Lactococci

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 . The relatively low activity of the alkaline endopeptidase is further suppressed in cheese by the highly competitive actions of NOP and CEP.

Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 139 - 46
Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production; Broadbent JR et al.; Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria . Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells . Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454 . Intergeneric transfer of nisin and sucrose genes between L . lactis and a dairy Enterococcus sp . was also investigated . Erythromycin-resistant Enterococcus sp . recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM beta 1 . Matings between L . lactis 11454 and an Enterococcus sp . recipient that contained pAM beta 1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 x 10(-9) transconjugants per donor cfu . Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA.DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.

Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 100 - 5
A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp . cremoris Wg2; Niven GW et al.; A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp . cremoris Wg2 to hydrolyse different peptide substrates . The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine . There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates . It was also observed that the enzyme tended to have higher affinities but lower Vmax values for tripeptides with hydrophobic C-terminal residues . The values determined for Km and Vmax increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly)n, the optimum, as determined from Vmax/Km, being when n = 4 . Typical Km values for the most effective substrates were in the range 0.2-0.6 mM.

Appl Environ Microbiol, 1995 Dec, 61(12), 4348 - 56
Sequencing and analysis of the prolate-headed lactococcal bacteriophage c2 genome and identification of the structural genes; Lubbers MW et al.; The 22,163-bp genome of the lactococcal prolate-headed phage c2 was sequenced . Thirty-nine open reading frames (ORFs), early and late promoters, and a putative transcription terminator were identified . Twenty-two ORFs were in the early gene region, and 17 were in the late gene region . Putative genes for a DNA polymerase, a recombination protein, a sigma factor protein, a transcription regulatory protein, holin proteins, and a terminase were identified . Transcription of the early and late genes proceeded divergently from a noncoding 611-bp region . A 521-bp fragment contained within the 611-bp intergenic region could act as an origin of replication in Lactococcus lactis . Three major structural proteins, with sizes of 175, 90, and 29 kDa, and eight minor proteins, with sizes of 143, 82, 66, 60, 44, 42, 32, and 28 kDa, were identified . Several of these proteins appeared to be posttranslationally modified by proteolytic cleavage . The 175- and 90-kDa proteins were identified as the major phage head proteins, and the 29- and 60-kDa proteins were identified as the major tail protein and (possibly) the tail adsorption protein, respectively . The head proteins appeared to be covalently linked multimers of the same 30-kDa gene product . Phage c2 and prolate-headed lactococcal phage bIL67 (C . Schouler, S . D . Ehrlich, and M.-C . Chopin, Microbiology 140:3061-3069, 1994) shared 80% nucleotide sequence identity . However, several DNA deletions or insertions which corresponded to the loss or acquisition of specific ORFs, respectively, were noted . The identification of direct nucleotide repeats flanking these sequences indicated that recombination may be important in the evolution of these phages.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Dec, 61(12), 4321 - 8
Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40; Garvey P et al.; The lactococcal plasmid pNP40, from Lactococcus lactis subsp . lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L . lactis subsp . lactis MG1614 . A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712 . Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity . Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs) . Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712 . Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity . DNA sequence analysis of this region revealed the presence of a single complete ORF . The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity . The mechanisms encoded by pPG01 and pCG1 in L . lactis subsp . lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively . Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition . The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.

J Bacteriol, 1995 Dec, 177(24), 7026 - 32
Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon; Kahn K et al.; A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene . Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues) . Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence . On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein . A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1) . In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751 . The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons . The two remaining ORFs on Tn5469 encode proteins of unknown function . Southern blot analysis showed that wild-type F . diplosiphon harbors five genomic copies of Tn5469 . In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene . Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.

