of AcDLC.

J Biol Chem, 2000 Apr 28, 275(17), 12367 - 73
Electron paramagnetic resonance evidence for a novel interconversion of ¿3Fe-4S(+) and ¿4Fe-4S(+) clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro; Liu A et al.; We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis . The activase (nrdG gene) together with S-adenosyl-L-methionine (AdoMet) give rise to a glycyl radical in the NrdD component . A semi-reduced {4Fe-4S}(+) cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase . Air exposure of the reduced enzyme gave a {3Fe-4S}(+) cluster . The Fe(3)S(4) cluster was convertible to the EPR-active {4Fe-4S}(+) cluster by renewed treatment with reducing agents, demonstrating a reversible {3Fe-4S}(+)- to-{4Fe-4S}(+) cluster conversion without exogenous addition of iron or sulfide . Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced {4Fe-4S}(+) cluster . Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive . Both reduced states gave the {3Fe-4S}(+) EPR signal after air exposure . The iron-sulfur cluster interconversion was also studied in the presence of AdoMet . The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the {3Fe-4S}(+) cluster after air exposure was unchanged . The results indicate that an AdoMet-mediated {4Fe-4S}(+) center is the native active species that induces the formation of a glycyl radical in the NrdD component.

Postgrad Med J, 2000 May, 76(895), 301 - 3
Osteomyelitis and possible endocarditis secondary to Lactococcus garvieae: a first case report; James PR et al.; Although osteomyelitis is commonly caused by staphylococcal infection, the first case of a lumbar osteomyelitis secondary to Lactococcus garvieae is reported . The case was complicated by possible endocarditis of an aortic valve prosthesis.

Virology, 2000 Apr 25, 270(1), 65 - 75
Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain; Bouchard JD et al.; Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics . Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes . Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages . In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L . lactis host . They occurred by homologous recombination with phage-related sequences present in the host chromosome . Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication . We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages .

Microbiol Res, 2000 Mar, 154(4), 321 - 31
Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food; El-Shafei HA et al.; 100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production . Twenty six strains producing a nisin-like bacteriocin were selected . Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis . The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied . Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3 . The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied . Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.

Biochemistry, 2000 Apr 25, 39(16), 4855 - 62
Kinetics and structural requirements for the binding protein of the Di-tripeptide transport system of Lactococcus lactis; Sanz Y et al.; The gene (dppA) encoding the binding protein of the di-tripeptide ABC transporter of Lactococcus lactis (DppA) was cloned under the control of the nisin promoter . Amplified expression ( approximately 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purification of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chromatography . Ligand binding to DppA-(His)(6) elicited an electrophoretic mobility shift, a decrease in the intrinsic fluorescence, and a blue shift of the emission maximum . Each of these parameters detected conformational changes in the protein that reflect ligand binding, and these were used to determine the structural requirements of DppA-(His)(6) for binding peptides . The major features of peptide binding include (i) high affinity for di- and tripeptides, (ii) requirement of a free N-terminal alpha-amino group and an alpha-peptide bound contiguous with the N-terminal amino group, (iii) stereospecificity for L-isomers, and (iv) preference for dipeptides containing methionine or arginine, followed by hydrophobic tripeptides consisting of leucine or valine residues . Maximal binding affinity was detected at pH 6.0, and the K(d) for binding increased 1 order of magnitude for every unit increase in pH . This suggests that the ionization of protein residues (pK > 6.0) in or in close proximity to the binding site is critical in the binding mechanism.

Mol Membr Biol, 1999 Oct-Nov, 16(4), 297 - 304
Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis; Fang G et al.; The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin . The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E . coli phospholipid/phosphatidylcholine . A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity . The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes . Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations . This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes . Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used . The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.

J Bacteriol, 2000 May, 182(9), 2530 - 5
Kinetics and substrate specificity of membrane-reconstituted peptide transporter DtpT of Lactococcus lactis; Fang G et al.; The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes . The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-{(14)C}Ala as a reporter peptide in the presence of various peptides or peptide mimetics . The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue . The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides . Free amino acids, omega-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT . For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds . Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT(1) and PepT(2) shows that the bacterial transporter is more restrictive in its substrate recognition.

J Bacteriol, 2000 May, 182(9), 2481 - 91
Genome plasticity among related ++Lactococcus strains: identification of genetic events associated with macrorestriction polymorphisms; Le Bourgeois P et al.; The genomic diversity of nine strains of the Lactococcus lactis subsp . cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis . These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage . Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns . The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions . Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.

J Biol Chem, 2000 Jun 30, 275(26), 19443 - 8
Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4; Andersson J et al.; Ribonucleotide reductase (RNR) is an essential enzyme in all organisms . It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides . The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools . We have characterized the allosteric regulation of the class III RNR from bacteriophage T4 . Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively . The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range . ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction . dATP is not a general negative allosteric effector . These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis . The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo . Surprisingly, we did not observe any significant UTP reduction under the conditions used . Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.

Lett Appl Microbiol, 2000 Mar, 30(3), 249 - 53
Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin; Modi KD et al.; Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes . A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry . The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes . The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L . monocytogenes Scott A was also studied . When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L . monocytogenes at 55, 60 and 65 degrees C . However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells . The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min . When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.

Appl Environ Microbiol, 2000 Apr, 66(4), 1354 - 9
Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds; Rijnen L et al.; The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor . We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd . In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha-ketoglutarate from glutamate, which is present at high levels in cheese . Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers . The GDH-producing lactococcal strain degraded amino acids without added alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate . Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds . Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese.

Appl Environ Microbiol, 2000 Apr, 66(4), 1253 - 8
A food-grade cloning system for industrial strains of Lactococcus lactis; Sorensen KI et al.; We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker . This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested . As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides . Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis . The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region . Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk . Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L . lactis strains . The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.

Pharmacol Ther, 2000 Mar, 85(3), 245 - 9
Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis; van Veen HW et al.; The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms . A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein . Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms . Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell . Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism . The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Carbohydr Res, 2000 Feb 25, 324(3), 170 - 81
Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp . cremoris B39; van Casteren WH et al.; Lactococcus lactis subsp . cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2 . The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide . Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> . 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: {structure: see text}.

Gene, 2000 Jan 25, 242(1-2), 347 - 56
The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis; Ravn P et al.; We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis {Israelsen et al., Appl . Environ . Microbiol . 61 (1995) 2540-2547} . We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion . Transposition of TnNuc into the L . lactis chromosome allows the generation of fusions in-frame with the nuc gene . TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e . clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L . lactis chromosome . The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay . TnNuc was used for the identification of novel secretion signals from L . lactis . The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence . In one case, the gene identified codes for a transmembrane protein . The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 {Madsen et al., Mol . Microbiol . 32 (1999) 75-87} . In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.

Genes Dev, 2000 Mar 1, 14(5), 559 - 73
Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences; Mohr G et al.; Group II intron homing occurs primarily by a mechanism in which the intron RNA reverse splices into a DNA target site and is then reverse transcribed by the intron-encoded protein . The DNA target site is recognized by an RNP complex containing the intron-encoded protein and the excised intron RNA . Here, we analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group II intron in vitro and in vivo . Our results suggest a model similar to yeast mtDNA introns, in which the intron-encoded protein first recognizes a small number of nucleotide residues in double-stranded DNA and causes DNA unwinding, enabling the intron RNA to base-pair with the DNA for reverse splicing . Antisense-strand cleavage requires additional interactions between the protein and 3' exon . Key nucleotide residues are recognized directly by the intron-encoded protein independent of sequence context, and there is a stringent requirement for fixed spacing between target site elements recognized by the protein and RNA components of the endonuclease . Experiments with DNA substrates containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in the 3' exon but not the distal 5' exon region . Finally, by applying the target-site recognition rules, we show that the L1.LtrB intron can be modified to insert at new sites in a plasmid-borne thyA gene in Escherichia coli . This strategy should be generally applicable to retargeting group II introns and to delivering foreign sequences to specific sites in heterologous genomes.

Mol Microbiol, 2000 Mar, 35(5), 1042 - 51
HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing; Poquet I et al.; We identified an exported protease in Lactococcus lactis ssp . lactis strain IL1403 belonging to the HtrA/DegP family . Inactivation of the chromosomal gene (htrALl) encoding this protease (HtrALl) results in growth thermo-sensitivity at very high temperatures (above 37 degrees C for L . lactis) . The role of htrALl in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e . fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations . In the wild-type (wt) strain, degradation products, including the C-terminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total . In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased . These results suggest that HtrALl is the sole extracellular protease that degrades abnormal exported proteins . In addition, our results reveal that HtrALl is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein . We propose that in lactococci, and possibly in other Gram-positive organisms with small sized-genomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.

Microbiology, 2000 Feb, 146 ( Pt 2), 445 - 53
Microbiological and molecular impacts of AbiK on the lytic cycle of Lactococcus lactis phages of the 936 and P335 species; Boucher I et al.; The lactococcal abortive infection mechanism AbiK was previously shown to be highly effective against the small isometric-headed bacteriophage ul36 of the P335 species, as evidenced by an efficiency of plaquing (e.o.p.) of 10(-6), a 14-fold reduction in the burst size and an efficiency at which centres of infection form (e.c.o.i.) of 0.5% . No phage DNA was detected in the infected AbiK+ cells {Emond, E., Holler, B . J., Boucher, I., Vandenbergh, P . A., Vedamuthu, E . R., Kondo, J . K . & Moineau, S . (1997) . Appl Environ Microbiol 63, 1274-1283} . Here, the effects of AbiK are compared on the small isometric-headed phages p2 and P008 (936 species) and on the phage P335 (P335 species) . The microbiological impacts of AbiK on p2 were relatively similar to those reported for ul36, with an e.o.p . of 10(6), an 11-fold reduction in the burst size and an e.c.o.i . of 5% . Contrary to phage ul36, replication of phage p2 DNA was observed in the AbiK+ cells . Only immature forms (concatemeric and circular DNA) of phage p2 DNA were found, indicating that the presence of AbiK prevented phage DNA maturation . These distinct molecular consequences of AbiK were also observed for phages P335 and P008, two phages that propagate on the same host . To the knowledge of the authors, this is the first time that different phage responses towards an Abi system have been reported.

