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