J Biol Chem, 1995 Nov 10, 270(45), 27299 - 304
Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction; Kuipers OP et al.; The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG . When a 4-base pair deletion is introduced into the structural nisA gene (delta nisA), transcription of delta nisA is abolished . Transcription of the delta nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides . Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of two-component regulators, transcription of delta nisA was no longer inducible by nisin . Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L . lactis NZ9800 (delta nisA) only upon induction with nisin species . The expression level of gusA was directly related to the amount of inducer that was added extracellularly . These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.

Gene, 1995 Nov 7, 165(1), 9 - 15
The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis; Waterfield NR et al.; 18 different promoter elements, encompassing a 71-fold range of activity, were isolated from the chromosome of Lactococcus lactis (Ll) MG1363 and from an uncharacterised small isometric bacteriophage of Ll . The Vibrio fischeri (Vf) luciferase-encoding gene (lux) was used as a reporter in Ll, so that the promoters could be identified strictly on the basis of their activity in the homologous host . Sequence and primer extension analysis of six of the promoters has provided a new consensus sequence for the -35 and -10 hexanucleotide motifs present upstream from lactococcal transcription start points . When the nucleotide sequence of the most active promoter (P15) was compared with that of the highly expressed Ll usp45 gene, a novel 8-bp region of homology was identified which corresponded to the newly derived consensus -35 sequence element; this element may therefore be of general importance in Ll gene expression . The isolation of these promoters has also enabled us to investigate the characteristics of the Vf Lux activity in Ll under different physiological conditions using promoters of different strengths . Lux activity in Ll is critically dependent upon the phase of cell growth . Luminescence falls sharply in stationary phase, possibly due to a lack of FMNH2 . In contrast to the kinetics of Lux function in Escherichia coli (Ec), Lux activity in Ll declines rapidly after addition of the substrate; the rate of decay is dependent both on the growth phase and on the strength of the promoter . It is apparent that the previously reported thermal instability of Lux is in fact a function of the host organism in which Lux is expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Nov 3, 270(44), 26092 - 8
The Lactococcal lmrP gene encodes a proton motive force-dependent drug transporter; Bolhuis H et al.; To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide . Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium . The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments . Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E . coli . Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules . Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate . These observations are indicative for a drug-proton antiport system . A lmrP deletion mutant was constructed via homologous recombinant using DNA fragments of the flanking region of the gene . The L . lactis (delta lmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate . The results indicate that in the absence of the functional drug-proton anti-porter LmrP, L . lactis is able to overexpress another, ATP-dependent, drug extrusion system . These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L . lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H . W., Poolman, B., Driessen, A . J . M., and Konings, W . N . (1994) J . Bacteriol . 176, 6957-6964).

Plasmid, 1995 Nov, 34(3), 234 - 5
Nucleotide sequence of a plasmid pCL2.1 from Lactococcus lactis ssp . lactis ML8; Chang HC et al.; The nucleotide sequence of a small cryptic plasmid, pCL2.1, from Lactococcus lactis ssp . lactis ML8 was determined . Sequence analysis of the pCL2.1 revealed that it contained 2112 bp, 33.9% GC, and two open reading frames that encoded polypeptides of 26 and 14 kDa . In vitro transcription-translation of the pCL2.1 confirmed the existence of two polypeptides . Based on sequence homology, it is deduced that the ORF2 product functions in plasmid replication by a rolling circle mechanism.

J Dairy Res, 1995 Nov, 62(4), 629 - 40
Water-soluble peptides in Cheddar cheese: isolation and identification of peptides in the diafiltration retentate of the water-soluble fraction; Singh TK et al.; The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes . Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl . pH 8.6, and reversed-phase HPLC or electroblotting from urea-PAGE gels . Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry . Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from beta-casein, especially from a short region in the N-terminal half of the molecule . Only six peptides originated from alpha s1-casein; beta-lactoglobulin was also identified in the retentate . The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.