Microbiology, 2000 Feb, 146 ( Pt 2), 435 - 43
Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain; Seegers JF et al.; A 6.1 kb plasmid from the Lactococcus lactis subsp . cremoris strain UC509.9, named pCIS3, was found to mediate a restriction/modification (R/M) phenotype . Nucleotide sequence analysis of pCIS3 revealed the presence of an hsdS gene, typical of type I R/M systems . The presence of this plasmid resulted in a 10(4)-fold reduction in the efficiency of plating (e.o.p.) of unmodified phage . In addition to the hsdS gene of pCIS3, two more hsdS genes were identified in strain UC509.9, one located on the chromosome downstream of a gene highly homologous to hsdM genes and a third on the smallest (4 kb) plasmid, named pCIS1 . The replication region of pCIS3 was highly similar to that of a large family of lactococcal theta replicons . In addition, pCIS3 was found to encode a member of the CorA family of magnesium transporters.

Appl Environ Microbiol, 2000 Mar, 66(3), 1223 - 7
Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp . lactis C2; Wang H et al.; A functional pyc gene was isolated from Lactococcus lactis subsp . lactis C2 and was found to complement a Pyc defect in L . lactis KB4 . The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria . The pyc gene was also detected in Lactococcus lactis subsp . cremoris and L . lactis subsp . lactis bv . diacetylactis strains.

Appl Environ Microbiol, 2000 Mar, 66(3), 987 - 94
Multiplex PCR for detection and identification of lactococcal bacteriophages; Labrie S et al.; Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species . The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates . Three sets of primers, one for each species, were designed based on conserved regions of their genomes . The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target . The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67 . An 86.4% identity was found over the five mcp genes . The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages . The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1 . The comparison of the six msp genes revealed an 82 . 2% identity . A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised . Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries . This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40) . An identity of 93.4% within a 739-bp region of the five phages was found . The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step . The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.

Appl Environ Microbiol, 2000 Mar, 66(3), 895 - 903
Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages; Durmaz E et al.; Recombinant phages are generated when Lactococcus lactis subsp . lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species . These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations . They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L . lactis chromosomal DNA . The objective of this study was to characterize four recombinant variants that arose from infection of L . lactis NCK203 (Abi(+)) with phage phi31 . HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA . The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31 . This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes . A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin . Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203 . Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages . The chromosomal insertions did not affect an inducible prophage present in NCK203 . Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses . Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.

J Bacteriol, 2000 Mar, 182(6), 1600 - 8
Specificity mutants of the binding protein of the oligopeptide transport system of Lactococcus lactis; Picon A et al.; The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined . To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L . lactis to produce a strain that was totally defective in oligopeptide transport . Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level . The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp . Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L . lactis, taking into account the known structure of the serovar Typhimurium protein . Putative peptide binding-site residues were mutated . All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin . Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected . Cells expressing OppA(D471R) had a similar K(m) for transport, whereas the V(max) was increased more than twofold as compared to the wild-type protein . The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

Infect Immun, 2000 Mar, 68(3), 1359 - 65
Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin; Love RM et al.; Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci . These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules . Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease . Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8 . An isogenic noninvasive mutant of S . gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P . gingivalis . Heterologous expression of the S . mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P . gingivalis or the ability to promote P . gingivalis coinvasion of dentin . An isogenic afimbrial mutant of P . gingivalis had 50% reduced binding to S . gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S . gordonii wild-type cells . Expression of the S . gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P . gingivalis, penetrate dentinal tubules, and promote P . gingivalis coinvasion of dentin . The results demonstrate that collagen-binding and P . gingivalis-binding properties of antigen I/II polypeptides are discrete functions . Specificity of antigen I/II polypeptide recognition accounts for the ability of P . gingivalis to coinvade dentinal tubules with S . gordonii but not with S . mutans . This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology of pulpal and periapical diseases.

J Appl Microbiol, 1999 Dec, 87(6), 849 - 855
Diversity among lactococci isolated from ewes' raw milk and cheese; Gaya P et al.; The technological and genetic characteristics of lactococci present in ewes' raw milk and 1-d-old ewes' raw milk cheeses sampled over a 1-year period were investigated . The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1.25 units after 6 h incubation at 30 degrees C reached 14.5% in spring vs 10.7% in summer, 8.3% in autumn and 3.0% in winter . In 1-d-old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1.25 units increased up to 32.3% in spring vs 23.4% in summer, 8.0% in autumn and 10.3% in winter . Fast acid-producing lactic acid bacteria mainly belonged to the genus Lactococcus . Using polymerase chain reaction protocols, fast acid-producing lactococci were grouped as 61 Lactococcus lactis subsp . lactis, 13 L . lactis subsp . cremoris and 14 L . lactis subsp . lactis biovar diacetylactis . Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid-producing lactococci, using two primers, resulted in 21 different RAPD patterns for L . lactis subsp . lactis isolates, nine RAPD patterns for L . lactis subsp . cremoris isolates and three RAPD patterns for L . lactis subsp . lactis biovar diacetylactis isolates . Up to 19 different RAPD patterns were found for L . lactis isolates from cheeses made in a particular month.

Plasmid, 2000 Mar, 43(2), 111 - 22
Nucleotide sequence and analysis of plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) involved in pediocin A production; Giacomini A et al.; The complete sequence of the 19515-bp plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) has been determined . This plasmid is involved in Pediocin A production, a bacteriocin active against a wide range of gram-positive bacteria . It appears to replicate via a theta mechanism, with structures closely related to those of many lactococcal plasmids . Genes homologous to mobilization functions are also present, which are similar in sequence and arrangement to mobA, mobB, and mobC of some staphylococcal plasmids, although the last one contains a deletion in its central part . The region involved in bacteriocin activity has been limited to a 9.4-kb fragment, containing 10 open reading frames organized in a single operon . Since Pediocin A has a molecular weight of about 80 kDa (Piva and Headon, Microbiology, 140, 697-702, 1994), and a gene long enough to encode it is not present in pMD136, it is proposed that genes residing on the plasmid are responsible for the regulation of bacteriocinogenic activity . Gene arrangement and sequence homologies suggest the presence of a two-component-like regulatory mechanism .

Biochim Biophys Acta, 2000 Feb 29, 1490(3), 279 - 90
Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM; Yarmus M et al.; The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp . lactis EZ26, is associated with the presence of a 6-kb plasmid, pHU1 . The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI-Eco47III fragment . Sequencing analysis of this fragment revealed the presence of six open reading frames (ORFs) . Deletion and mutation analyses showed that orfX and orfY are not required for lacticin RM production or immunity, whereas the other ORFs (lacA, lacF, lacG and lacI) are necessary for the bacteriocin's production . Transcription analysis indicated that lacA, lacF and lacG are organized in an operon . lacA is probably the lacticin RM structural gene . It putatively encodes a 134-amino acid peptide, and it does not share homology with known bacteriocins . The deduced LacG protein is hydrophobic and consists of six potential trans-membrane helices . lacF encodes a conserved ATP-binding domain homologous to ABC transporters known in bacteriocin immunity systems . LacF and LacG may form an active ABC transporter . Gene-disruption mutations indicated that both are required for immunity against lacticin RM . lacI encodes a small cationic protein, which is required for the production of and immunity to lacticin RM . Protection was obtained only when lacF, lacG and lacI were present together.

FEMS Microbiol Lett, 2000 Feb 15, 183(2), 229 - 34
Expression of green fluorescent protein in Lactococcus lactis; Fernandez de Palencia P et al.; The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp . lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host . GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies . Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy . Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP . Our results indicate that GFP can be used as a reporter in L . lactis.

J Biotechnol, 2000 Jan 28, 77(1), 17 - 23
Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR; Hugenholtz J et al.; Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR . Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose . EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former . They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium . They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls . By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars . The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

Mol Microbiol, 2000 Feb, 35(3), 517 - 28
Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals; Rallu F et al.; Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication . Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH . In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L . lactis strain MG1363 . Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism . None of these genes was identified previously as involved in lactococcal acid tolerance . The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains . Our results reveal that L . lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance . In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction . Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

Lett Appl Microbiol, 1999 Nov, 29(5), 313 - 6
Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile; Kimoto H et al.; There have been few studies on the probiotic activity of Lactococcus strains although they are commonly used as starter bacteria in manufacturing many kinds of fermented dairy products . Nine strains of the genus Lactococcus were examined for their probiotic properties, such as adherence to human enterocyte-like Caco-2 cells and tolerance to acid and bile . Six strains were adhesive and the highest adhesion was observed with Lactcoccus lactis ssp . lactis NIAI527 . This strain adhered to the microvilli of cells as observed by scanning electron microscopy and also tolerated low pH and bile . These properties should make strain 527 a potential new probiotic strain.

Appl Environ Microbiol, 2000 Feb, 66(2), 588 - 98
Expression of the Staphylococcus hyicus lipase in Lactococcus lactis; Drouault S et al.; The extracellular Staphylococcus hyicus lipase was expressed under the control of different promoters in Lactococcus lactis and Bacillus subtilis . Its expression at high and moderate levels is toxic for the former and the latter hosts, respectively . In L . lactis, the lipase was expressed at a high level, up to 30% of the total cellular proteins, under the control of the inducible promoter PnisA . About 80% of the lipase remained associated with the cells . Close to half of this amount remained associated with the inner side of the cytoplasmic membrane as unprocessed pre-pro-lipase . The other half was trapped by the cell wall and partially degraded at the N-terminal end . This result suggests that extracellular proteases degrade the lipase . Surprisingly, the kinetics and the pattern of lipase degradation were different in the two L . lactis subspecies, L . lactis subsp . cremoris and L . lactis subsp . lactis . The extracellular proteolytic systems that degrade lipase are thus different in these closely related subspecies . The incorrect export of the lipase is not due to an inappropriate leader peptide but may be due to an inefficiency of several steps of lipase secretion . We propose that (i) the S . hyicus lipase may require a special accessory system to be correctly exported or (ii) the kinetics of lipase synthesis may be a critical factor for proper folding.

Appl Environ Microbiol, 2000 Feb, 66(2), 571 - 7
Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp . cremoris NCDO 763; Yvon M et al.; In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases . We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT . In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT . We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription . BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5'-phosphate-dependent aminotransferases . In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric . It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively . The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine . However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors . Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.

