FEMS Microbiol Lett, 1996 Oct 15, 144(1), 89 - 93 Evidence for a role of NisT in transport of the lantibiotic nisin produced by Lactococcus lactis N8; Qiao M et al.; The biosynthesis, immunity and regulation of nisin, a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis, is encoded by two gene clusters, nisA/ZBTCIPRK and nisFEG . The mutant strain LAC46 with a deletion in the translocator gene nisT could not secrete nisin but nisin activity was detected from cell lysates . The nisT mutation was complemented by a NisT-expression plasmid resulting in restored capacity to secrete nisin . These results demonstrate that NisT is the transport protein dedicated to translocate nisin and that dehydration and lanthionine formation in nisin maturation can occur independently of transport. J Biol Chem, 1996 Oct 11, 271(41), 25582 - 9 Membrane topology of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae . Evidence for a new structural class of secondary transporters; van Geest M et al.; The predicted secondary structure model of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) presents the 12-transmembrane helix motif observed for many secondary transporters . Biochemical evidence presented in this paper is not consistent with this model . N-terminal and C-terminal fusions of CitS with the biotin acceptor domain of the oxaloacetate decarboxylase of K . pneumoniae catalyze citrate transport, showing the correct folding of the CitS part of the fusion proteins in the membrane . Proteolysis experiments with these fusion proteins revealed that the N terminus of CitS is located in the cytoplasm, while the C terminus faces the periplasm . The membrane topology was studied further by constructing a set of 20 different fusions of N-terminal fragments of the citrate transporter with the reporter enzyme alkaline phosphatase (CitS-PhoA fusions) . Most fusion points were selected in hydrophilic areas flanking the putative transmembrane-spanning domains in CitS that are predicted from the hydropathy profile of the primary sequence . The alkaline phosphatase activities of cells expressing the CitS-PhoA fusions suggest that the polypeptide traverses the membrane nine times and that the C-terminal half of the protein is characterized by two large hydrophobic periplasmic loops and two large hydrophilic cytoplasmic loops . CitS belongs to the family of the 2-hydroxycarboxylate transporters in which also the citrate carriers, CitPs, of lactic acid bacteria and the malate transporter, MleP, of Lactococcus lactis are found . Since the hydrophobicity profile of CitS is very similar to the hydrophobicity profiles of CitP and MleP, it is most likely that the new structural motif of nine transmembrane segments is shared within this new transporter family. Gene, 1996 Oct 3, 174(2), 259 - 63 Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain; Ward LJ et al.; The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence . The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp . dextranicum NZDRI 2218 . The DNA sequence of this ISS1-like element (IS1297) and the Le . mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements . No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site . IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1 . An overlapping ORF of 174 bp in the same orientation was also present . A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297 . IS1297 was shown to hybridize with other dairy Leuconostoc strains . This is the first sequence of an ISS1-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1. Microbiology, 1996 Oct, 142 ( Pt 10), 2825 - 30 Mutational analysis and chemical modification of Cys24 of lactococcin B, a bacteriocin produced by Lactococcus lactis; Venema K et al.; Using site-directed mutagenesis the single cysteine residue at position 24 of lactococcin B was replaced by all other possible amino acids . Most of these mutant molecules retained bacteriocin activity, with the exception of those in which cysteine was replaced by a positively charged amino acid . This would seem to be in agreement with the authors' earlier observation that treatment of the wild-type molecule with HgCl2 resulted in its inactivation . The factor that causes inactivation of lactococcin B seems to be the introduction of a positive charge at position 24 by HgCl2 rather than oxidation of this residue, as treatment of the bacteriocin with other oxidative chemicals did not interfere with the ability of lactococcin B to dissipate the membrane potential of sensitive cells . Results are also reported which imply that inactive lactococcin B can still bind to its receptor . It can be replaced by an active bacteriocin molecule, resulting in dissipation of the membrane potential. Antonie Van Leeuwenhoek, 1996 Oct, 70(2-4), 253 - 67 Physiology of pyruvate metabolism in Lactococcus lactis; Cocaign-Bousquet M et al.; Lactococcus lactis, a homofermentative lactic acid bacterium, has been studied extensively over several decades to obtain sometimes conflicting concepts relating to the growth behaviour . In this review some of the data will be examined with respect to pyruvate metabolism . It will be demonstrated that the metabolic transformation of pyruvate can be predicted if the growth-limiting constraints are adequately established . In general lactate remains the major product under conditions in which sugar metabolism via a homolactic fermentation can satisfy the energy requirements necessary to assimilate anabolic substrates from the medium . In contrast, alternative pathways are involved when this energy supply becomes limiting or when the normal pathways can no longer maintain balanced carbon flux . Pyruvate occupies an important position within the metabolic network of L . lactis and the control of pyruvate distribution within the various pathways is subject to co-ordinated regulation by both gene expression mechanisms and allosteric modulation of enzyme activity. Antonie Van Leeuwenhoek, 1996 Oct, 70(2-4), 243 - 51 Lactococcus lactis and stress; Rallu F et al.; It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory . Natural stresses like starvation and acidity are generated by cell growth itself . Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment . It is now clear that defense mechanisms to withstand different stresses must be present in all organisms . The exploration of stress responses in lactic acid bacteria has just begun . Several stress response genes have been revealed through homologies with known genes in other organisms . While stress response genes appear to be highly conserved, however, their regulation may not be . Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits . The first part of this report addresses the available information on stress response in Lactococcus lactis . Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death . The second part of this report will focus on progress made in acid stress response, particularly in L . lactis and on factors which may affect its regulation . Acid tolerance is presently under study in L . lactis . Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5 . Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes . These results show that L . lactis possesses an inducible response to acid stress in exponential phase . To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37 degrees C for transposition insertional mutants which were able to survive . About thirty mutants resistant to low pH conditions were characterized . The interrupted genes were identified by sequence homology with known genes . One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH . Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis . Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10668 - 72 Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1; van Veen HW et al.; Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells . Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1 . LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site . LmrA is similar to each of the two halves of MDR1 and may function as a homodimer . The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding . LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil . As LmrA can be functionally expressed in E . coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters. Appl Environ Microbiol, 1996 Oct, 62(10), 3662 - 7 Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin; de Ruyter PG et al.; The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes . In the nisin-producing L . lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached . Expression of the gusA gene was also studied in L . lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L . lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome . In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium . Without nisin, no beta-glucuronidase production was observed . To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene . Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin . In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold . The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800 . This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein . These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins. J Biol Chem, 1996 Sep 27, 271(39), 24123 - 8 Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP; Bolhuis H et al.; The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli . The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E . coli . LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-{4-(trimethylamino)phenyl}-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism . Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs . Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer . This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium . This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA. Plasmid, 1996 Sep, 36(2), 125 - 41 Molecular analysis of the replication origin of the Lactococcus lactis plasmid pCJ305; Foley S et al.; The replication origin region, ori, of the Lactococcus lactis subsp . lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2 . These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (RepB) essential for plasmid replication . Gel retardation assays, using lactococcal crude cell extracts in which RepB was overproduced, were used to demonstrate that the replication protein interacts with DNA sequences within the origin region . IR1 was identified as a RepB binding site . The -35 region of the repB promoter is contained within the loop of the potential stem-loop structure of IR1, suggesting autoregulation of repB . The pCI305 RepB failed to interact with DNA sequences within the minimal replicons of nine other members of the pCI305 family of plasmids and it was concluded that this DNA-protein interaction was replicon specific . In vivo studies were performed to determine the role of the three-and-one-half copies of the 22-bp sequences . When this sequence was provided in trans on a compatible vector, it resulted in the loss of pCI305 from the cell population (incompatibility). Int J Food Microbiol, 1996 Sep, 32(1-2), 27 - 34 Production of antifungal substance by Lactococcus lactis subsp . lactis CHD-28.3; Roy U et al.; Six of the 2100 colonies of lactic acid bacteria isolated from 4 month old Cheddar cheese and raw buffalo milk showed antifungal activity against Aspergillus flavus IARI when tested by the well agar diffusion assay on Potato Dextrose Agar containing 0.1% Triton X-100 . Out of these, the most promising isolate having a broad spectrum of antifungal activity including Aspergillus flavus IARI, A . flavus NCIM 555, A . parasiticus NCIM 898 and Fusarium spp . was identified as Lactococcus lactis subsp . lactis CHD-28.3 . Among the mold cultures used as indicator strains, the most sensitive towards antifungal substance produced by the test culture was A . flavus IARI . The cell-free supernatant of the test culture in Elliker's broth adjusted to pH 6.8 produced an inhibition zone of 15-19 mm against A . flavus IARI, A . flavus NCIM555 and A . parasiticus NCIM898 . The isolate when grown at 30 degrees C for 48 h in Elliker's broth showed optimum antifungal activity . When the supernatant was neutralized to pH 7.0 or 7.5, there was little reduction in activity . However, after enzymatic treatment of supernatant with chymotrypsin, trypsin and pronase E, the antifungal activity disappeared which indicated the proteinaceous nature of the antifungal substance. Microbiology, 1996 Sep, 142 ( Pt 9), 2385 - 92 Molecular analysis of the regulation of nisin immunity; Dodd HM et al.; The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue . The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism . This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis . Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component . Changes throughout the molecule also affected its induction capacity . The production of certain variant nisins by engineered lactococcal strains is reduced in parallel with the strains' immunity to nisin . This can be attributed to inefficient induction by the variant molecule . Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains. FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 295 - 9 Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp . lactis biovar . diacetylactis S94; Prevots F et al.; A gene which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis ssp . lactis biovar . diacetylactis S94 . This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I of homology) and total resistance to the small isometric-headed bacteriophage phi 59 (group III of homology) . The cloned gene is predicted to encode a polypeptide of 346 amino acid residues with a deduced molecular mass of 41 455 Da . No homology with any previously described genes was found . A probe was used to determine the presence of this gene in two strains on 31 tested. Arch Biochem Biophys, 1996 Sep 1, 333(1), 121 - 6 Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin); Lian W et al.; A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I . Mierau et al., J . Bacteriol . 175, 2087-2096, 1993) . The gene for the neutral endopeptidase from L . lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus . The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure . A number of peptides were studied as substrates for the enzyme . The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin . In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP . Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates . As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine . Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine . This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme . Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme . These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme. Appl Environ Microbiol, 1996 Sep, 62(9), 3494 - 8 Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp . cremoris W56; Nyengaard NR et al.; The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp . cremoris W56 and sequenced . The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa) . A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L . lactis. Appl Environ Microbiol, 1996 Sep, 62(9), 3075 - 82 AbiG, a genotypically novel abortive infection mechanism encoded by plasmid pCI750 of Lactococcus lactis subsp . cremoris UC653; O'Connor L et al.; AbiG is an abortive infection (Abi) mechanism encoded by the conjugative plasmid pCI750 originally isolated from Lactococcus lactis subsp . cremoris UC653 . Insensitivity conferred by this Abi manifested itself as complete resistance to phi 712 (936 phage species) with only partial resistance to phi c2 (c2 species) . The mechanism did not inhibit phage DNA replication . The smallest subclone of pCI750 which expressed the Abi phenotype contained a 3.5-kb insert which encoded two potential open reading frames . abiGi (750 bp) and abiGii (1,194 bp) were separated by 2 bp and appeared to share a single promoter upstream of abiGi . These open reading frames showed no significant homology to sequences of either the DNA or protein databases; however, they did exhibit the typical low G+C content (29 and 27%, respectively) characteristic of lactococcal abi genes . In fact, the G+C content of a 7.0-kb fragment incorporating the abiG locus was 30%, which may suggest horizontal gene transfer from a species of low G+C content . In this context, it is notable that remnants of IS elements were observed throughout this 7.0-kb region. J Bacteriol, 1996 Sep, 178(17), 5164 - 73 A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1; Christiansen B et al.; The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp . cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B . Christiansen, M . G . Johnsen, E . Stenby, F . K . Vogensen, and K . Hammer, J . Bacteriol . 176:1069-1076, 1994) . The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP . This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3 By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process . Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration . Orf1 contains 485 amino acids and is located just upstream of attP . The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end . Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages . The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency. Curr Microbiol, 1996 Sep, 33(3), 194 - 9 The Lactic Acid Stress Response of Lactococcus lactis subsp . lactis Hartke A, Bouche S, Giard JC, Benachour A, Boutibonnes P, Auffray Y. The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp . lactis has been studied . A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5 . Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells . Among these are the major heat shock proteins DnaK and GroEL . In conjunction with a previous report (Hartke et al . 1994), the results establish that L . lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons . Both systems are independent of de novo protein synthesis. EMBO J, 1996 Aug 15, 15(16), 4239 - 45 Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane; Bolhuis H et al.; Lactococcus lactis possesses an ATP-dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P-glycoprotein . One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds . It has been suggested that P-glycoprotein removes drugs directly from the membrane . Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF-AM and cationic diphenylhexatriene (DPH) derivatives from the membrane . The non-fluorescent probe BCECF-AM can be converted intracellularly into its fluorescent derivative, BCECF, by non-specific esterase activities . The development of fluorescence was decreased upon energization of the cells . These and kinetic studies showed that BCECF-AM is actively extruded from the membrane before it can be hydrolysed intracellularly . The increase in fluorescence intensity due to the distribution of TMA-DPH into the phospholipid bilayer is a biphasic process . This behaviour reflects the fast entry of TMA-DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane . The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet . Taken together, these results demonstrate that the lactococcal MDR transporter functions as a 'hydrophobic vacuum cleaner', expelling drugs from the inner leaflet of the lipid bilayer . Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters. FEBS Lett, 1996 Aug 12, 391(3), 317 - 22 The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges; van den Hooven HW et al.; The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp . lactis . This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine . Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin . Here we report the first complete structure of this type of lantibiotic . The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26. Int J Food Microbiol, 1996 Aug, 31(1-3), 99 - 106 Induction of IFN-gamma and IL-1 alpha production in macrophages stimulated with phosphopolysaccharide produced by Lactococcus lactis ssp . cremoris; Kitazawa H et al.; The induction of interferon (IFN) and interleukin-1 (IL-1) production in murine macrophages by a phosphopolysaccharide, produced by a dairy lactic acid bacteria, Lactococcus lactis ssp . cremoris, was investigated . When the phosphopolysaccharide was added into macrophage cultures at concentrations from 1 to 200 micrograms/ml, substantial IFN titers (6.2-79.2 IU/ml) were detected . Using the reverse transcription-polymerase chain reaction (RT-PCR), the expression of mRNA encoding IFN-gamma was verified in spleen macrophage cultures . Macrophages stimulated with the phosphopolysaccharide also produced IL-1 alpha at a concentration of 50 micrograms/ml . This study showed for the first time that phosphopolysaccharide derived from a dairy lactic acid bacterium can induce IFN-gamma and IL-1 alpha production in macrophages . These findings strongly suggest that the phosphopolysaccharide is a type of 'biological response modifier' and the fermented dairy foods containing Lactococcus lactis ssp . cremoris can be designated as a physiologically functional food. J Bacteriol, 1996 Aug, 178(16), 5005 - 12 Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis including a novel gene, pyrK, involved in pyrimidine biosynthesis; Andersen PS et al.; Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon in Lactococcus lactis . Two of the genes are the well-known pyr genes pyrDb and pyrF, encoding dihydroorotate dehydrogenase and orotidine monophosphate decarboxylase, respectively . The third gene encodes a protein which was shown to be necessary for the activity of the pyrDb-encoded dihydroorotate dehydrogenase; we propose to name the gene pyrK . The pyrK-encoded protein is homologous to a number of proteins which are involved in electron transfer . The lactococcal pyrKDbF operon is highly homologous to the corresponding part of the much-larger pyr operon of Bacillus subtilis . orf2, the pyrK homolog in B . subtilis, has also been shown to be necessary for pyrimidine biosynthesis (A . E . Kahler and R . L . Switzer, J . Bacteriol . 178:5013-5016, 1996) . Four genes adjacent to the operon, i.e., orfE, orfA, orfC, and gidB, were also sequenced . Three of these were excluded as members of the pyr operon by insertional analysis (orfA) or by their opposite direction of transcription (orfE and gidB) . orfC, however, seems to be the distal gene in the pyrKDbF-orfC operon. Infect Immun, 1996 Aug, 64(8), 3401 - 6 Characterization of PepB, a group B streptococcal oligopeptidase; Lin B et al.; Group B streptococci were recently reported to possess a cell-associated collagenase . Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-{2-furyl}acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase . We cloned and sequenced the gene for the enzyme (pepB) . The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases . The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin. Appl Environ Microbiol, 1996 Aug, 62(8), 2966 - 9 Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5; Chan WC et al.; A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized . It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis . These observations, which parallel those of W . Liu and J . N . Hansen (Appl . Environ . Microbiol . 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics. Appl Environ Microbiol, 1996 Aug, 62(8), 2692 - 700 Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene; Kawai S et al.; Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined . The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed . The enzymatic properties of the S . bovis malic enzyme were almost identical to those of other malic enzymes previously reported . However, we found that the S . bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate . The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity . A sequence analysis of the cloned fragment revealed the presence of two open reading frames that were 1,299 and 1,170 nucleotides long . The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis. FEBS Lett, 1996 Jul 22, 390(2), 129 - 32 Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin; Chan WC et al.; The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166 . These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity . Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease . There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action . Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin . The fragment nisin1-12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin . These results are discussed in terms of current models for the mechanism of action of nisin. Mol Microbiol, 1996 Jul, 21(1), 123 - 31 Fate of peptides in peptidase mutants of Lactococcus lactis; Kunji ER et al.; The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN, pepC, pepO, pepX and pepT were deleted . Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu-Gly-Gly, Gly-Phe-Leu, Leu-Gly-Pro, Ala-Pro-Leu and Gly-Leu-Gly-Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu . In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation . The mutant lacking all five peptidases could still grow on Gly-Leu and Tyr-Gly-Gly-Phe-Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO-like endopeptidase . These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides . In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala-Pro-Leu . The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen. Mol Microbiol, 1996 Jul, 21(1), 45 - 53 Splicing of a group II intron in a functional transfer gene of Lactococcus lactis; Shearman C et al.; A chromosomally located sex factor that controls conjugation in Lactococcus lactis 712 has been cloned and sequenced, leading to the discovery of an open reading frame with homology to the maturases of group II self-splicing introns . Reverse transcriptase polymerase chain reaction amplification was used to demonstrate that the intron was spliced out of mRNA in vivo, and sequence analysis revealed the site of splicing . The intron was inserted within a sex-factor gene which encodes a protein with homology to proteins involved in rolling-circle DNA replication . Gene-disruption experiments were used to demonstrate that this mobA gene was essential for sex-factor transfer and this suggests that intron splicing is a necessary part of the conjugation process . The sequence of the intron was modelled to produce a secondary structure that exhibited several features characteristic of the IIA subgroup . Here we report the characterization of a new group II intron in the Gram-positive bacterium L . lactis and demonstrate for the first time in bacteria both splicing in vivo and an active role for the gene carrying the intron. Int J Syst Bacteriol, 1996 Jul, 46(3), 664 - 8 Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffalos with subclinical mastitis and confirmation of L . garvieae as a senior subjective synonym of Enterococcus seriolicida; Teixeira LM et al.; During a survey of bacterial agents that cause subclinical mastitis in water buffalos, we isolated several strains of gram-positive cocci that appeared to be enterococci except that they grew very slowly at 45 degrees C and grew slowly in broth containing 6.5% NaCl . On the basis of the results of conventional physiologic tests, these strains were identified as Enterococcus durans . However, none of the strains reacted with the AccuProbe Enterococcus genetic probe . The whole-cell protein profiles of these organisms were compared with the profiles of Enterococcus and Lactococcus reference strains . apart from minor quantitative differences, the mastitis isolates had indistinguishable protein profiles that were similar to the profiles of the Lactococcus garvieae and Enterococcus seriolicida type strains . The results of DNA relatedness studies performed by using the hydroxyapatite method at 55 and 70 degrees C indicated that all of the mastitis isolates were related to the type strain of L . garvieae at the species level, despite the fact that they exhibited several uncommon phenotypic characteristics (growth at 45 degrees C, growth in broth containing 6.5% NaCl, and failure to produce acid from mannitol and sucrose) . The high levels of DNA relatedness between strains of L . garvieae is a senior synonym of E . seriolicida, L . garvieae should be retained as the species name and strain ATCC 43921 should remain the type strain of this species. Appl Environ Microbiol, 1996 Jul, 62(7), 2641 - 3 Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis; Swindell SR et al.; Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria . Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate . This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363 . Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination . The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated . Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl. Appl Environ Microbiol, 1996 Jul, 62(7), 2636 - 40 Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains; Goupil N et al.; Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen . L . lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene . We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium . Industrial dairy strains of L . lactis subsp . lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized . Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk . Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains. Microbiology, 1996 Jul, 142 ( Pt 7), 1685 - 91 Analysis of heat shock gene expression in Lactococcus lactis MG1363; Arnau J et al.; The induction of the heat shock response in Lactococcus lactis subsp . cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation . Cloning of the dnaJ and groEL homologues was carried out . Northern blot analysis showed a similar induction pattern for dnaK, dnaJ and groELS after transfer from 30 degrees C to 43 degrees C when MG1363 was grown in defined medium . The dnaK gene showed a 100-fold induction level 15 min after temperature shifting . Induction of the first two genes in the dnaK operon, orf1 and grpE, resembled the pattern observed for the above genes, although maximum induction was observed earlier for orf1 and grpE . Novel transcript sizes were detected in heat-shocked cells . The induction kinetics observed for ftsH suggested a different regulation for this gene . Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L . lactis MG1363 is presented . A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock. Eur J Biochem, 1996 Jul 1, 239(1), 156 - 64 Interaction of the lantibiotic nisin with membranes revealed by fluorescence quenching of an introduced tryptophan; Martin I et al.; Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp . lactis . The target for nisin action is the cytoplasmic membrane of gram-positive bacteria . To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue . Activity of the site-directed variant containing tryptophan was established to be similar to that of the wild-type peptide . Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment . Quenching experiments with aqueous and membrane-restricted quenchers (iodide and spin-labelled lipids, respectively) both confirmed a non-aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity . The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles . This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes. J Bacteriol, 1996 Jul, 178(13), 3689 - 94 Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614; Madsen SM et al.; Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced . These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms . The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine . Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene . A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified . A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L . lactis promoter probe vector . In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription. Curr Microbiol, 1996 Jul, 33(1), 35 - 9 Cloning vectors for lactococci based on a plasmid encoding resistance to cadmium; Liu CQ et al.; An 8.8-kb plasmid (pND302) was identified in Lactococcus lacti spp lactis M71 which encodes cadmium resistance (CdR) . Most of the commercial lactococcal strains tested were sensitive to cadmium . Therefore, CdR should provide a useful selectable marker for constructing cloning vectors in lactococci . pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning . Two E . coli/L lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E . coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (NisR) of lactococcal origin into the EcoRI site of pND302, separately . The E . coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing NisR and CdR, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning . Both pND302 and pND625 can be transformed by electroporation into L . lactis LMO230 at 10(3)/micrograms DNA and maintained stably in LMO230 . The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. FEMS Microbiol Lett, 1996 Jun 15, 140(1), 23 - 8 Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling; Sheehan MM et al.; An active chimeric cell wall lytic enzyme (Tsl) has been constructed by fusing the region coding for the N-terminal half of the lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain of the major pneumococcal autolysin . The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumococcal cell walls . This experimental approach demonstrated that the Tuc2009 lysin possesses a modular structure and further supports the hypothesis that many cell wall lytic enzymes have evolved by the fusion of preexisting catalytic and peptidoglycan-binding domains. FEMS Immunol Med Microbiol, 1996 Jun, 14(2-3), 167 - 77 Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis; Norton PM et al.; The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052) . Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies . When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein. J Appl Bacteriol, 1996 Jun, 80(6), 626 - 34 Regulation of the nisin operons in Lactococcus lactis N8; Qiao M et al.; The antibiotic peptide nisin produced by Lactococcus lactis is used as a food preservative due to its activity against spores and vegetative cells of Gram-positive bacteria . The post-translational maturation of this secreted peptide includes dehydration of serine and threonine residues, lanthionine formation and a proteolytic processing of 23 amino acids from the N-terminus . Mutations in the nisZ, nisB and nisP genes of the biosynthetic nisZBTCIPRK nisin operon were made by gene replacement or integration of a plasmid . The mutations caused a drastic decrease of the transcription from the promoters upstream of the nisZBTCIPRK and nisFEG operons resulting in loss of nisin production and nisin immunity . The transcription of the nisin operons and nisin immunity could be partially restored by adding nisin to the growth medium of the cells . Nisin induction of the mutant strains also increased the level of the putative immunity NisI protein . These results showed that the nisZBTCIPRK operon is positively autoregulated and that the nisFEG operon is in the same regulon. J Bacteriol, 1996 Jun, 178(12), 3557 - 63 Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr; Ye JJ et al.; By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars . Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase . Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L . lactis or B . subtilis or in intact cells of B . subtilis . Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion. J Bacteriol, 1996 Jun, 178(12), 3531 - 8 Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci; Mills DA et al.; Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria} group II intron . Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01 . Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases . Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria . Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype . These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria. J Bacteriol, 1996 Jun, 178(12), 3434 - 9 Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis; de Ruyter PG et al.; The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA) . Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L . lactis NZ9700, were identified . The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0 . P . Kuipers, M . M . Beerthuyzen, P . G . G . A . de Ruyter, E . J . Luesink, and W . M . de Vos, J . Biol . Chem . 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated . The nisR promoter was shown to direct nisin-independent gusA expression in L . lactis MG 1363, which is a nisin-transposon- and plasmid-free strain . L . lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin . A similar regulation was found in L . lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster . In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin . These results show that, like the nisA promoter, the nisF promoter is nisin inducible . The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control. J Biol Chem, 1996 May 24, 271(21), 12294 - 301 Biosynthesis of lantibiotic nisin . Posttranslational modification of its prepeptide occurs at a multimeric membrane-associated lanthionine synthetase complex; Siegers K et al.; The lantibiotic nisin of Lactococcus lactis is matured from a ribosomally synthesized prepeptide by postranslational modification . Genetic and biochemical evidence suggests that genes nisB and nisC of the nisin gene cluster encode proteins necessary for prenisin modification . Inactivation of both genes resulted in complete loss of nisin production . The preparation of membrane vesicles revealed that NisB and NisC are attached to the cellular membrane, and co-immunoprecipitation experiments showed that they are associated with each other . By using the yeast two-hybrid system, which is a highly sensitive method to unravel protein-protein interactions, we could show that the nisin prepeptide physically interacts with the NisC protein, suggesting that NisC contains a binding site for prenisin . This was also confirmed by co-immunoprecipitation of the NisC protein and the NisA prepeptide by antibodies directed against the leader sequence of the nisin prepeptide . The two-hybrid analysis also confirmed the interaction between NisB and NisC as well as the interaction between NisB and NisC as well as the interaction between NisC and the NisT ABC transporter . A minor interaction was also indicated between prenisin and the NisB protein . Furthermore, the two-hybrid investigations also revealed that at least two molecules of NisC and two molecules of NisT are part of the modification and transport complex . Our results suggest that lantibiotic maturation and secretion occur at a membrane-associated multimeric lanthionine synthetase complex consisting of proteins NisB, NisC, and the ABC transporter molecules NisT. Plasmid, 1996 May, 35(3), 145 - 55 Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation; Yu W et al.; Fast milk-coagulating (Fmc+) strains of lactococci are known to segregate slow milk-coagulating (Fmc-) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA . It was found that the Fmc- phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system . In Lactococcus lactis subsp . lactis (L . lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430 . In Lactococcus lactis subsp . cremoris SK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system . The Lac plasmid in L . lactis C2 encoded both the Prt and Opp systems . Fmc- derivatives of L . lactis C2 were missing the prt genes and had Opp integrated into the chromosome, possibly due to transposition events . Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc+ phenotype. Protein Sci, 1996 May, 5(5), 852 - 6 Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme; Nielsen FS et al.; Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms . In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A . In solution, the enzyme is bright yellow . It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each . The enzyme shows optimal function in the pH range 7.5-9.0 . It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive . The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5 . The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments . The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees . A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2% . Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes. J Clin Microbiol, 1996 May, 34(5), 1296 - 8 Antimicrobial susceptibilities of Lactococcus lactis and Lactococcus garvieae and a proposed method to discriminate between them; Elliott JA et al.; The MICs of antimicrobial agents contained in the SCEPTOR Streptococcus MIC panels (Becton Dickinson Microbiology Systems) were determined for Lactococcus lactis, L . garvieae, and unknown Lactococcus species . Several isolates had reduced susceptibilities to many of the antimicrobial agents contained in the panel . For L . garvieae, the MICs of penicillin and, possibly, cephalothin were higher than for L . lactis, and unlike L . lactis, L . garvieae was resistant to clindamycin, indicating that knowledge of the Lactococcus species causing an infection might influence the choice of antimicrobial therapy . Susceptibility to clindamycin can also be used to differentiate between L . lactis and L . garvieae. Microbiology, 1996 May, 142 ( Pt 5), 1281 - 8 Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactoccocus lactis N8; Ra SR et al.; Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria . Northern blots and RT/PCR analysis of the nisin-producing strain N8 revealed that the nisZBTCIPRKFEG gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTCIPRK and nisFEG . The promoter of the nisFEB operon was mapped . The -35 to -1 region upstream of the transcription start of the nisFEG promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene . In contrast to earlier reports, nisin was found to be secreted during the early stages of growth was well as later in the growth cycle . The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 5.5 . In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost . Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin . Nisin induction of the mutant strains also resulted in an increased amount of the NisI protein and an increase in the level of immunity . Induction using higher concentrations of nisin yielded a higher level of immunity . These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules. Appl Environ Microbiol, 1996 May, 62(5), 1799 - 802 Variacin, a new lanthionine-containing bacteriocin produced by Micrococcus varians: comparison to lacticin 481 of Lactococcus lactis; Pridmore D et al.; A new lanthionine-containing bacteriocin, variacin, displaying a broad host range of inhibition against gram-positive food spoilage bacteria, has been identified from two strains of Micrococcus varians isolated from meat fermentations . The new bacteriocin was purified, and its amino-terminal end and total amino acid composition were determined . The structural gene was isolated and analyzed . Variacin is resistant to heat and pH conditions from 2 to 10 . Its primary sequence shows significant homology to lacticin 481 to Lactococcus lactis, which is more pronounced for the probacteriocin than for the leader sequence . Variacin, like lacticin 481, contains lanthionine and beta-methyllanthionine residues, but its leader sequence clearly resembles nonlantibiotic leader sequences . In particular, the prepeptide contains glycine residues at positions -1 and -2 of the processing site. Appl Environ Microbiol, 1996 May, 62(5), 1689 - 92 The genes for secretion and maturation of lactococcins are located on the chromosome of Lactococcus lactis IL1403; Venema K et al.; Southern hybridization and PCR analysis were used to show that Lactococcus lactis IL1403, a plasmid-free strain that does not produce bacteriocin, contains genes on its chromosome that are highly homologous to lcnC and lcnD and encode the lactococcin secretion and maturation system . The lcnC and lcnD homologs on the chromosome of IL1403 were interrupted independently by Campbell-type integrations . Both insertion mutants were unable to secrete active lactococcin . Part of the chromosomal lcnC gene was cloned and sequenced . Only a few nucleotide substitutions occurred, compared with the plasmid-encoded lcnC gene, and these did not lead to changes in the deduced amino acid sequence . No genes homologous to those for lactococcin A, B, or M could be detected in IL1403, and the strain does not produce bacteriocin activity. J Bacteriol, 1996 May, 178(10), 2794 - 803 Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk; Mierau I et al.; To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed . In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN . Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated . The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain . In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell . No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed . Also, the expression of the peptidases still present in the various mutants was not detectably affected . Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides . These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins . Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components. Gene, 1996 Apr 17, 170(1), 151 - 2 Sequence of a Lactococcus lactis DNA fragment homologous to the recF gene of Bacillus subtilis; MacCormick CA et al.; The recF gene of Lactococcus lactis ATCC 7962 is located 3 kb downstream from the lacZ gene and is transcribed in the opposite orientation . The recF gene is immediately preceded by a 121-codon ORF, and both recF and orf121 may be transcribed from the same promoter . The deduced RecF amino-acid sequence shows high homology to that of the Bacillus subtilis and Streptococcus pyogenes RecF proteins. Virology, 1996 Apr 15, 218(2), 306 - 15 A genomic region of lactococcal temperate bacteriophage TP901-1 encoding major virion proteins; Johnsen MG et al.; Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome . This genomic region was sequenced . The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure . Upstream of each ORF, except ORF b2 and ORF x, potential ribosome-binding sites were found, suggesting independent translation . However, coupled translation is suggested for ORF x and as a possibility for ORF b3 and ORF c2, which have ribosome-binding sites located more distant from their start codons . ORF b2 may be translationally fused with mhp at a low frequency . The mhp and mtp genes are transcribed as a 3.7-kb mRNA with at least six additional ORFs . The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs. J Biol Chem, 1996 Apr 12, 271(15), 8779 - 85 The ribonucleotide reductase system of Lactococcus lactis . Characterization of an NrdEF enzyme and a new electron transport protein; Jordan A et al.; Escherichia coli contains the genetic information for three separate ribonucleotide reductases . Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen . The NrdAB enzyme is physiologically active . The function of the nrdEF gene is not known . The third enzyme (class III), coded by nrdDG, operates during anaerobiosis . The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes . Surprisingly, an nrdD- mutant of L . lactis grew well under standard anaerobic growth conditions . The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein . The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein . The same gene organization is present in E . coli . We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E . coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib) . The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes . The two classes also differ in their allosteric regulation by dATP. Lett Appl Microbiol, 1996 Apr, 22(4), 307 - 10 Tween 80 effect on bacteriocin synthesis by Lactococcus lactis subsp . cremoris J46; Huot E et al.; Sorbitan polyoxyethylene monooleate (Tween 80) suppressed bacteriocin cell adhesion . Within the range 0-1% (v/v), there was an increase in bacteriocin production in regulated (pH 5.5 or 6.0) batch cultures with increasing Tween 80 concentration . For example, at pH 5.5 and in the presence of 1% Tween 80, bacteriocin production was about fourfold higher than in its absence . However, further increase in Tween 80 concentration did not result in a significant modification of the bacteriocin titre . It was shown that the increase was not linked to an activating effect of the surfactant on preformed enzyme, to an increase of bacteriocin availability or to a sensitization of the target cell, demonstrating that Tween 80 promoted bacteriocin production. Appl Environ Microbiol, 1996 Apr, 62(4), 1481 - 6 Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector; Wyckoff HA et al.; A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized . The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305 . Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein . No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range . A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon. Appl Environ Microbiol, 1996 Apr, 62(4), 1452 - 3 An origin of DNA replication from Lactococcus lactis bacteriophage c2; Waterfield NR et al.; An origin of DNA relication was identified in the intergenic region between the early and late gene regions of prolate lactococcal phage c2 . A DNA fragment containing this origin, designated ori, was shown to direct DNA replication in Lactococcus lactis but not in Escherichia coli . A comparison of ori with the corresponding regions of other prolate phages revealed strict conservation of the nucleotide sequence in one half of this intergenic region . This conserved region alone would not support DNA replication . No open reading frames were identified in the ori fragment, suggesting that host factors alone are sufficient to initiate DNA replication at ori . A novel class of lactococcal vectors and E . coli-L . lactis shuttle vectors based on ori have been constructed. Appl Environ Microbiol, 1996 Apr, 62(4), 1274 - 82 Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos; Labarre C et al.; Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank . Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments . Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure . The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present . The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells . The gene was expressed in E . coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species . The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core . Energy-dependent L-{14C}malate transport was observed with E . coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector . Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster. J Appl Bacteriol, 1996 Apr, 80(4), 453 - 7 Improved method for quantification of the bacteriocin nisin; Wolf CE et al.; Nisin, a bacteriocin produced by Lactococcus lactis subsp . lactis, is used in some types of food preservation due to its inhibitory action on Gram-positive bacteria and their spores . A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision . Several variables were evaluated . Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0 x 75% compared 1 x 5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na2HPO4 buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions . This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method. Z Lebensm Unters Forsch, 1996 Apr, 202(4), 329 - 33 Peptides inhibitory to endopeptidase and aminopeptidase from Lactococcus lactis ssp . lactis MG1363, released from bovine beta-casein by chymosin, trypsin or chymotrypsin; Stepaniak L et al.; Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm of Lactococcus lactis ssp . lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and alpha-chymotryptic hydrolysates of beta-casein (beta-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry . Chymosin released beta-CN f193-209, kinetic constant (Ki) of which for inhibition of PepO was 60 microM . This peptide also inhibited (Ki = 1700 microM) the 95-kDa aminopeptidase (PepN) from L . lactis ssp . lactis MG 1363 . Trypsin released two PepO-inhibitory peptides: one, beta-CN f69-97, was not degradable by PepO (Ki = 4.7 microM), while the other, beta-CN f141-163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO . A peptide, beta-CN f69-84, which inhibited PepO with a Ki of 8.1 microM, was isolated from the alpha-chymotryptic hydrolysate . Peptides released from beta-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN . PepO degraded beta-CN f193-209 very slowly compared with the hydrolysis of methionine enkephalin . All four inhibitory peptides (beta-CN f193-209, f69-97, f69-84, f141-163) were readily degraded by thermolysin. Yeast, 1996 Mar 15, 12(3), 215 - 25 Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe; Ansanay V et al.; The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology . The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe . The heterologous protein is expressed at a high level in cell extracts of a S . cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid . Malolactic enzyme specific activity is three times higher than in L . lactis extracts . Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of L-lactate during fermentation on glucose-rich medium in the presence of malic acid . Isotopic filiation was used to demonstrate that 75% of the L-lactate produced originates from endogenous L-malate and 25% from exogenous L-malate . Moreover, although a small amount of exogenous L-malate was degraded by S . cerevisiae transformed or not by mleS, all the exogenous degraded L-malate was converted into L-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways . These results indicate that the sole limiting step for S . cerevisiae in achieving malolactic fermentation is in malate transport . This was confirmed using a different model, S . pombe, which efficiently degrades L-malate . Total malolactic fermentation was obtained in this strain, with most of the L-malate converted into L-lactate and CO2 . Moreover, L-malate was used preferentially by the malolactic enzyme in this strain also. Mol Gen Genet, 1996 Mar 7, 250(4), 428 - 36 Transcriptional activation of the citrate permease P gene of Lactococcus lactis biovar diacetylactis by an insertion sequence-like element present in plasmid pCIT264; Lopez de Felipe F et al.; The lactococcal plasmid pCIT264 contains a cluster of three genes (citQ, citR and citP) involved in the transport of citrate in Lactococcus lactis biovar diacetylactis . The cit cluster contains a copy of a newly discovered insertion sequence (IS)-like element located between its promoter P1 and the first gene of the cluster . In this report, we show that this IS-like element can act as a mobile switch for the downstream genes, creating two new transcriptional promoters named P2 and P2' . The P2 promoter is recognized by the lactococcal RNA polymerase in vivo . This is a hybrid promoter composed of a -35 region reading outwards 12bp from the right end of the IS-like element, and a nucleotide sequence from the recipient plasmid, adjacent to the element, which provides an appropriately spaced -10 region . Transcription of the citQRP cluster from this promoter takes place during the exponential and stationary phases of growth in L . lactis . Promoter P2' is included in the IS-like element and is the only promoter responsible for expression of citP in E . coli . Thus, it appears that the introduction of this element into pCIT264 allows expression of the citQRP cluster in E . coli, and increases its levels of expression in L . lactis. Microbiologia, 1996 Mar, 12(1), 61 - 74 Genetic determinants for the biosynthesis of nisin, a bacteriocin produced by Lactococcus lactis; Rodriguez JM et al.; In the past, the genetic determinants for nisin biosynthesis were thought to be plasmid-located . However, it has been shown that production of nisin, immunity to nisin, and other properties such as the fermentation of sucrose, are encoded on 70 kb conjugative transposons that are chromosomally located . The extrachromosomal location of the nisin genes has not been substantiated by experiments that unequivocally show plasmid transfer . Two natural variants of nisin have been identified, nisin A and nisin Z, encoded by the genes nisA and nisZ, respectively . Both genes have been cloned and sequenced and differ only in a single base pair . Approximately 12 kb downstream from the structural gene has been cloned and sequenced, and a further 10 genes involved in the biosynthesis of nisin have been identified . The nisB and nisC gene products are involved in nisin maduration, the nisT in its secretion and the nisP in its processing . The nisR and nisK gene products have a regulatory role and the nisI, nisF, nisE and nisG are involved in immunity to nisin . All these genes display significant homology to the corresponding genes of the related lantibiotics subtilin and epidermin. Appl Environ Microbiol, 1996 Mar, 62(3), 1008 - 13 Food-grade cloning and expression system for Lactococcus lactis; Platteeuw C et al.; A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis . The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon . In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA . The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L . lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene . Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell . All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium . The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression . While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 36 - 40 Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth; Matsusaki H et al.; The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied . Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail . Production of nisin Z was optimal at 30 degrees C and in the pH range 5.0-5.5 . The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z . A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2 . It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production. FEMS Microbiol Lett, 1996 Mar 1, 136(3), 289 - 95 Characterization of an insertion sequence-like element identified in plasmid pCIT264 from Lactococcus lactis subsp . lactis biovar diacetylactis; Magni C et al.; Plasmid pCIT264 from Lactococcus lactis subsp . lactis biovar diacetylactis (L . diacetylactis) contains an insertion sequence (IS)-like element located in the citrate utilization (citQRP) cluster . This 967-nucleotide long element is bounded by 17 bp perfect inverted repeats and contains an open reading frame (ORF1) composed of 296 codons, which could encode a transposase . Expression of the IS from pCIT264 generates two mRNAs of 2900 and 1900 nucleotides . The transcription is driven by the P3 promoter, composed of a -10 region located at the right end of the IS and of a -35 region positioned downstream of this element . The IS-like element (IS982) is present in seven copies in the L.diacetylactis genome . The copy present in pCIT264 is highly stable and does not promote rearrangements of the cit cluster . We suggest that the stable maintenance of the IS-like element in pCIT264 could be due to a translational control of the putative transposase by an antisense RNA. Mol Microbiol, 1996 Mar, 19(6), 1343 - 55 Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t; van Sinderen D et al.; The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis . The linear r1t genome is composed of 33,350 bp and was shown to possess 3' staggered cohesive ends . Fifty open reading frames (ORFs) were identified which are, probably, organized in a life-cycle-specific manner . Nucleotide sequence comparisons, N-terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs . In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified . One ORF seems to be contained within a self-splicing group I intron . In addition, the bacteriophage att site required for site-specific integration into the host chromosome was determined. Mol Microbiol, 1996 Mar, 19(6), 1331 - 41 Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t; Nauta A et al.; A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized . It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec . Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences . The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators . Two of these repeats partially overlap the two promoter sequences . The distant third repeat is located within the tec coding sequence . Gel mobility shift assays demonstrated that Rro specifically binds to this sequence . To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed . Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low . Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle . In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion. J Bacteriol, 1996 Mar, 178(6), 1766 - 9 Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis; Franke CM et al.; Four in-frame translational fusions to both the reporter proteins beta-galactosidase and alkaline phosphatase support a topological model of LcnD, a protein implicated in the transport of several bacteriocins from Lactococcus lactis, in which the N-terminal part is located intracellularly and one transmembrane helix spans the cytoplasmic membrane. J Bacteriol, 1996 Mar, 178(6), 1525 - 31 Identical transcriptional control of the divergently transcribed prtP and prtM genes that are required for proteinase production in lactococcus lactis SK11; Marugg JD et al.; We have investigated transcriptional regulation of the divergently transcribed genes required for proteinase production (prtP and prtM) of Lactococcus lactis SK11 . Their promoters partially overlap and are arranged in a face-to-face configuration . The medium-dependent activities of both prtP and prtM promoters were analyzed by quantitative primer extension studies and beta-glucuronidase assays with L . lactis MG1363 cells harboring transcriptional gene fusions of each promoter with the promoterless beta-glucuronidase gene (gusA) from Escherichia coli . High-level production of prtP- or prtM-specific mRNAs was found after the growth of cells in media with low peptide concentrations, while increases in peptide concentrations resulted in an approximately eightfold decrease in mRNA production . Furthermore, prtP and prtM promoters exhibited similar efficiencies under different growth conditions . Deletion analysis of the prt promoter region showed that all the information needed for full activity and regulation of the prtP and prtM promoters is retained within a 90-bp region which includes both transcription initiation sites . An inverted repeat sequence positioned around the prtP and prtM transcription initiation sites was disrupted by either deletion or insertion of a small DNA sequence to analyze their effects on the activities of both prtP and prtM promoters . The mutations affected the activities of these promoters only marginally at low peptide concentrations but resulted in 1.5- to 5-fold derepression at high peptide concentrations . These results indicate that the expression of both prtM and prtP genes is controlled in an identical manner via a control mechanism capable of repressing transcription initiation at high peptide concentrations. FEMS Microbiol Lett, 1996 Feb 1, 136(1), 19 - 24 Exploitation of a chromosomally integrated lactose operon for controlled gene expression in Lactococcus lactis; Payne J et al.; Lactococcus lactis MG5267 is a plasmid-free strain in which the lactose operon is integrated in the bacterial chromosome . The chromosomal lacG gene which encodes phospho-beta-galactosidase was inactivated by a double cross-over integration event . Unexpectedly, the resultant mutant was shown to retain a Lac-positive phenotype . The lysin gene from Listeria monocytogenes bacteriophage LM-4 was subsequently integrated into the chromosome of this strain such that expression of the heterologous gene was mediated by the lactose operon promoter . Expression of the lysin gene was shown to be regulated by growth on lactose . This represents an important strategy for the controlled and stabilised expression of biotechnologically useful genes in L . lactis. Antonie Van Leeuwenhoek, 1996 Feb, 69(2), 151 - 9 Immunity to lantibiotics; Saris PE et al.; Bacteria producing bacteriocins have to be protected from being killed by themselves . This mechanism of self-protection or immunity is especially important if the bacteriocin does not need a specific receptor for its action, as is the case for the type A lantibiotics forming pores in the cytoplasmic membrane . At least two different systems of immunity have evolved in this group of bacteriocins containing modified amino acids as a result of posttranslational modification . The immunity mechanism of Pep5 in Staphylococcus epidermidis is based on inhibition of pore formation by a small 69-amino acid protein weakly associated with the outer surface of the cytoplasmic membrane . In Lactococcus lactis and Bacillus subtilis the putative immunity lipoproteins NisI and SpaI, respectively, are also located at the outer surface of the cytoplasmic membrane, suggesting that a similar mechanism might be utilized by the producers of nisin and subtilin . In addition an ABC-transport system consisting of two membrane proteins, (NisEG, SpaG and the hydrophobic domain of SpaF, and EpiEG) and a cytoplasmic protein (NisF, the cytoplasmic domain of SpaF, and EpiF) play a role in immunity of nisin, subtilin and epidermin by import, export or inhibition of pore formation by the membrane components of the transport systems . Almost nothing is known of the immunity determinants of newly described and other type of lantibiotics. Antonie Van Leeuwenhoek, 1996 Feb, 69(2), 109 - 17 Genetics of subtilin and nisin biosyntheses: biosynthesis of lantibiotics; Entian KD et al.; Several peptide antibiotics have been described as potent inhibitors of bacterial growth . With respect to their biosynthesis, they can be divided into two classes: (i) those that are synthesized by a non-ribosomal mechanism, and (ii) those that are ribosomally synthesized . Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics . They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyllanthionine . They are derived from prepeptides which are post-translationally modified and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al . 1988) . Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984) . It is produced by Lactococcus lactis strains belonging to serological group N . The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes . Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released . A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al . 1987) . Nisin occurs as a partially amphiphilic molecule (Van de Ven et al . 1991) . Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al . 1980) . In several countries nisin is used to prevent the growth of clostridia in cheese and canned food . The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al . 1988; Kaletta & Entian 1989) . Nisin has two natural variants, nisin A, and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al . 1991; De Vos et al . 1993) . Subtilin is produced by Bacillus subtilis ATCC 6633 . Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988) . Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig . 1), and both lantibiotics possess similar antibiotic activities . Due to its easy genetic analysis B . subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis . The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al . 1995b) . The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig . 2) . These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium . In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein. J Dairy Res, 1996 Feb, 63(1), 131 - 40 Purification of tributyrin esterase from Lactococcus lactis subsp . cremoris E8; Holland R et al.; A tributyrin esterase was purified from Lactococcus lactis subsp . cremoris E8 using FPLC chromatography . This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa . The enzyme was active against tributyrin and p-nitrophenyl butyrate . The N-terminal sequence of the enzyme was determined . The enzyme had a pH optimum in the neutral range, was stable on freezing at -20 degrees C, and had a half life of 1 h at 50 degrees C. Appl Environ Microbiol, 1996 Feb, 62(2), 612 - 9 An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147; Ryan MP et al.; Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition . The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci . On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147 . The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L . lactis subsp . cremoris DPC4268 . The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147 . The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials . Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains . The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria . Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products. Curr Microbiol, 1996 Feb, 32(2), 85 - 8 Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish; Eldar A et al.; The reference strains of Enterococcus seriolicida (ATCC 49156T) (T = type strain) and of Lactococcus garvieae (ATCC 43921T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar . The high DNA-DNA homologies (70-100%) and the low delta Tme (less than 1.1 degrees C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species . E . seriolicida strains should be classified as L . garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution. J Bacteriol, 1996 Feb, 178(3), 600 - 5 Lactococcin G is a potassium ion-conducting, two-component bacteriocin; Moll G et al.; Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta . Peptide synthesis of the alpha and beta peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis . Approximately equivalent amounts of both peptides were required for optimal bactericidal effect . No effect was observed with either the alpha or beta peptide in the absence of the complementary peptide . The combination of alpha and beta peptides (lactococcin G) dissipates the membrane potential (delta omega), and as a consequence cells release alpha-aminoisobutyrate, a non-metabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism . In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake . Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient . Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions . However, rapid release of potassium was observed in the presence of lactococcin G . These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the alpha and beta peptides in the target bacterial membrane. Mol Microbiol, 1996 Jan, 19(2), 221 - 30 Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB; Parreira R et al.; The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403 . Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10-15 min after infection . The decay of the transcripts is the probable cause of the arrest of the sensitive phage development . Mapping of the 5' end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites . We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell. Enzyme Microb Technol, 1996 Jan, 18(1), 52 - 8 Detection of cleavage of a prenisin-mimicking decapeptide by Lactococcus lactis subsp . lactis endoproteinase activity; Nelis HJ et al.; As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp . lactis strain . Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products . Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond . This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate. Adv Exp Med Biol, 1996, 379, 63 - 73 Modelling and engineering of enzyme/substrate interactions in subtilisin-like enzymes of unknown 3-dimensional structure; Siezen RJ; Homology modelling was used to predict enzyme-substrate interactions in three entirely different subtilisin-like enzymes of unknown three-dimensional structure . i.e . (a) cell-envelope proteinase of Lactococcus lactis, (b) putative leader peptidase for pre-nisin from L . lactis, and (c) human furin . Models were based on known three-dimensional structures of subtilisins and thermitase in complex with inhibitors . Detailed analysis of interactions of the P1-P4 residues of model substrates with the S1-S4 binding sites in each enzyme suggest that electrostatic interactions at all four binding sites can contribute to binding and hence to specificity . In particular, one or more negative charges in the S1 or S4 pockets can lead to a high selectivity for Arg residues in the substrate . Many of the predicted interactions have been confirmed by engineering of either enzyme, substrate or both. J Dairy Sci, 1996 Jan, 79(1), 20 - 6 Beta-casomorphins: analysis in cheese and susceptibility to proteolytic enzymes from Lactococcus lactis ssp . cremoris; Muehlenkamp MR et al.; Commercial cheese products were surveyed for beta-casomorphin peptides . Two extraction methods were compared: 1) water and 2) chloroform and methanol . Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection . beta-Casomorphin standards were used for comparison with cheese peptide profiles . Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for beta-casomorphins . However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to beta-casomorphins . Therefore, beta-casomorphins were absent, or concentrations were below the HPLC detection threshold for beta-casomorphin of 2 micrograms/ml of cheese extract . The susceptibility of beta-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp . cremoris was investigated . beta-Casomorphin standards were incubated at 4 degrees C with bacterial cell lysate at pH 5.0, 5.2, 5.4, and 5.7 . Salt concentrations varied among 0, 1.5, and 5% . The concentration of added beta-casomorphins and the degradation products were monitored over 15 wk using HPLC . Enzymatic degradation of beta-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures . Therefore, if formed in cheese, beta-casomorphins may be degraded under conditions common for Cheddar cheese. Lett Appl Microbiol, 1996 Jan, 22(1), 76 - 9 Comparative effectiveness of nisin and bacteriocin J46 at different pH values; Huot E et al.; A comparative study of the inhibitory activity of nisin, the well-known lantibiotic produced by certain strains of Lactococcus lactis subsp . lactis, and of the bacteriocin produced by L . lactis subsp . cremoris J46, a strain previously isolated from fermented milk, was conducted . For both bacteriocins, the activity against L . lactis subsp . cremoris decreased with increasing pH . In addition, the bacteriocin preparations were more stable at 4 degrees than at 20 degrees C . The influence of the storage temperature was more crucial for nisin . Essentially the same activity was observed for bacteriocin J46 stored for 3 h at 4 degrees or 20 degrees C . More interesting was the observed stability of bacteriocin J46 at pH values between 5.8 and 6.8 . For example, about 23% of nisin activity was lost at pH 6.4 whereas no loss of bacteriocin J46 activity was observed. Microbiology, 1996 Jan, 142 ( Pt 1), 47 - 55 A gene replacement strategy for engineering nisin; Dodd HM et al.; A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin (nisA) genes . This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the nisA gene . The wild-type chromosomal gene is effectively replaced with a variant nisA gene, by the technique of gene replacement . The recovery of full nisin immunity was employed as a means of directly selecting strains that had acquired an intact nisA gene by the gene replacement process . With this approach the other genes required for pre-nisin maturation are not affected and any alterations to DNA sequences are restricted to only those specific mutations introduced in the nisA gene . The effectiveness of the system was demonstrated by the expression of a number of variant nisA genes leading to the successful production and characterization of nisins containing the substitutions Dha5A, Dha33A, Dha5, 33A, H27K, 130W and K12L . The enhanced yields of these engineered nisin molecules, when compared to their production in a plasmid-complementation system, underlines the improvement offered by this gene replacement strategy. Infect Immun, 1996 Jan, 64(1), 380 - 3 Interactions of human mannose-binding protein with lipoteichoic acids; Polotsky VY et al.; We explored the interaction of human recombinant mannose-binding protein and lipoteichoic acids (LTAs) by enzyme-linked immunosorbent assay . The best ligand was Micrococcus luteus lipomannan, followed by Enterococcus spp . LTA containing mono-, di-, and oligoglucosyl substituents . LTAs lacking terminal sugars (those of Streptococcus pyogenes and Staphylococcus aureus) or containing galactosyl substituents (those of Listeria spp . and Lactococcus spp.) were poor ligands . These results are consistent with known structural requirements for binding through the mannose-binding protein carbohydrate recognition domain. Mol Biotechnol, 1995 Dec, 4(3), 297 - 314 Bacteriophage resistance in Lactococcus; Dinsmore PK et al.; Lactic acid bacteria are industrial microorganisms used in many food fermentations . Lactococcus species are susceptible to bacteriophage infections that may result in slowed or failed fermentations . A substantial amount of research has focused on characterizing natural mechanisms by which bacterial cells defend themselves against phage . Numerous natural phage defense mechanisms have been identified and studied, and recent efforts have improved phage resistance by using molecular techniques . The study of how phages overcome these resistance mechanisms is also an important objective . New strategies to minimize the presence, virulence, and evolution of phage are being developed and are likely to be applied industrially. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 413 - 8 Lactococcin A overexpression in a Lactococcus lactis subsp . lactis transformant containing a Tn5 insertion in the lcnD gene; Requena T et al.; Lactococcin A production in lactococci has recently been linked to a signal-sequence-independent secretory system consisting of a four-gene cluster . Lactococcus lactis subsp . lactis LLM23L-A1 has been obtained after Tn5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production . In contrast to other Tn5-generated mutants, strain LLM23L-Al exhibited a 12-fold increase in lactococcin A production . Overproduction of lactococcin A was not linked to an increased pLLM23 copy number . Restriction-enzyme analysis indicated the site of Tn5 insertion to be at the 3' end of lcnD, and upstream of the lcnA structural gene . From DNA sequencing, the Tn5 insertion was located -79 bp upstream of the transcription start site of the lcnA and lciA genes, eliminating eight amino acids from the C-terminal end of lactococcin D . Northern blots revealed overproduction of a 500-base transcript in strain LLM23L-A1, which corresponded to that predicted from the positions of the lactococcin A operon transcriptional start site and the termination structures . This result suggests that the overproduction of lactococcin A in strain LLM23L-A1 is at the transcriptional level and provides further impetus for elucidating the complete regulatory mechanism for lactococcin A expression. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 386 - 92 The occurrence of two intracellular oligoendopeptidases in Lactococcus lactis and their significance for peptide conversion in cheese; Baankreis R et al.; Two intracellular oligopeptide-preferring endopeptidases have been detected in Lactococcus lactis . A neutral thermolysin-like oligoendopeptidase (NOP) has been purified to homogeneity and an alkaline oligoendopeptidase has been partially purified . The specificity of the oligoendopeptidases towards important intermediary cheese peptides, produced by chymosin action on the caseins, clearly differs from that of the cell-envelope proteinase (CEP) . NOP is active under conditions prevailing in cheese and contributes to initial proteolysis in a young cheese . It probably plays a crucial role in the degradation of an important bitter peptide in cheese, the beta-casein 193-209 fragment .