Mol Gen Genet, 1995 Nov 1, 249(1), 43 - 50
Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01; Seegers JF et al.; The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized . The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro . In addition, the effect of this SSO on plasmid maintenance was determined . The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs) . The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO . One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174 . This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro . The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro . The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event . The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Microbiology, 1995 Nov, 141 ( Pt 11), 2873 - 81
A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk; l'Anson KJ et al.; Degenerate PCR primers were designed from the N-terminal amino acid sequence of a glutamyl aminopeptidase (PepA) from Lactococcus lactis . These primers were used to screen a lambda library for clones containing the gene (pepA) encoding PepA . The DNA sequence of a 2.1 kb fragment containing pepA was determined . The sequence revealed the presence of one complete and two incomplete open reading frames (ORFs) . The complete ORF encodes a putative protein of 353 amino acids with a predicted N-terminal sequence identical to that determined for purified PepA . The pepA gene was subcloned on an Escherichia coli plasmid vector and production of active PepA was confirmed by means of a zymogram . Mutants of L . lactis in which the pepA gene was inactivated grew to normal cell densities in milk but exhibited a reduced growth rate during the exponential phase . Thus whilst PepA is required for optimal growth it is not essential.

Appl Environ Microbiol, 1995 Nov, 61(11), 4105 - 9
Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site; Boyce JD et al.; Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp . cremoris H2 . One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int) . The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L . lactis strains.

Appl Environ Microbiol, 1995 Nov, 61(11), 4099 - 104
Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T; Boyce JD et al.; Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp . cremoris . Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared . The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T . Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor . Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions . By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro.

Appl Environ Microbiol, 1995 Nov, 61(11), 4089 - 98
Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends; Boyce JD et al.; The Lactococcus lactis temperate bacteriophage BK5-T is a type phage in the lactococcal phage classification (A . W . Jarvis, G . F . Fitzgerald, M . Mata, A . Mercenier, H . Neve, I . B . Powell, C . Ronda, M . Saxelin, and M . Teuber, Intervirology 32:2-9, 1991) . The nucleotide sequence of 18,935 bp of the genome of BK5-T was determined and analyzed for the presence of open reading frames and other structural features . Thirty-two open reading frames longer than 60 codons were identified, and these appeared to be grouped into at least seven transcriptional units . A search of the nucleotide sequence for restriction sites identified a small number of discrepancies with the previously published physical map of the BK5-T genome (G . Lakshmidevi, B . E . Davidson, and A . J . Hillier, Appl . Environ . Microbiol . 54:1039-1045, 1988) . Subsequent analysis of restriction digests of BK5-T DNA which were heated prior to electrophoresis indicated that BK5-T DNA was not terminally redundant as previously reported but contained cohesive ends.

Appl Environ Microbiol, 1995 Nov, 61(11), 3967 - 71
Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions; Platteeuw C et al.; The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L . lactis lacA promoter . More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L . lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene . The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L . lactis strains was studied under different fermentation conditions . Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted . However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L . lactis NZ2700 . Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate . When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L . lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed . Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol . These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L . lactis.

Appl Environ Microbiol, 1995 Nov, 61(11), 3934 - 9
Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases; Reid JR et al.; Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme . The sequences of the oligopeptides were based on two regions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to PI- and PIII-type lactococcal proteinases . In each series, one oligopeptide had an identical sequence to that of the kappa-casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His<-->Glu . Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of kappa-casein resulted in reduced cleavage of susceptible bonds by the PI-type proteinase and increased cleavage of susceptible bonds by the PIII-type proteinase . In the case of the oligopeptide corresponding to residues 153 to 169 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the PI-type enzyme) . Substitution of Glu by His in this oligopeptide, even in the P7 position, resulted in increased cleavage of the bond by the PI-type proteinase and reduced cleavage by the PIII-type proteinase . C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins, 1995 Oct, 23(2), 278 - 81
Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis; Chich JF et al.; The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli . The enzyme was isolated in its active form in two purification steps . Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0 . The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit . The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