Eur J Biochem, 2000 Feb, 267(3), 901 - 9
Engineering a disulfide bond and free thiols in the lantibiotic nisin Z; van Kraaij C et al.; The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues . To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C . Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics . NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C . The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity . The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol . Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment . Their ability to permeabilize lipid vesicles was not thiol-dependent . Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide . These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 191 - 5
Synergistic effects of nisin and thymol on antimicrobial activities in Listeria monocytogenes and Bacillus subtilis; Ettayebi K et al.; Nisin Z and thymol were tested, alone and in combination, for antibacterial activity against Listeria monocytogenes ATCC 7644 and Bacillus subtilis ATCC 33712 . The antibacterial effect of nisin Z, produced by Lactococcus lactis KE3 isolated from the traditional Moroccan fermented milk, was greatly potentiated by sub-inhibitory concentrations of thymol in both bacterial strains . Our data showed that the concentration of nisin required for effective control of food-borne pathogenic bacteria could be considerably lowered by the use of thymol in combination . The use of low concentrations of nisin could lead to a less favourable condition for the occurrence of nisin-resistant bacterial sub-populations.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 177 - 82
Identification and characterisation of a gene encoding aminoacylase activity from Lactococcus lactis MG1363; Curley P et al.; Analysis of the sequence of a randomly cloned chromosomal DNA fragment (3.2 kb) from Lactococcus lactis revealed the presence of part of an open reading frame, designated amd1, which specifies a protein displaying significant similarity to aminoacylases from various bacteria . The presence of an immobilised copy of an IS982 element immediately upstream of the coding region of amd1 has probably resulted in the displacement of amd1's native promoter . This genetic organisation was shown to be retained in seven other dairy strains, one of which was only slightly different . The amd1 gene was overexpressed in L . lactis NZ9800 under the control of the inducible nisA promoter and the deacetylating capacity of its gene product was measured on a number of substrates.

J Bacteriol, 2000 Feb, 182(4), 1136 - 43
The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways; Novak L et al.; Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network . The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways . The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways . Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored . The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux . Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction . Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.

DNA Seq, 1999, 10(3), 163 - 72
Cloning, DNA sequence analysis, and deletion of a gene encoding diacetyl-acetoin reductase from Lactococcus lactis; Aungpraphapornchai P et al.; Diacetyl is produced by strains of lactic acid bacteria used in the dairy industry . Production of this important flavour compound could be increased by genetic manipulation of genes encoding enzymes involved in diacetyl metabolism . This paper reports the cloning and sequencing of the gene (dar) encoding diacetyl-acetoin reductase from Lactococcus lactis . Analysis of the DNA sequence of the dar gene and surrounding area revealed the presence of a putative operon with similarity to the family of ABC transporter systems . The dar gene has been deleted from the chromosome by double cross-over homologous recombination.

Arch Microbiol, 2000 Jan, 173(1), 21 - 8
P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity; Schubert K et al.; A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L . monocytogenes with an apparent molecular mass of 45 kDa . This protein was detected in the culture supernatant and at the cell surface of L . monocytogenes . Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L . grayi . The structural gene was cloned in Escherichia coli and sequenced . Translation of the gene starts at a TTG initiation codon . The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa . It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa . Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L . monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis . P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property).

J Biol Chem, 2000 Jan 28, 275(4), 2463 - 71
The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis . Catalytic properties and allosteric regulation of the pure enzyme system; Torrents E et al.; Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase . Strict anaerobic growth depends on the activity of these genes . Both were sequenced, cloned, and overproduced in Escherichia coli . The corresponding proteins, NrdD and NrdG, were purified close to homogeneity . The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E . coli proteins . Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate . EPR experiments demonstrated a {4Fe-4S}(+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E . coli NrdD and NrdG proteins . Different from E . coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated . Also the FeS cluster was easily lost from NrdG . The substrate specificity and overall activity of the L . lactis enzyme was regulated according to the general rules for ribonucleotide reductases . Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP . The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects . The results with the L . lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Appl Microbiol Biotechnol, 1999 Dec, 53(1), 36 - 42
Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis; Duan K et al.; A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp . lactis LL57-1 . Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE . The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal theta-type replicons of the pWV02/pCI305 family . pND324 was stable at both 30 degrees C and 37 degrees C, whereas derivatives that lack cisE were highly unstable at 37 degrees C, indicating that cisE is essential for thermostability . Sequences that are similar to orfX1 are commonly present in the lactococcal theta-type plasmids . The orfX2 product is homologous to TrfA, a 43-kDa protein of the E . coli theta-type plasmid RK2 required for replication and maintenance . Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324 . Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed . In L . lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 degrees C for 40 generations . The integrated plasmid was totally stable for at least 100 generations without selection at 30 degrees C.

Int J Food Microbiol, 1999 Dec 15, 53(2-3), 105 - 13
Effect of chelators and nisin produced in situ on inhibition and inactivation of gram negatives; Boziaris IS et al.; The ability of chelators and nisin generated in situ to inhibit and inactivate E . coli and other gram negatives in a model substrate was investigated . The effect of various chelators and different concentrations of exogenous nisin on inhibition of E . coli in broth medium showed that only EDTA and pyrophosphates were able to cause appreciable inhibition of E . coli by nisin . In a broth where L . lactis NCFB 497 produced nisin in a concentration of 250-300 IU/ml, pyrophosphates were unable to inactivate E . coli . Under the same conditions, addition of EDTA led to inactivation of E . coli at neutral and slightly acidic pH only . A cocktail of strains of E . coli was less sensitive than E . coli ATCC 25922 alone . Pseudomonas aeruginosa was more sensitive and salmonellae more resistant . EDTA also caused a slight reduction in the L . lactis population and its biochemical activity as regards pH drop and acid production . Some of the inhibition of E . coli could be ascribed to the physical presence of Lactococcus cells rather than their metabolites excreted into the medium . Failure to observe any inhibition in fermented broths at their natural pH (4.0) was ascribed to the poor chelating power of EDTA under acid conditions.

J Dairy Sci, 1999 Dec, 82(12), 2625 - 31
The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows; Ryan MP et al.; Lacticin 3147 is a broad-spectrum bacteriocin produced by the food-grade organism Lactococcus lactis . Lacticin 3147 is active at a neutral pH and has been shown to be bactericidal to streptococci and staphylococci in vitro . The effectiveness of an intramammary teat seal formulation, and a teat seal containing lacticin 3147 was evaluated at drying off in 68 uninfected quarters of 18 cows . Following infusion of either teat seal or lacticin 3147 combined with teat seal, a deliberate infection challenge of Streptococcus dysgalactiae (approximately equal to 1.5 x 10(4) cfu per teat) was administered by direct inoculation into the teat sinus . During an 8-d experimental period following inoculation, 61% of control quarters and 6% of the treatment quarters either developed clinical mastitis or were shedding the challenge organism . Randomly amplified polymorphic DNA polymerase chain reaction genetic typing was used to confirm that both the new infections and the bacteria surviving in the teats at the end of the experiment were the challenge strain . The combination of teat seal and lacticin 3147 was well tolerated within the udder and elicited only a temporary increase in somatic cell count to 5.7 x 10(5)/ml (88 h after infusion) in a previously uninfected lactating udder quarter . Therefore, we concluded that this nonantibiotic approach to mastitis prevention may contribute to a reduction in the routine application of antibiotics at drying off in the future.

J Food Prot, 2000 Dec, 63(12), 1707 - 12
Identification and partial characterization of lacticin BH5, a bacteriocin produced by Lactococcus lactis BH5 isolated from Kimchi; Hur JW et al.; Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240 . Strain BH5 was identified tentatively as Lactococcus lactis by API test . Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method . The activity of lacticin BH5, named tentatively as the bacteriocin produced by L . lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase . Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method . Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV . The inhibitory activities of lacticin BH5 were detected during treatments up to 100 degrees C for 30 min . Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined . It demonstrated a typical bactericidal mode of inhibition against M . flavus ATCC 10240 . The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

J Dairy Res, 2000 Nov, 67(4), 585 - 96
Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk; Govindasamy-Lucey S et al.; The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp . cremoris starter strains and eight Lc . lactis subsp . cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h . Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I . This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc . lactis subsp . cremoris MG1363 (Buist et al . Journal of Bacteriology 180 5947-5953 1998) . In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc . lactis subsp . cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation . The distribution of autolysins within the cell envelope of Lc . lactis subsp . cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis . AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity . An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity . These results show that autolysis of commercial Lc . lactis subsp . cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

J Dairy Res, 2000 Nov, 67(4), 571 - 83
Metabolism of lactose and citrate by mutants of Lactococcus lactis producing excess carbon dioxide; El Attar A et al.; Mutants of Lactococcus lactis producing excess carbon dioxide could be isolated on LDHA-20 agar (described by El Attar et al . Journal of Dairy Research 67 641-646 2000) . The use of these mutants in the manufacture of Roquefort cheese has the potential to improve the formation of openings in this cheese . The aim of this work was to examine the stability of these mutants, their enzymic activities and their metabolism of lactose and citrate during growth in milk . They produced less L-lactate than the parent strain and their lactate dehydrogenase activity was lower . Nevertheless none of the mutants produced no L-lactate at all and the most active gas generators among them generally produced 30-50 mM-L-lactate . Unexpectedly, all the strains produced some D-lactate, some > 10 mM . We found that carbon dioxide production by the mutants could be determined indirectly by assaying acetoin, citrate and 2,3-butanediol by high-performance liquid chromatography . Generally, spontaneous mutants were more stable than those obtained after treating with nitrosoguanidine or u.v . irradiation.

FEMS Microbiol Lett, 2000 Jan 15, 182(2), 249 - 54
Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp . cremoris MG1363; Dobric N et al.; This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp . cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

FEMS Microbiol Lett, 2000 Jan 15, 182(2), 207 - 11
Metabolic flux in glucose/citrate co-fermentation by lactic acid bacteria as measured by isotopic ratio analysis; Goupry S et al.; The flux of carbon into lactic acid, diacetyl and acetoin during the co-metabolism of glucose and citrate by Lactococcus lactis subsp . lactis biovar . diacetylactis has been determined using natural abundance isotopic ratio analysis . During fermentation in the conditions used (glucose, 27.8 mM; citric acid, 13.9 mM; initial pH 6.2-6.4, anaerobic) it is shown that approximately 65% of the carbon source used for the aroma compounds is derived from the carbohydrate . Equally, citrate contributes approximately 30% of the carbon recovered in lactic acid . Thus, there is no evidence for a metabolic separation of the catabolism of these two carbon sources.

Appl Environ Microbiol, 2000 Jan, 66(1), 42 - 8
Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine beta-lyase; Fernandez M et al.; The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development . Cystathionine beta-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an alpha, gamma elimination . With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese . The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds . The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase . The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity . A disruption of the metC gene was achieved in strain MG1363 . Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via alpha, gamma elimination to form volatile aroma compounds.

Vet Immunol Immunopathol, 1999 Dec 15, 72(1-2), 203 - 12
Recent developments in fish vaccinology; Gudding R et al.; During the last 10 to 20 years vaccination has become established as an important method for prevention of infectious diseases in farmed fish, mainly salmonid species . So far, most commercial vaccines have been inactivated vaccines administered by injection or immersion . Bacterial infections caused by Gram-negative bacteria such as Vibrio sp., Aeromonas sp., and Yersinia sp . have been effectively controlled by vaccination . With furunculosis, the success is attributed to the use of injectable vaccines containing adjuvants . Vaccines against virus infections, including infectious pancreatic necrosis, have also been used in commercial fish farming . Vaccines against several other bacterial and viral infections have been studied and found to be technically feasible . Pasteurellosis, streptococcosis (lactococcosis) and infections with iridoviruses are candidate diseases for control by immunoprophylaxis in the near future . The overall positive effect of vaccination in farmed fish is reduced mortality . However, for the future of the fish farming industry it is also important that vaccination contributes to a sustainable biological production with negligible consumption of antibiotics . A potential side-effect associated with injectable vaccines is local reactions in the peritoneal cavity . The paper presents recent developments in immunoprophylaxis of fish and some problems that should be addressed by the research community in the years to come.

J Bacteriol, 2000 Jan, 182(1), 203 - 6
Glycine betaine transport in Lactococcus lactis is osmotically regulated at the level of expression and translocation activity; van Der Heide T et al.; Microorganisms react upon hyperosmotic stress by accumulating compatible solutes . Here we report that Lactococcus lactis uses a transport system for glycine betaine that, contrary to earlier observations (D . Molenaar et al., J . Bacteriol . 175:5438-5444, 1993), is osmotically regulated at the levels of both expression and transport activity.

J Bacteriol, 2000 Jan, 182(1), 30 - 7
Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000; Kearney K et al.; The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed . The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs) . One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode . Deletion analysis showed that this ORF, designated repA, is indeed required for replication . The results also suggest that the origin of replication is located outside repA . Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified . Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans . This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.

FEMS Microbiol Lett, 2000 Jan 1, 182(1), 185 - 91
Identification of a DNA region from lactococcal phage sk1 protecting phage 712 from the abortive infection mechanism AbiF; Rince A et al.; Bacteriophage 712 is a small isometric-headed phage which is sensitive to the lactococcal abortive infection mechanism AbiF . Its 29.6-kb DNA genome was characterized by restriction mapping and transcriptional analysis . Construction of a gene bank of lactococcal phage sk1, which is insensitive to the action of AbiF, in Lactococcus lactis containing AbiF resulted in the identification of a 324-bp DNA fragment which reduced the effect of the abortive infection mechanism on phage 712 . Analysis of this region provided evidence that the action of AbiF is related to the cos ends of small isometric-headed phages . Sequence analysis of a 3.2-kb segment containing the middle operon and the cos ends of phage 712 genome allowed comparison of this part of the phage 712 genome with the equivalent sequences of four other small isometric-headed phages.

FEMS Microbiol Lett, 2000 Jan 1, 182(1), 149 - 54
Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes; Cvitkovitch DG et al.; Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone . In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S . mutans strain NG8 . Tn917-lac transposition occurred at a frequency of ca . 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression . Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated . Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion . The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose . Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium . Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters . These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S . mutans and possibly in other medically relevant streptococcal species.

Gene, 2000 Jan 4, 241(1), 157 - 66
The pyrH gene of Lactococcus lactis subsp . cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1; Wadskov-Hansen SL et al.; The pyrH gene of Lactococcus lactis subsp . cremoris MG1363, encoding UMP kinase, has been sequenced and cloned . It encodes a polypeptide of 239 amino acid residues (deduced molecular weight of 25951), which was shown to complement a temperature sensitive pyrH mutation in Escherichia coli, thus establishing the ability of the encoded protein to synthesize UDP . The pyrH gene in L . lactis is flanked downstream by frr1 encoding ribosomal recycling factor 1 and upstream by an open reading frame, orfA, of unknown function . The three genes were shown to constitute an operon transcribed in the direction orfA-pyrH-frr1 from a promoter immediately in front of orfA . This operon belongs to an evolutionary highly conserved gene cluster, since the organization of pyrH on the chromosomal level in L . lactis shows a high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other prokaryotes

J Biol Chem, 1999 Dec 31, 274(53), 37544 - 50
Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147; Ryan MP et al.; Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147 . In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity . The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da . Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides . Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product . Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.

J Bacteriol, 1999 Dec, 181(24), 7430 - 8
The genetic switch regulating activity of early promoters of the temperate lactococcal bacteriophage TP901-1; Madsen PL et al.; A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters p(R) and p(L) . ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters . Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1 . ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter p(L) by ORF4 . Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either p(L) is open and p(R) is closed or vice versa . The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation . Induction of a repressed p(L) fusion containing orf4 and orf5 was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site . Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages.

Microb Pathog, 1999 Dec, 27(6), 407 - 17
Identification of genes in a KG- phenotype of Lactococcus garvieae, a fish pathogenic bacterium, whose proteins react with antiKG- rabbit serum; Hirono I et al.; Five different clones (SA1B05, SA1B10, SA2F01, SA8A11 and SA9H10) were isolated from the gene library of the Lactococcus garvieae SA8201 (KG-) strain by immunological screening using rabbit serum against L . garvieae (KG-) phenotype cells . A Western blot analysis indicated that the molecular sizes of immunologically detected proteins of SA1B05, SA1B10, SA2F01, SA8A11 and SA9H10, which were fused with LacZ protein, were 25, 30, 28, 26 and 13 kDa, respectively . The amino acid sequences of the immunologically detected proteins of SA1B05, SA1B10, SA2F01 and SA8A11 were homologous to a processing protease of Bacillus subtilis (36.6%), dihydropteroate synthase of Escherichia coli (34.6%), trigger factor of B . subtilis (45.8%) and N-acetylglucosamine-6-phosphate deacetylase of Vibrio furnissii (37.1%), respectively . There was no significant homologous sequence of SA9H10 in DDBJ/EMBL/GenBank and SwissProt . We cloned and sequenced a longer DNA fragment (SA9H10L) of SA9H10 from the gene library . The predicted amino acid sequence of this clone was weak homology to M protein of Streptococcus pyogenes (22.7%) . Five genes were specifically expressed in the KG- phenotype strains . However, SA8A11 and SA9H10 was expressed in the mutated strain SA8201-TTC, whose serological phenotype was changed from KG- to KG+ by 2,3,5-triphenyltetrazolium chloride .

Microb Comp Genomics, 1999, 4(3), 173 - 86
Phylogenetic analyses of proton-translocating transhydrogenases; Studley WK et al.; The proton-translocating nicotinamide nucleotide transhydrogenases (TH) provide a simple model for understanding chemically coupled transmembrane proton translocation . To further our understanding of TH structure-function relationships, we have identified all sequenced homologous of these vectorial enzymes and have conducted sequence comparison studies . The NAD-binding domains of TH are homologous to bacterial alanine dehydrogenases (ADH) and eukaryotic saccharopine dehydrogenases (SDH) as well as N5(carboxyethyl)-L-ornithine synthase of Lactococcus lactis and dipicolinate synthase of Bacillus subtilis . A multiple alignment, a phylogenetic tree, and two signature sequences for this family, designated the TH-ADH-SDH or TAS superfamily, have been derived . Additionally, the TH family has been characterized . Phylogenetic analyses suggested that these proteins have evolved without inter-system shuffling . However, interdomain splicing-fusion events have occurred during the evolution of several of these systems . Analyses of the multiple alignment for the TH family revealed that domain conservation occurs in the order: NADP-binding domain (domain III) > NAD-binding domain (domain I) > proton-translocating transmembrane domain (domain II) . A topologic model for the proton-translocating transmembrane domain consistent with published data is presented, and a possible involvement of specific transmembrane alpha-helical segments in channel formation is suggested.

Microbiology, 1999 Nov, 145 ( Pt 11), 3185 - 94
Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection; Wouters JA et al.; Low-temperature adaptation and cryoprotection were studied in the lactic acid bacterium Lactococcus lactis MG1363 . An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 degrees C for 4 h compared to mid-exponential-phase cells grown at 30 degrees C, indicating an active protection against freezing . Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L . lactis MG1363 (Wouters et al., 1998, Microbiology 144, 2885-2893) . The 7 kDa CSPs appeared to be the most strongly induced proteins upon cold shock to 10 degrees C . Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 degrees C, while induction was lower at 20 and 4 degrees C . However, pre-incubation at 20 and 4 degrees C, as well as stationary-phase conditions, also induced cryoprotection (approx . 30-, 130- and 20-fold, respectively, compared to 30 degrees C mid-exponential phase) . For all treatments leading to an increased freeze survival (exposure to 4, 10 and 20 degrees C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested . Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions . However, the level of observed freeze protection does not directly correlate with the csp gene expression levels . In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied . This revealed that L . lactis cells overproducing CspD at 30 degrees C show a 2-10-fold increased survival after freezing compared to control cells . This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that other factors supply additional cryoprotection.

Microbiology, 1999 Nov, 145 ( Pt 11), 3155 - 61
Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon; Martinez B et al.; Synthesis of lactococcin 972 is plasmid-encoded . An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified . The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin . The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains . Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

Biochemistry, 1999 Dec 7, 38(49), 16298 - 306
The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids; Margolles A et al.; Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs . A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography . The histidine-tagged protein was overexpressed in L . lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter . This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein . For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L . lactis phospholipids . The detergent was removed by adsorption onto polystyrene beads . The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes . Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine . These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L . lactis.

J Appl Microbiol, 1999 Oct, 87(4), 500 - 10
Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains; Bouksaim M et al.; Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp . lactis biovar diacetylactis UL 719 . Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z . Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum . The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively . In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount . In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected . In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.

Biochim Biophys Acta, 1999 Dec 6, 1461(2), 201 - 6
Structure-function analysis of multidrug transporters in Lactococcus lactis; van Veen HW et al.; The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms . A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein . Another multidrug transporter in L . lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms . Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell . Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism . The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Biotechnol Bioeng, 1999, 66(4), 211 - 8
Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction; Planas J et al.; A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid . Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized . Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch . With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained . The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate . Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems . In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate . In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp . lactis, was 0.45 . The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems .

J Bacteriol, 1999 Dec, 181(23), 7291 - 7
Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1; Breuner A et al.; In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified . The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present . However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase . Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases . Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon . This location of an excisionase gene of a temperate bacteriophage has never been described before . The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis . Excision of the vectors was investigated in the presence of different TP901-1 genes . In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil . By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured . The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.