Virology, 1995 Oct 1, 212(2), 595 - 606
Virion positions and relationships of lactococcal temperate bacteriophage TP901-1 proteins; Johnsen MG et al.; The major proteins of phage TP901-1 virion were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and structural relations were determined using specific antibodies, obtained by affinity purification from a polyclonal serum . A 23-kDa protein was identified as the major tail protein, and a 31-kDa molecule as the major head protein, respectively . Labeling experiments with antibodies against two proteins, with molecular masses of 20 and 19 kDa, indicated that they were baseplate-related components . A 72-kDa protein was found to be part of a neck passage structure, which includes a collar . Evidence for the presence of attached whiskers was also obtained . T7 RNA polymerase-mediated expression of the two major proteins confirmed the gene location of the previously sequenced region of the phage genome . The relation to other lactococcal phages was determined by DNA hybridization and antibody probing, showing that despite low DNA similarity, TP901-1 NPS epitopes were detected in both related and unrelated small isometric-headed phages.

Appl Environ Microbiol, 1995 Oct, 61(10), 3573 - 9
Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum; van Belkum MJ et al.; Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187 . A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J . W . Hastings, M . Sailer, K . Johnson, K . L . Roy, J . C . Vederas, and M . E . Stiles, J . Bacteriol . 173:7491-7500, 1991) . When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected . Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L . gelidum UAL187-13 and Lactococcus lactis IL1403 . Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene . The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production . The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters . The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A . Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A . The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L . lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, 1995 Sep 15, 3(9), 961 - 8
The three-dimensional structure of 6-phospho-beta-galactosidase from Lactococcus lactis; Wiesmann C et al.; BACKGROUND: The enzyme 6-phospho-beta-galactosidase hydrolyzes phospholactose, the product of a phosphor-enolpyruvate-dependent phosphotransferase system . It belongs to glycosidase family 1 and no structure has yet been published for a member of this family . RESULTS: The crystal structure of 6-phospho-beta-galactosidase was determined at 2.3 A resolution by multiple isomorphous replacement, using the wild-type enzyme and a designed cysteine mutant . A second crystal form, found with the mutant enzyme, was solved by molecular replacement, yielding the conformation of two chain loops that are invisible in the first crystal form . The active center, located through catalytic residues identified in previous studies, cannot be accessed by the substrate if the two loops are in their defined conformation . The enzyme contains a (beta alpha)8 barrel and the relationship of its chain fold to that of other glycosidases has been quantified . As a side issue, we observed that a cysteine point mutant designed for X-ray analysis crystallized mainly as a symmetric dimer around an intermolecular disulfide bridge formed by the newly introduced cysteine . CONCLUSIONS: The presented analysis provides a basis on which to model all other family 1 members and thereby will help in elucidating the catalytic mechanisms of these sequence-related enzymes . Moreover, this enzyme belongs to a superfamily of glycosidases sharing a (beta alpha)8 barrel with catalytic glutamates/aspartates at the ends of the fourth and the seventh strands of the beta barrel.

Mol Microbiol, 1995 Sep, 17(6), 1121 - 31
The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress; Duwat P et al.; The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain . This single alteration had a surprisingly pleiotropic effect . In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses . We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain . Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition . These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur . Unexpectedly, thermal stress arrested growth of the recA strain . Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE . Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain . In contrast, levels of a novel heat-shock protein, HfIB, are elevated . In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32 . We propose that recA regulates the heat-shock response via HfIB . This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

Plasmid, 1995 Sep, 34(2), 105 - 18
Characterization of the replicon from the lactococcal theta-replicating plasmid pJW563; Gravesen A et al.; The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kb EcoRI fragment . This DNA fragment was sequenced ans a 1155-bp open reading frame, repB563, encoding a putative protein RepB563 of 385 amino acids was found . An AT-rich noncoding region, repA563, was found upstream of repB563 . This segment included several direct and inverted repeats . A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled to repB563, RepB563 supplied in trans could support replication of a plasmid containing repA563 and a truncated repB563 . This observation suggests that RepB563 is a trans-acting replication protein, and repA563 the cis-acting origin of replication, repA563, repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids . The repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated . The comparison revealed highly conserved areas among this family of plasmids . In addition, variable domains emerged, presumably having a plasmid specific function, pVS40 and pC1305 were plasmids with replication proteins showing high homology to RepB563 . Despite this homology, replication from repA563 could not be supported by the pVS40 or pC1305 replication protein supplied in trans . Likewise the pJW563 protein could not support replication from the pVS40 origin . pJW563 was found to be compatible with the pVS40 and pC1305 replicons . The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating pladmids . Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific in many cases rendering the plasmids compatible.

J Bacteriol, 1995 Sep, 177(18), 5254 - 60
Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene; Sanders JW et al.; In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected . One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis . The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain . The deduced amino acid sequence of L . lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus . A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA . The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene . The induction of beta-galactosidase activity occurred in aerated cultures . Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration . The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.

Can J Microbiol, 1995 Sep, 41(9), 832 - 41
Characterization of diacetin B, a bacteriocin from Lactococcus lactis subsp . lactis bv . diacetylactis UL720; Ali D et al.; Fourteen Lactococcus lactis strains showing inhibitory activity against Listeria innocua SICC 4202 were isolated from different French raw milks and raw milk cheeses and screened for bacteriocin production by the triple layer method under conditions that eliminate the effects of lactic acid and hydrogen peroxide . Three bacteriocinogenic strains (two Lactococcus lactis subsp . lactis bv . diacetylactis UL719 and UL720 and one Lactococcus lactis subsp . lactis UL730) were selected for their high capacity to inhibit the growth of various food pathogens, including Listeria monocytogenes, Staphylococcus aureus, and clostridial strains . The inhibitory compounds from these three strains are inactivated by selected proteases, indicating their protein nature . They retained their antibacterial activity after heat treatments of 100 degrees C for 60 min and 121 degrees C for 20 min, and in the pH range from 2 to 11 . The bacteriocin diacetin B produced by strain UL720 has been purified by a pH-dependent adsorption-desorption procedure, followed by reverse-phase high performance liquid chromatography, with a yield of 1.25% of the original activity . Mass spectrometry analysis indicates that the pure peptide has a molecular mass of 4292.32 or 4490.28 Da, while amino acid sequencing allowed the identification of the primary structure of the bacteriocin composed of 37 amino acid residues . The structure of the peptide did not show similarity with other known bacteriocins from lactic acid bacteria.

Lett Appl Microbiol, 1995 Sep, 21(3), 183 - 9
Expression of lactococcin A and pediocin PA-1 in heterologous hosts; Chikindas ML et al.; Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0 . The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes . Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter . Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.

Microbiology, 1995 Sep, 141 ( Pt 9), 2071 - 9
Expression of foreign genes and selection of promoter sequences in Acholeplasma laidlawii; Jarhede TK et al.; The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors . In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii . Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A . laidlawii . One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids . This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A . laidlawii . To increase the expression of foreign genes, random A . laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis alpha-amylase gene as a probe . Selection was done in Escherichia coli as well as directly in A . laidlawii . Active recombinants from E . coli were also able to express alpha-amylase activities and an enzyme of native size in A . laidlawii . The highest activity was obtained from a recombinant selected directly in A . laidlawii . This is the first example of a promoter sequence selected in a mollicute . Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A . laidlawii, Acholeplasma oculi, Lactococcus and E . coli . Vectors related to pNZ18 should be useful for the genetic analysis of specific A . laidlawii proteins and functions.