Protein Sci, 1999 Oct, 8(10), 2121 - 9
N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues; Sackett DL et al.; N5-(L-1-carboxyethyl)-L-ornithine synthase {E.C . 1.5.1.24} (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods . The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers . The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins . CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues . Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5 . All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3 . The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer . In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely . The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain . Chymotrypsin specifically cleaves native CEOS after Phe255 . Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained . Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

Biochemistry, 1999 Nov 2, 38(44), 14440 - 50
Kinetics and consequences of binding of nona- and dodecapeptides to the oligopeptide binding protein (OppA) of Lactococcus lactis; Lanfermeijer FC et al.; The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters . This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues . Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand . The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides . This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA . Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants . These results are discussed in relation to the affinity constant for transport of these substrates . Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.

Appl Environ Microbiol, 1999 Nov, 65(11), 5151 - 3
PCR amplification of the gene acmA differentiates Lactococcus lactis subsp . lactis and L . lactis subsp . cremoris; Garde S et al.; The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined . Isolates were genotypically identified to the subspecies level with a PCR technique . On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies . L . lactis subsp . lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L . lactis subsp . cremoris exhibits a single 1,131-bp fragment.

Appl Environ Microbiol, 1999 Nov, 65(11), 5003 - 8
Regulation of exopolysaccharide production by Lactococcus lactis subsp . cremoris By the sugar source; Looijesteijn PJ et al.; Lactococcus lactis produced more exopolysaccharide (EPS) on glucose than on fructose as the sugar substrate, although the transcription level of the eps gene cluster was independent of the sugar source . A major difference between cells grown on the two substrates was the capacity to produce sugar nucleotides, the EPS precursors . However, the activities of the enzymes required for the synthesis of nucleotide sugars were not changed upon growth on different sugars . The activity of fructosebisphosphatase (FBPase) was by far the lowest of the enzymes involved in precursor formation under all conditions . FBPase catalyzes the conversion of fructose-1, 6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose . By overexpression of the fbp gene, which resulted in increased EPS synthesis on fructose, it was proven that the low activity of FBPase is indeed limiting not only for EPS production but also for growth on fructose as a sugar source.

Appl Environ Microbiol, 1999 Nov, 65(11), 4881 - 6
Survival, physiology, and lysis of Lactococcus lactis in the digestive tract; Drouault S et al.; The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration . For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria . Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells . The green fluorescent protein was used to assess the bacterial lysis independently of death . We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact . Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival) . In contrast, only 10 to 30% of bacteria survive in the duodenum . Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis . This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm . This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.

Appl Environ Microbiol, 1999 Nov, 65(11), 4873 - 80
Genetic characterization of the major lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds; Rijnen L et al.; In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds . In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a Lactococcus lactis subsp . cremoris strain . Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds . Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily gamma; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass . We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids . It is also highly involved in methionine and leucine conversion . AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine . However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium.

Vet Microbiol, 1999 Sep 29, 69(4), 287 - 300
Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms; Schmidtke LM et al.; Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes . Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser . Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin . The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction . Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development . Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells . Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells . L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa . Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates . Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue . Fish were susceptible to infection when challenged with parent cells of L . garvieae.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 357 - 65
Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?
Kleerebezem M, van Kranenburg R, Tuinier R, Boels IC, Zoon P, Looijesteijn E, Hugenholtz J, de Vos WM.
Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis . From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised . They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination . The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering . In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars . The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L . lactis . Finally, model development has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 347 - 52
Multidrug resistance in lactic acid bacteria: molecular mechanisms and clinical relevance; van Veen HW et al.; The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms . The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms . Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters . Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell . Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism . The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 337 - 46
Developing applications for lactococcal bacteriocins; Ross RP et al.; While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential . An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against gram-positive bacteria . Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters . The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora . The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate . Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications . Field trials have demonstrated its efficacy in the prevention of mastitis infections in dairy cows . In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci . Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 207 - 15
Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties; Meisel H et al.; The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g . during gastrointestinal digestion or food processing . The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides . In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from beta- and alpha-caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides . The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products . However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria . Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, angiotensin-I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium . Bioactive peptides can interact with target sites at the luminal side of the intestinal tract . Furthermore, they can be absorbed and then reach peripheral organs . Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 77 - 88
Group II introns and expression of conjugative transfer functions in lactic acid bacteria; Dunny GM et al.; The homologous lactococcal conjugative elements pRS01 and the sex factor of Lactococcus lactis strain 712 both contain a Group II intron within a gene believed to encode a conjugative relaxase enzyme . This enzyme is responsible for nicking of DNA at the origin of transfer (oriT) sequence of the sex factor DNA to initiate the strand transfer process . Group II introns have been studied in eukaryotes, and several of these elements in yeast mitochondrial genes have received considerable attention . These introns are relatively large in size and generally encode a protein within the intron sequence . In addition to splicing activity . Group II introns are mobile genetic elements . The intron-encoded proteins (IEPs) contain endonuclease and reverse transcriptase domains believed to play an enzymatic role in genetic mobility reactions, while a putative maturase domain is thought to promote splicing by stabilizing the folding of the intron RNA into an active ribozyme structure which carries out the splicing reaction . The lactococcal introns represent the first examples of Group II introns shown to be functional in vivo in prokaryotes . Because of the advantages of a bacterial system for genetic and molecular studies, the Ll.ltrB intron from pRS01 has attracted the attention of several laboratories interested in Group II intron biology . Recently, it has been shown that the system can be adapted to function in Escherichia coli (although at somewhat reduced efficiency) . In addition, it has been recently proven that the best studied form of mobility, the homing of the intron into an intronless allele of the cognate exon gene, occurs via an RNA intermediate and does not require DNA homology or generalized host recombination functions . Current efforts are analysis of the role Ll.ltrB splicing in regulating expression of pRS01 conjugation functions . The lactococcal Group II introns represent the first demonstrated genetically mobile prokaryotic retroelements, and they also have considerable potential as genetic engineering tools for Lactic Acid Bacteria (LAB) and other organisms.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 27 - 76
Low-redundancy sequencing of the entire Lactococcus lactis IL1403 genome; Bolotin A et al.; Lactococcus lactis is an AT-rich gram positive bacterium phylogenetically close to the genus Streptococcus . Various strains of L . lactis are used in dairy industry as starters for cheese making . L . lactis is also one of the well characterized laboratory microorganisms, widely used for studies on physiology of lactic acid bacteria . We describe here a low redundancy sequence of the genome of the strain L . lactis IL1403 . The strategy which we followed to determine the sequence consists of two main steps . First, a limited number of plasmids and lambda-phages that carry random segments of the genome were sequenced . Second, sequences of the inserts were used for production of novel sequencing templates by applying Multiplex Long Accurate PCR protocols . Using of these PCR products allowed to determine the sequence of the entire 2.35 Mb genome with a very low redundancy, close to 2 . The error rate of the sequence is estimated to be below 1% . The correctness of the sequence assembly was confirmed by PCR amplification of the entire L . lactis IL1403 genome, using a set of 266 oligonucleotides . Anotation of the sequence was undertaken by using automatic gene prediction computer tools . This allowed to identify 1495 protein-encoding genes, to locate them on the genome map and to classify their functions on the basis of homology to known proteins . The function of about 700 genes expected to encode proteins that lack homologs in data bases cannot be reliably predicted in this way . The approach which we used eliminates high redundancy sequencing and mapping efforts, needed to obtain detailed and comprehensive genetic and physical maps of a bacterium . Availability of detailed genetic and physical maps of the L . lactis IL1403 genome provides many entries to study metabolism and physiology of bacteria from this group . The presence of 42 copies of five different IS elements in the IL1403 genome confirms the importance of these elements for genetic exchange in Lactococci . These include two previously unknown elements, present at seven and fifteen copies and designated IS1077 and IS983, respectively . Five potential or rudimentary prophages were identified in the genome by detecting clusters of phage-related genes . The metabolic and regulatory potential of L . lactis was evaluated by inspecting gene sets classified into different functional categories . L . lactis has the genetic potential to synthesise 20 standard amino acids, purine and pyrimidine nucleotides and at least four cofactors . Some of these metabolites, which are usually present in chemically defined media, can probably be omitted . About twenty compounds can be used by L . lactis as a sole carbon source . Some 83 regulators were revealed, indicating a regulatory potential close to that of Haemophilus influenzae, a bacterium with a similar genome size . Unexpectedly, L . lactis has a complete set of late competence genes, which may have concerted transcriptional regulation and unleadered polycistronic mRNAs . These findings open new possibilities for developing genetic tools, useful for studies of gene regulation in AT-rich gram positive bacteria and for engineering of new strains for the diary industry.

J Dairy Sci, 1999 Oct, 82(10), 2108 - 14
The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows; Ryan MP et al.; Lacticin 3147 is a broad-spectrum bacteriocin produced by the food-grade organism Lactococcus lactis . Lacticin 3147 is active at a neutral pH and has been shown to be bactericidal to streptococci and staphylococci in vitro . The effectiveness of an intramammary teat seal formulation, and a teat seal containing lacticin 3147 was evaluated at drying off in 68 uninfected quarters of 18 cows . Following infusion of either teat seal or lacticin 3147 combined with teat seal, a deliberate infection challenge of Streptococcus dysgalactiae (approximately equal to 1.5 x 10(4) cfu per teat) was administered by direct inoculation into the teat sinus . During an 8-d experimental period following inoculation, 61% of control quarters and 6% of the treatment quarters either developed clinical mastitis or were shedding the challenge organism . Randomly amplified polymorphic DNA polymerase chain reaction genetic typing was used to confirm that both the new infections and the bacteria surviving in the teats at the end of the experiment were the challenge strain . The combination of teat seal and lacticin 3147 was well tolerated within the udder and elicited only a temporary increase in somatic cell count to 5.7 x 10(5)/ml (88 h after infusion) in a previously uninfected lactating udder quarter . Therefore, we concluded that this nonantibiotic approach to mastitis prevention may contribute to a reduction in the routine application of antibiotics at drying off in the future.

Biochemistry, 1999 Oct 19, 38(42), 13900 - 5
The secondary multidrug transporter LmrP contains multiple drug interaction sites; Putman M et al.; The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane . Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L . lactis showed that the LmrP-mediated H(+)/Hoechst 33342 antiport reaction obeyed Michaelis-Menten kinetics, with a low apparent affinity constant of 0.63 microM Hoechst 33342 (= 0.5 mmol Hoechst 33342/mol phospholipid) . Several drugs significantly inhibited LmrP-mediated Hoechst 33342 transport through a direct interaction with the protein rather than through dissipation of the proton motive force or reduction of the membrane partitioning of Hoechst 33342 . The characterization of the mechanism of inhibition of LmrP-mediated Hoechst 33342 transport indicated competitive inhibition by quinine and verapamil, noncompetitive inhibition by nicardipin and vinblastin, and uncompetitive inhibition by TPP(+) . The three types of inhibition of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles indicate for the first time the presence of multiple drug interaction sites in a secondary multidrug transporter.

Arch Biochem Biophys, 1999 Nov 1, 371(1), 115 - 23
Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine . HCl-induced unfolding and a low temperature requirement for refolding; Ruvinov SB et al.; Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases . The enzyme was inactivated at approximately 1 M GdnHCl . A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces . A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data . GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C . Refolding and reconstitution of the enzyme were optimal at </=15 degrees C and yields of active tetramer increased as the concentration of unfolded subunits decreased . Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 degrees C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 microM subunit, respectively) when incubated at 15 degrees C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl(2), and KCl .

DNA Seq, 1998, 9(5-6), 263 - 74
Characterization of the nisFEG operon of the nisin Z producing Lactococcus lactis subsp . lactis N8 strain; Immonen T et al.; Biosynthesis of the food additive nisin, a posttranslationally modified peptide antibiotic existing as two natural variants (A and Z), requires eleven genes (nisA/ZBTCIPRKFEG) involved in modification, secretion, regulation and self-immunity . The suggested self-immunity genes (nisFEG) of the nisin Z producer Lactococcus lactis subsp . lactis N8 were cloned and sequenced . Putative binding sites of the NisR transcription factor were recognized upstream of the nisF promoter . The hydrophilic NisF protein was expressed in Escherichia coli and shown to be associated with the membrane . Expression of the nisF gene from a plasmid in L . lactis MG1614, a strain lacking the nisin operons, did not increase the nisin resistance of the cells . This showed that NisF alone does not protect against nisin . Overexpression of the nisF gene in the N8 nisin producer did not affect the level of nisin immunity, indicating that the wild-type amount of NisF is not limiting the level of nisin immunity . Production of antisense-nisEG or antisense-nisG RNA in L . lactis N8 resulted in severe reduction in the level of nisFEG mRNA and a clearly reduced immunity showing that the nisFEG transcript is important for development of nisin self-immunity.

DNA Seq, 1998, 9(5-6), 245 - 61
Evidence for a mosaic structure of the Tn5481 in Lactococcus lactis N8; Immonen T et al.; The sequences of the left end of the nisin-sucrose transposon Tn5481 in Lactococcus lactis subsp . lactis N8, the adjacent 1.5 kb chromosomal region upstream of the junction site as well as a 5.0 kb region downstream of the nisZBTCIPRKFEG genes within the transposon have been determined . In the upstream chromosomal region, an incomplete open reading frame encoding a protein with strong N-terminal homology to the low-affinity branched chain amino acid carriers was identified . Within the transposon, downstream of the nisin gene cluster, a 186 bp almost identical copy of the left hand sequence was located . Further downstream, four new open reading frames were found . The codon usage in these reading frames as well as the G+C content of the region are clearly different from those of the nisin genes, suggesting that the functionally unrelated areas of Tn5481 are gathered from different origins during the evolution of the transposon.

FEMS Microbiol Lett, 1999 Oct 15, 179(2), 461 - 6
Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions; Lopez de Felipe F et al.; The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined . In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions . However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions . A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction . The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions . A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates . NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased . Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux . Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase . At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation . These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.

J Bacteriol, 1999 Oct, 181(20), 6238 - 46
Genetic and biochemical characterization of a high-affinity betaine uptake system (BusA) in Lactococcus lactis reveals a new functional organization within bacterial ABC transporters; Obis D et al.; The cytoplasmic accumulation of exogenous betaine stimulates the growth of Lactococcus lactis cultivated under hyperosmotic conditions . We report that L . lactis possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters . Through transposon mutagenesis, a mutant deficient in betaine transport was isolated . We identified two genes, busAA and busAB, grouped in an operon, busA (betaine uptake system) . The transcription of busA is strongly regulated by the external osmolality of the medium . The busAA gene codes for the ATP-binding protein . busAB encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the Bacillus subtilis betaine-binding protein, OpuAC . BusA of L . lactis displays a high affinity towards betaine (K(m) = 1.7 microM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of L . lactis to be under dual control at the biochemical and genetic levels . A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of L . lactis . The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.

J Dairy Sci, 1999 Sep, 82(9), 1897 - 903
Production of menaquinones by lactic acid bacteria; Morishita T et al.; Lactic acid bacteria were examined for their ability to produce quinone compounds, which may include dietary sources of menaquinones . Isoprenyl quinones in bacterial cells grown in a synthetic medium were extracted and analyzed by thin layer chromatography . Lactococcus lactis ssp . cremoris (three strains), Lactococcus lactis ssp . lactis (two strains), and Leuconostoc lactis were selected as high producers of quinone that synthesized more than 230 nmol of quinones/g of dried cells . The quinones were presumed to be menaquinone-7 to -10 by high performance liquid chromatography . Precise molecular weights were determined by mass spectrometry for Lactococcus lactis ssp . cremoris YIT 2011 and Leuconostoc lactis YIT 3001 and identified as menaquinone-8 and -9 for the former and menaquinone-9 and -10 for the latter . Those strains, when grown either in reconstituted nonfat dry milk or a soymilk medium, produced a beneficial quantity for dietary supplement (i.e., 29 to 123 micrograms of menaquinones/L of the fermented medium).

Biopolymers, 1999 Nov, 50(6), 641 - 6
Concentration and shear-rate dependence of the viscosity of an exocellular polysaccharide
Tuinier R, Zoon P, Stuart MA, Fleer GJ, de Kruif CG.
The viscosity of an exocellular polysaccharide (EPS) produced by the bacterium Lactococcus lactis subsp . cremoris B40 was studied in aqueous solution at an ionic strength of 0.10M . First, the zero-shear viscosity was determined as a function of the concentration . From the data in the low concentration range, the intrinsic viscosity was determined . In addition, the shear-thinning behavior was measured at several concentrations . By combining existing theories, a new equation is proposed that describes and predicts the intrinsic viscosity and the concentration dependence of the (zero-shear) viscosity of B40 EPS solutions from the molar mass and the hydrodynamic radius of the polysaccharide . Based on the Rouse theory, the shear-rate dependence of the viscosity also could be described and predicted from the molecular characteristics, i.e., molar mass and radius of gyration . It is shown that these equations can be applied to all random coil polysaccharides .

Curr Opin Biotechnol, 1999 Oct, 10(5), 492 - 7
Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations; Hugenholtz J et al.; Lactic acid bacteria such as Lactococcus lactis are the microorganisms of choice for performing metabolic engineering in relation to food fermentation . These bacteria are used extensively in food fermentations, they have a simple and therefore controllable metabolism and the molecular genetics of these food bacteria is well-developed . There have been recent successes in metabolic engineering in these lactic acid bacteria, including examples of changes in both primary metabolism (diacetyl and alanine) and secondary metabolism (exopolysaccharides and flavour).

Appl Environ Microbiol, 1999 Oct, 65(10), 4443 - 50
Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis; Horn N et al.; The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides . Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A . The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N . Horn, M . I . Martinez, J . M . Martinez, P . E . Hernandez, M . J . Gasson, J . M . Rodriguez, and H . M . Dodd, Appl . Environ . Microbiol . 64:818-823, 1998) . This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed . The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host . Highest yields were achieved with L . lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems . The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L . lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced . The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.

Dis Aquat Organ, 1999 Jul 30, 37(2), 121 - 6
The protective immune response of yellowtail Seriola quinqueradiata to the bacterial fish pathogen Lactococcus garvieae; Ooyama T et al.; Yellowtail Seriola quinqueradiata were immunized with 2 different Lactococcus garvieae bacterin, formalin-killed KG- phenotype cells (capsulated phenotype) and formalin-killed KG+ phenotype cells (unencapsulated phenotype) . These 2 injected vaccines conferred long-term protection to yellowtail against an artificial infection of an encapsulated Lactococcus garvieae strain with long-lasting agglutinating titres against KG+ phenotype cells . However, no agglutinating titres or low agglutinating titres against KG- phenotype cells were detected in fish given each of these bacterin . These results suggested that a capsule in KG- phenotype cells apparently affects their immunogenicity, but the antigens which conferred protection to fish against lactococcal infection may be located on the surface of KG+ phenotype cells, and are not cell capsules in KG- phenotype cells . The protection offered by a formalin-killed KG+ phenotype cell vaccine would not appear to be strain specific . Encapsulated L . garvieae cells were well phagocytosed, and fimbrie-like appendages were seen in KG- phenotype cells after treatment with yellowtail immune serum.

J Food Prot, 1999 Sep, 62(9), 1011 - 6
Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens; Morgan SM et al.; The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms . A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5 . The fermentate was spray dried, and the resulting powder exhibited inhibitory activity . The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH . In addition, the ability of the powder to inhibit L . monocytogenes Scott A in an infant milk formulation was assessed . Resuspension of approximately 10(8) midexponential phase L . monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C . In the case of S . aureus 10, resuspension of 2.5 x 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C . The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L . monocytogenes within 3 h at 30 degrees C . These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.

Mol Cell, 1999 Aug, 4(2), 239 - 50
A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA; Wank H et al.; Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT) . Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF . This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT . Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 11 - 9
Identification of a virulence-associated protein homolog gene and ISRa1 in a plasmid of Riemerella anatipestifer; Weng S et al.; Riemerella anatipestifer is the causative agent of polyserositis of ducks and geese . We have previously reported that a 3.9-kb plasmid, pCFC1, carries protein genes (vapD1 and vapD2) that are similar to virulence-associated genes of other bacteria . In the present study, we report the complete sequence of a second plasmid of 5.6 kb, pCFC2 . pCFC2 has a 28% G-C content and three large open reading frames (ORFs) . One of the ORFs (designated asVapD1) encodes a polypeptide that shares 53.9, 53.9, 48.3, 48.3 and 46.1% identity with virulence-associated proteins of Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae, Helicobacter pylori and Haemophilus influenzae, respectively . The second ORF encodes a putative DNA replication protein (RepA3) with 309 amino acids and a molecular mass of approximately 36 kDa . A novel insertion sequence (IS) element, designated ISRa1, was found on the plasmid pCFC2 . ISRa1 was flanked by 15-bp imperfect inverted repeats (only one mismatched nucleotide) . ISRa1 contained an ORF encoding a putative transposase of 292 amino acids . Southern blot analysis indicated that in R . anatipestifer strains examined, ISRa1 was present with 2-20 copies (at least) . ISRa1 displayed a sequence approximately 35% homologous to the putative IS982 and RSBst-alpha from Lactococcus lactis ssp . cremoris SK11 and Bacillus stearothermophilus CU21 . Three hybridization patterns of genomic DNA of eight R . anatipestifer strains with an ISRa1 probe indicated that ISRa1 might be a useful tool for epidemiological studies.