Gene, 1995 Aug 30, 162(1), 59 - 62
Construction of a Streptococcus pyogenes recA mutant via insertional inactivation, and cloning and sequencing of the complete recA gene; Tao L et al.; To facilitate future genetic studies with Streptococcus pyogenes (Sp), a recA mutant (Rec11) was constructed using a streptococcal integration vector carrying a PCR-derived internal recA fragment . The insertion of the plasmid in the mutant chromosome was identified by Southern hybridization . Resistance to UV and the ability to accept linear DNA transformation by Rec11 were greatly decreased, confirming its RecA phenotype . Using the PCR-derived fragment as a probe, we cloned and sequenced the complete Sp recA gene, which is highly homologous to the recA of S . pneumoniae and Lactococcus lactis.

FEMS Microbiol Lett, 1995 Aug 15, 131(1), 75 - 80
The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L . lactis; Qiao M et al.; Lactococcus lactis cells secreting the lantibiotic nisin, commercially used for food preservation, must protect their cell membrane against the pore-forming activity of extracellular nisin . The nisI gene product has been suggested to be a lipoprotein, which due to the location on the extracellular surface would be an ideal candidate for an immunity protein . In vivo labelling of NisI from L . lactis N8 expressed in Escherichia coli proved that NisI is a lipoprotein . Expression of nisI in the nisin-sensitive L . lactis MG1614 strain resulted in immunologically active protein on the cytoplasmic membrane in comparable amounts to the immune strain L . lactis N8, but only to slightly increased nisin immunity, suggesting that additional proteins are needed for full immunity.

Mol Microbiol, 1995 Aug, 17(3), 427 - 37
Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria; de Vos WM et al.; Lantibiotics form a family of highly modified peptides which are secreted by several Gram-positive bacteria . They exhibit antimicrobial activity, mainly against other Gram-positive bacteria, by forming pores in the cellular membrane . These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences . Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance . For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self-protection proteins, processing enzymes and regulatory proteins have been identified . This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production . Most of the attention is focused on the lantibiotic nisin, which is produced by the food-grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry.

Trends Microbiol, 1995 Aug, 3(8), 299 - 304
Lactococcal bacteriocins: mode of action and immunity; Venema K et al.; Bacteriocins are antimicrobial peptides produced by bacteria . Some of those synthesized by Lactococcus lactis have been characterized in great detail recently . The lactococcal bacteriocins are hydrophobic cationic peptides, which form pores in the cytoplasmic membrane of sensitive cells.

J Bacteriol, 1995 Aug, 177(16), 4652 - 7
Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides; Foucaud C et al.; A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363 . Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L . lactis . The specificity of DtpP partially overlaps that of DtpT . DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides . The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background . This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides . The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids . The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.

Appl Environ Microbiol, 1995 Aug, 61(8), 3172 - 6
Characterization of plasmid-encoded citrate permease (citP) genes from Leuconostoc species reveals high sequence conservation with the Lactococcus lactis citP gene; Vaughan EE et al.; The citrate permease determinant (citP) in several Leuconostoc strains was demonstrated to be plasmid encoded by curing experiments and hybridization studies with a DNA fragment containing the citP gene from Lactococcus lactis subsp . lactis biovar diacetylactis NCDO176 . Cloning and nucleotide sequence analysis of Leuconostoc lactis NZ6070 citP revealed almost complete identity to lactococcal citP.