Carbohydr Res, 1999 Apr 30, 317(1-4), 131 - 44
Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp . cremoris B40; van Casteren WH et al.; EPS B40 from Lactococcus lactis subsp . cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-{alpha-L-Rhap-(1 -->2)}{alpha-D-Galp-1-PO4-3}-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> . A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment . Purified endoV from T . viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed . After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers . Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted . The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV {J.-P . Vincken, G . Beldman, A.G.J . Voragen, Carbohydr . Res., 298 (1997) 299-310}.

J Bacteriol, 1999 Sep, 181(17), 5521 - 6
Acetate utilization in Lactococcus lactis deficient in lactate dehydrogenase: a rescue pathway for maintaining redox balance; Hols P et al.; Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase . 13C and 1H nuclear magnetic resonance studies using {2-13C}glucose and {2-(13)C}acetate as substrates demonstrated that acetate was exclusively converted to ethanol . This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase.

Cryobiology, 1999 Aug, 39(1), 88 - 102
Effect of heat shock or cold shock treatment on the resistance of lactococcus lactis to freezing and lyophilization
Broadbent JR, Lin C.
This study investigated the effect of heat shock or cold shock treatment on the resistance of commercial Lactococcus lactis ssp . lactis and L . lactis ssp . cremoris cheese starter bacteria to freezing and freeze-drying . The ability to withstand freezing at -60 degrees C for 24 h was variable among lactococci, but could be significantly (P < 0.05) improved in most strains by a 25-min heat shock at 42 degrees C (L . lactis ssp . lactis) or 39 degrees C (L . lactis ssp . cremoris) or by a 2-h cold shock at 10 degrees C . In addition, stress treatments that improved lactococcal cryotolerance significantly (P < 0.05) enhanced the resistance of most strains to lyophilization . Greater resistance to freezing or lyophilization was not detected when stress treatments were performed in broth that contained erythromycin, which indicated that stress-induced proteins were required for cell protection . Membrane fatty acid analysis of stress-treated cells suggested that enhanced resistance to freezing and lyophilization may be related to changes in cell membrane lipid composition . Heat-shocked cells had a higher 19:0 cyclopropane fatty acid content than control cells, and cold-shocked cells contained a lower ratio of saturated to unsaturated fatty acids . Other factors must also be involved in cell protection, however, because similar changes in membrane lipid composition were detected in strains whose resistance to freezing and lyophilization was not improved by heat or cold shock treatment .

J Microbiol Methods, 1999 Aug, 37(2), 183 - 5
An improved method for screening alpha-acetolactate producing mutants; Monnet C et al.; Alpha-Acetolactate-deficient Lactococcus lactis ssp . lactis biovar . diacelylactis are utilised in several industrial processes for producing diacetyl and alpha-acetolactate . They can be selected by screening after random mutagenesis . We improved a previously described screening method {Monnet, C., Schmitt, P., Divies, C., 1997 . Appl . Environ . Microbiol . 63, 793-795}, which makes it possible to screen up to 1000 colonies per agar plate, whereas the previous method allowed to screen only 60 colonies per agar plate . The new screening method facilitates selection of alpha-acetolactate-deficient mutants.

Biotechnol Prog, 1999 Jul, 15(4), 646 - 54
Long-term mechanical and biological stability of an immobilized cell reactor for continuous mixed-strain mesophilic lactic starter production in whey permeate
Lamboley L, Lacroix C, Artignan JM, Champagne CP, Vuillemard JC.
The aim of this study was to characterize the biological and rheological stability of continuous immobilized cell fermentation for the production of mixed-strain mesophilic starters . Three strains of Lactococcus were immobilized separately in kappa-carrageenan-locust bean gum gel beads . Continuous fermentation was carried out in a 1 L pH-controlled stirred tank reactor, operated at 30 degrees C, pH = 6.2, and D = 2 h(-)(1) in whey permeate medium supplemented with yeast extract (1.5%) and 0.1 M KCl and inoculated with 30% (v/v) bead inoculum (strain ratio 1:1:1) . The continuous mixed-strain immobilized cell fermentation demonstrated a high biological stability, and no strain became dominant or was eliminated during the 52 day fermentation . The total and specific free cell populations showed high time stability . All immobilized populations, except MD, were unchanged, but a cross contamination of gel beads initially immobilizing a pure culture occurred, leading to a redistribution of immobilized population in individual beads . After initial modifications of bead rheological properties during colonization batches, the beads demonstrated a high mechanical stability, even with reduced KCl supplementation of the broth medium, in the range 0.1-0 M . This work emphasizes the potential of immobilized cell technology for producing mixed lactic starters in continuous fermentation.

Biochemistry, 1999 Aug 10, 38(32), 10352 - 60
Stereoselectivity of the membrane potential-generating citrate and malate transporters of lactic acid bacteria; Bandell M et al.; The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively . Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR(2)-COO(-) {Bandell, M., et al . (1997) J . Biol . Chem . 272, 18140-18146} . In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues . Modification of the OH or the COO(-) groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition . Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers . The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding . MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group . However, only the S-enantiomers were translocated by MleP . CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups . These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP . The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.

Dis Aquat Organ, 1999 Jun 23, 37(1), 33 - 41
Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail; Nakai T et al.; The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L . garvieae infection in yellowtail . Anti-L . garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity . In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later . Simultaneous administration of live L . garvieae and phage enhanced recovery of the phage from the spleen or intestine . The survival rate was much higher in yellowtail that received i.p . injection of the phage after i.p . challenge with L . garvieae, compared with that of control fish without phage injection . When fish were i.p . injected with phage at different hours after L . garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time . Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L . garvieae . Anal-intubated L . garvieae were detected constantly in the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later . On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later . These results indicate that intraperitoneally or orally administered anti-L . garvieae phage prevented fish from experimental L . garvieae infection, suggesting potential use of the phage for controlling the disease.

Microbiology, 1999 Jul, 145 ( Pt 7), 1519 - 30
Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis; Hoflack L et al.; Detailed functional analysis revealed the modular organization of pGI2, a 9672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp transposon Tn4430 . Whereas the pGI2 leading-strand replicon was identified through deletion experiments, sequence comparisons indicated the presence of an ssot-like single-strand origin commonly found among Bacillus plasmids . Southern hybridization confirmed the existence of ssDNA intermediates, but only in the case of plasmid derivatives lacking the ssot site . Moreover, the pGI2 replication protein Rep displayed significant similarity with that of pTX14-3, a 7-6 kb plasmid from B . thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolling-circle replicating (RCR) plasmids . In addition, both plasmids share a conserved 320 bp region downstream of their rep genes which, in the case of pGI2, appeared indispensable for replication . This region is therefore likely to correspond to, or to be part of, the actual double-strand origin of both plasmids . Another interesting feature of pGI2 is the presence of a mobilization (Mob) protein, as demonstrated by its ability to be mobilized by the conjugative plasmid pAW63 from B . thuringiensis serovar kurstaki HD73 . The same transfer system was also used to unambiguously demonstrate similar properties of the related Mob-like protein from pTX14-3 . A closer analysis of this family of related Mob proteins suggested a subdomanial organization among its members . Finally, the 270 residue pGI2 ORF2 was shown to be related to ORF43 of pMRC01, a 60 kb conjugative plasmid from Lactococcus lactis subsp . lactis . Although no function has been assigned to the putative ORF43 protein, it is located downstream of a bacteriocin operon, next to an IS946 element . pGI2 appears thus far as an assemblage of functional modules with no obvious metabolic function, presumably acting as a reservoir of carrier (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for its bacterial host.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8985 - 90
An aminoacyl-tRNA synthetase paralog with a catalytic role in histidine biosynthesis; Sissler M et al.; In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways . Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase (HisG) . Prediction of the function of HisZ in Lactococcus lactis was assisted by comparative genomics, a technique that revealed a link between the presence or the absence of HisZ and a systematic variation in the length of the HisG polypeptide . HisZ is required for histidine prototrophy, and three other lines of evidence support the direct involvement of HisZ in the transferase function . (i) Genetic experiments demonstrate that complementation of an in-frame deletion of HisG from Escherichia coli (which does not possess HisZ) requires both HisG and HisZ from L . lactis . (ii) Coelution of HisG and HisZ during affinity chromatography provides evidence of direct physical interaction . (iii) Both HisG and HisZ are required for catalysis of the ATP phosphoribosyltransferase reaction . This observation of a common protein domain linking amino acid biosynthesis and protein synthesis implies an early connection between the biosynthesis of amino acids and proteins.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 403 - 10
Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp . lactis; Farias ME et al.; The streptococcal plasmid pMV158 is not auto-transferable, but it can be mobilised between bacteria by the use of functions supplied by plasmids of the pIP501/pAM beta 1 family . Plasmid pMV158 encodes a protein, MobM, which is involved in its mobilisation . This process initiates when MobM specifically cleaves supercoiled pMV158 plasmid DNA at the origin of transfer, oriT . Plasmid pMV158 has been transferred to Lactococcus lactis by conjugation aided by plasmid pAM beta 1 . In the lactococcal host, MobM-mediated specific pMV158-relaxed molecules were detected . The intracellular amount of MobM has been quantified by immunoblot analyses and shown to be about 3500 molecules per cell . In the same host, we have mapped the initiation point of transcription of mobM . Transcription of this gene is directed from a promoter with an extended--10 region which overlaps with the pMV158-oriT.

Appl Environ Microbiol, 1999 Aug, 65(8), 3742 - 5
A nisin bioassay based on bioluminescence; Wahlstrom G et al.; A Lactococcus lactis subsp . lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed . By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains . As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay . This detection limit was lower than in previously described methods.

Appl Environ Microbiol, 1999 Aug, 65(8), 3681 - 9
Rapid fluorescence assessment of the viability of stressed Lactococcus lactis; Bunthof CJ et al.; The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp . lactis ML3 exposed to different stress treatments . The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70 degrees C very well . However, after treatment up to 60 degrees C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50 degrees C and was completely lost for those treated at 60 degrees C . In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared . The acidification capacity corresponded with the number of CFU . The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters . The cF labeling depended on membrane integrity, as was confirmed by PI exclusion . The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration) . When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment . This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L . lactis and has potential for physiological research and dairy applications related to lactic acid bacteria.