Appl Environ Microbiol, 1995 Aug, 61(8), 3024 - 30
Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk; Juillard V et al.; The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis . The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases . The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed . Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar . At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth . The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth . Oligopeptide transport-deficient strains (Opp-) of L . lactis were unable to utilize oligopeptides and grew poorly in milk . However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did . These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase . In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase . Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S . Kunji, A . Hagting, C.J . De Vries, V . Juillard, A.J . Haandrikman, B . Poolman, and W.N . Konings, J . Biol . Chem . 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Aug, 61(8), 2995 - 3001
Bacteriolytic activity caused by the presence of a novel lactococcal plasmid encoding lactococcins A, B, and M; Morgan S et al.; Lactococcus lactis subsp . lactis biovar diacetylactis DPC938 was identified as a bacteriocin-producing strain which exhibited a bacteriolytic effect on other lactococci . Lysis of such target strains was associated with decreases in optical density and release of the intracellular enzyme lactate dehydrogenase . DPC938 exhibits cross-immunity to L . lactis subsp . cremoris 9B4 (M.J . van Belkum, B.J . Hayema, A . Geis, J . Kok, and G . Venema, Appl . Environ . Microbiol . 55:1187-1191, 1989), a strain which produces the bacteriocins lactococcins A, B, and M . Genetic analyses revealed that a 15.5-kb region of DNA encoding these bacteriocins is highly conserved in 9B4, DPC938, and DPC3286, an overproducing derivative of DPC938 . This region is located on a 72- and a 78-kb nonmobilizable plasmid in DPC938 and DPC3286, respectively . The bacteriolytic effect exhibited by DPC938 and DPC3286 on sensitive cultures is most probably due to the concerted action of all three bacteriocins . Since these cultures exhibit a lytic effect on lactococci, they have a potential application in the dairy industry as accelerators of starter lysis and hence accelerators of cheese ripening.

J Biol Chem, 1995 Jul 14, 270(28), 16740 - 4
Purification and characterization of a small membrane-associated sugar phosphate phosphatase that is allosterically activated by HPr(Ser(P)) of the phosphotransferase system in Lactococcus lactis; Ye JJ et al.; In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion) . Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process . A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity . It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme . It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride . It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate . Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate . The enzyme exhibits a Ka for S46D HPr of 15 microM . Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.

Lett Appl Microbiol, 1995 Jul, 21(1), 10 - 3
Glucose metabolism and internal pH of Lactococcus lactis subsp . lactis cells utilizing NMR spectroscopy; Foucaud C et al.; The metabolism of glucose was studied in Lactococcus lactis subsp . CNRZ 125 by 13C NMR . The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells {150 vs 85 nmol g (dry wt)-1 s -1} . 31P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate) . The internal pHs of L . lactis subsp . lactis CNRZ 141 and CNRZ 125 were also measured by 31P NMR as a function of the external pH during growth . When the external pH was 6.8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7.4 . After the external pH had decreased to 5.5, the internal pH of strain CNRZ 141 had declined by 0.6 unit, whereas that of strain CNRZ 125 had decreased by only 0.2 unit of pH.

Appl Environ Microbiol, 1995 Jul, 61(7), 2771 - 4
Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract; Klijn N et al.; A human feeding study was performed with Lactococcus lactis TC165.5, which is genetically marked by insertion of the sucrose-nisin conjugative transposon Tn5276 and chromosomal resistance to rifampin and streptomycin . The fate of strain TC165.5 and its nucleic acids was monitored by conventional plating methods and by molecular detection techniques based on specific PCR amplification of the nisin (nisA) gene from DNA extracted from human feces . A method was developed for the efficient extraction of microbial DNA from human feces . The results show that a fraction of viable cells of L . lactis TC165.5 survived passage through the human gastrointestinal tract . Only cells that passed within 3 days of ingestion could be recovered from the feces of the volunteers, and they accounted for approximately 1% of the total number of cells consumed . The presence of nisA in DNA extracted from feces could be detected up to 4 days, when viable cells were no longer present.

Appl Environ Microbiol, 1995 Jul, 61(7), 2540 - 7
Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80; Israelsen H et al.; Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci . Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C . Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed . Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated . In most cases, the regulation observed on plates was reproducible in liquid medium . One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase . DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli . A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp . cremoris and the Lactococcus lactis subsp . lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted . Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis . When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.

J Bacteriol, 1995 Jul, 177(13), 3824 - 9
Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1; Bidnenko E et al.; Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1 . Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction . A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans . Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4 . The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence . Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1 . However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.