Infect Immun, 1999 Aug, 67(8), 4268 - 71
Identification of genes encoding two-component lantibiotic production in Staphylococcus aureus C55 and other phage group II S . aureus strains and demonstration of an association with the exfoliative toxin B gene; Navaratna MA et al.; The production of exfoliative toxin B (ET-B), but not ET-A, was shown to be specifically associated with production of a highly conserved two-component lantibiotic peptide system in phage group II Staphylococcus aureus . Two previously studied but incompletely characterized S . aureus bacteriocins, staphylococcins C55 and BacR1, were found to be members of this lantibiotic system, and considerable homology was also found with the two-component Lactococcus lactis bacteriocin, lacticin 3147 . sacalphaA and sacbetaA, the structural genes of the lantibiotics staphylococcins C55alpha and C55beta and two putative lantibiotic processing genes, sacM1 and sacT, were localized together with the ET-B structural gene to a single 32-kb plasmid in strain C55 . Irreversible loss of both ET-B and two-component lantibiotic production occurs during laboratory passage of ET-B-positive S . aureus strains, particularly at elevated temperatures.

Biochemistry, 1999 Jul 13, 38(28), 9069 - 83
RNA and protein catalysis in group II intron splicing and mobility reactions using purified components; Saldanha R et al.; Group II introns encode proteins with reverse transcriptase activity . These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription . Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity . Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of </=0.12 pM at 30 degrees C . This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur . Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.

Microbiology, 1999 Jun, 145 ( Pt 6), 1375 - 80
Lactococcus lactis contains only one glutamate decarboxylase gene; Nomura M et al.; Glutamate decarboxylase, which is associated with a glutamate-dependent acid-resistance mechanism, was purified from Lactococcus lactis subsp . lactis by a three-step procedure . The specific activity was increased about 114-fold with a yield of 16% . The N-terminal amino acid sequence of the enzyme was determined . The gene encoding this enzyme was cloned in Escherichia coli, and its nucleotide sequence was determined . The deduced amino acid sequence suggests that the enzyme is produced as a mature form (466 amino acid residues), not as a precursor protein . The subunit molecular mass of L . lactis glutamate decarboxylase was calculated to be 53 926 Da . The enzyme was maximally active at pH 4.7 and reacted only with L-glutamate among 20 alpha-amino acids . The apparent Km value was calculated to be 0.51 mM . The activity was stable at acidic pH values; there was no activity in the neutral pH range . At pH 4.1 the enzyme activity was retained at temperatures up to 70 degrees C in 10 min incubations . L . lactis glutamate decarboxylase behaved as a single protein when the enzyme was purified . A single band corresponding to the glutamate decarboxylase gene was detected on Southern blot analysis . These data suggest that there is one glutamate decarboxylase gene in L . lactis.

Dis Aquat Organ, 1999 May 31, 36(3), 227 - 31
Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases; Eldar A et al.; Clinical and macroscopic findings (anorexia, lethargy, loss of orientation and exophthalmia) indicate that Streptococcus iniae and Lactococcus garvieae infections of trout share some common features, but histopathology reveals notable differences between the 2 diseases . Meningitis and panophthalmitis are the main lesions among S . iniae infected trout, whereas L . garvieae infection results in a hyperacute systemic disease . Differences in the LD50s of the 2 pathogens and the sudden onset of signs and death correlate with the histopathological findings, indicating the severity of L . garvieae infection of trout.

Biotechnol Bioeng, 1999 Jul 20, 64(2), 200 - 12
In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis; Neves AR et al.; The metabolism of glucose by nongrowing cells of L . lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds . In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx . 3 min . The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis . In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts . A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps) . This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR . The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus . This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction . The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions .

Appl Environ Microbiol, 1999 Jul, 65(7), 2947 - 53
Specificity of lactococcus lactis subsp . cremoris SK11 proteinase, lactocepin III, in low-water-activity, high-salt-concentration humectant systems and its stability compared with that of lactocepin I
Reid JR, Coolbear T.
Marked changes in the specificity of hydrolysis of alphas1-, beta-, and kappa-caseins by lactocepin III from Lactococcus lactis subsp . cremoris SK11 were found in humectant systems giving the equivalent water activity (aw) and salt concentration of cheddar cheese . Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese . The stability of lactocepin III was compared with that of lactocepin I from L . lactis subsp . cremoris HP in the humectant systems at different pHs . Significant differences between the stability of each of the lactocepin types were evident . The relationship between stability and humectant type, aw, pH, and NaCl concentration was complex . Nevertheless, in those systems where aw, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III . It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 171 - 7
Nucleotide sequence of the streptococcin A-FF22 lantibiotic regulon: model for production of the lantibiotic SA-FF22 by strains of Streptococcus pyogenes; McLaughlin RE et al.; Streptococcin A-FF22 (SA-FF22) is a type AII linear lantibiotic produced by Streptococcus pyogenes strain FF22 . Sequence analysis of an approximate 10 kb region of DNA showed it to contain nine open reading frames arranged in three operons responsible for regulation, biosynthesis and immunity of SA-FF22 . This region is organized similarly to the Lactococcus lactis lacticin 481 region, however, unlike lacticin 481, a two-component regulatory system is essential for SA-FF22 production . Located immediately downstream of the scn region is a putative transposase gene, the presence of which supports earlier data that indicated a mobile nature to this region.

Nat Biotechnol, 1999 Jun, 17(6), 588 - 92
Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering; Hols P et al.; We report the engineering of Lactococcus lactis to produce the amino acid L-alanine . The primary end product of sugar metabolism in wild-type L . lactis is lactate (homolactic fermentation) . The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH) . We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O) . Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation) . Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

Arch Microbiol, 1999 Apr, 171(5), 343 - 9
Identification of biosynthetic intermediates of the extracellular polysaccharide viilian in Lactococcus lactis subspecies cremoris SBT 0495; Oba T et al.; Lactococcus lactis subspecies cremoris SBT 0495 produces the phosphopolysaccharide viilian, which consists of the repeating unit beta-D-glucosyl-(1-->4)-(alpha-L-rhamnosyl-(1-->2))-(alpha-D-galac tose-1- phosphoryl-(-->3)-beta-galactosyl-(1-->4)-beta-D-glucose . A lipid extract was prepared from cells in the late exponential phase of growth and was hydrolyzed by hydrochloric acid under mild conditions to split lipid-linked intermediates in the extract into lipid and sugar moieties . Both moieties were purified by chromatographic techniques and were characterized to identify intermediates of the viilian biosynthetic pathway . A polyisoprenoid isolated from the chloroform-soluble fraction of the hydrolyzed lipid extract was identified by mass spectrometry as undecaprenol . Saccharides isolated from the water-soluble fraction of the hydrolyzed lipid extract by anion-exchange chromatography, were characterized by glycosidic linkage analysis to discriminate sugar moieties of intermediates of viilian biosynthesis from compounds liberated from cell wall components . Some oligosaccharide analogues contain a glycerol residue, suggesting that these are fragments of glycosylglycerides and/or lipoteichoic acid . Three fragments were identified to be glucose, galactosyl-(1-->4)-glucose, and rhamnosyl-(1-->2)-galactosyl-(1-->4)-glucose, which are in agreement with the structure of the repeating unit of viilian . These saccharides most likely represent the first three steps of the sequential assembly of the repeating unit of the undecaprenol assembly.

Microbiology, 1999 May, 145 ( Pt 5), 1227 - 33
Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity; Ra R et al.; The lantibiotic nisin is produced by several strains of Lactococcus lactis subsp . lactis . The chromosomally located gene cluster nisABTCIPRKFEG is required for biosynthesis, development of immunity, and regulation of gene expression . Inframe deletions in the nisB and nisT genes, and disruption of nisC by plasmid integration, eliminated nisin production and resulted in a strongly reduced level of immunity of the strains . The transcription of two nisin operons was inactivated in these mutant strains, but could be restored by addition of small amounts of nisin to growing cultures . The immunity levels of the mutants were also raised by adding nisin to growing cultures, albeit not to wild-type level . A strain with an in-frame deletion in the nisI gene was still able to produce active nisin, but the production and immunity levels were markedly lower . By measuring immunity levels of the knock-out strains and determining mRNA levels, it is concluded that NisI has an important function for nisin immunity and must cooperate with nisFEG-encoded proteins to provide a high level of immunity . Maximal immunity could not be obtained in the mutant strains, probably because the wild-type transcription levels from nisA and nisF promoters are not reached when essential nis genes are disrupted . Using Southern hybridization with a consensus promoter probe, no other DNA sequences similar to the nisA and nisF promoters could be detected, indicating that these two elements are probably the only ones in the chromosome regulated by nisin and are thus the only ones involved in the regulation of producer immunity.

J Dairy Res, 1999 May, 66(2), 257 - 70
Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp . cremoris AM2; McDonnell M et al.; A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp . cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose . The purified aminopeptidase was found to have a native M(r) of 50,000-55,000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE . Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory . The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreitol were included in the purification buffers . The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus . While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr . This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly . The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.

Braz J Med Biol Res, 1999 Feb, 32(2), 191 - 8
Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system; Langella P et al.; Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms . Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles . To develop such live vaccines, a better control of protein secretion is required . We developed an efficient secretion system in the model LAB, Lactococcus lactis . Staphylococcal nuclease (Nuc) was used as the reporter protein . We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector . The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed . Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression . Secretion efficiency (SE) of Nuc in L . lactis was low, i.e., only 60% of the synthesized Nuc was secreted . Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc . On the basis of these results, we developed a secretion system and we applied it to the construction of an L . lactis strain which secretes a bovine coronavirus (BCV) epitopeprotein fusion (BCV-Nuc) . BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies . Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Appl Environ Microbiol, 1999 Jun, 65(6), 2287 - 93
Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis; O'Sullivan E et al.; The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp . cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo) . A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both . Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol . The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi . The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed . Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established . Each pHi below 6 . 6 was characterized by unique levels of ATR, H+-ATPase, and ATP . High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid . We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.

J Bacteriol, 1999 May, 181(10), 3277 - 80
Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis; le Coq D et al.; The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon . A B . subtilis ytvP mutant was auxotrophic for histidine . The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B . subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway . HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity . These observations demonstrated that HolPase is encoded by ytvP in B . subtilis and led us to rename this gene hisJ . Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L . lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.

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