J Bacteriol, 1995 Jul, 177(13), 3818 - 23
Characterization of the lactococcal abiD1 gene coding for phage abortive infection; Anba J et al.; Lactococcal phage abortive infection (AbiD1) determined by plasmid pIL105 is active on both prolate- and small-isometric-head phages of the C6A and 936 phage groups, respectively, which are considered two different species.The Abi phenotype was found to be encoded by a single gene, designated abiD1 . The abiD1-encoded protein (351 amino acids) does not show homology with any known protein and has a deduced isoelectric point of 10 . It also possesses two helix-turn-helix structures and an unusually high content of asparagine, isoleucine, and lysine . A consensual promoter with a TGy extension to the -10 box was mapped 76 bp upstream of the start codon . Transcription initiated at this strong promoter stops at a terminator located 48 bp downstream from the promoter . The termination process is very efficient, and transcripts corresponding to the abiD1 gene were not visible in our experimental conditions with or without phage infection . Expression of abiD1 under the control of a T7 promoter induced a lag phase in Lactococcus lactis cell growth, suggesting that overproduction of AbiD1 could be toxic for the cells . AbiD1 protein was visualized in Escherichia coli by using a tightly controlled expression system.

Appl Environ Microbiol, 1995 Jun, 61(6), 2193 - 202
Cloning and sequencing of LlaDCHI {corrected} restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system; Moineau S et al.; The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp . cremoris DCH-4 . It encodes a restriction/modification system named LlaDCHI {corrected} . When introduced into a phage-sensitive L . lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335 . The LlaDCHI {corrected} endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3' . It is an isoschizomer of Streptococcus pneumoniae DpnII . The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI {corrected} was localized . Cloning and sequencing of the entire LlaDCHI {corrected} system allowed the identification of three open reading frames . The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter . A putative terminator was found at the end of llaIIC . The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease . The LlaDCHI {corrected} system shares strong genetic similarities with the DpnII system . The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA . However, R.LlalII shared only 31% identity with R.DpnII.

J Bacteriol, 1995 Jun, 177(12), 3472 - 8
The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides; Juillard V et al.; The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp . cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry . After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained . The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface . Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide . All peptides corresponding to each of the MH+ calculated masses were determined . In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions . Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously . More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides . With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed . Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system . Uptake of these peptides could supply L . lactis with all amino acids, including the essential ones, indicating that growth of L . lactis might be possible on peptides released from beta-casein by proteinase only.

J Bacteriol, 1995 Jun, 177(11), 2982 - 9
Medium-dependent regulation of proteinase gene expression in Lactococcus lactis: control of transcription initiation by specific dipeptides; Marugg JD et al.; Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene (gusA) were used to study the medium- and growth-dependent expression of the divergently transcribed genes involved in proteinase production (prtP and prtM) of Lactococcus lactis SK11 . The results show that both the prtP and prtM genes are controlled at the transcriptional level by the peptide content of the medium and, to a lesser extent, by the growth rate . A more than 10-fold regulation in beta-glucuronidase activity was observed for both prtP and prtM promoters in batch and continuous cultures . The level of expression of the prtP and prtM promoters was high in whey permeate medium with relatively low concentrations of peptides, whereas at increased concentrations the expression of the promoters was repressed . The lowest level of expression was observed in peptide- and amino acid-rich laboratory media, such as glucose-M17 and MRS . The addition of specific dipeptides, such as leucylproline and prolylleucine, to the growth medium negatively affected the expression of the prtP-gusA fusions . The repression by dipeptides was not observed in mutants defective in the uptake of di-tripeptides, indicating that the internal concentration of dipeptides or derivatives is important in the regulation of proteinase production.

J Dairy Sci, 1995 Jun, 78(6), 1219 - 23
A milk-based method for detecting antimicrobial substances produced by lactic acid bacteria; Salama MS et al.; A new technique for the detection of antimicrobial substances produced by lactic acid bacteria has been developed . In this technique, milk agar plates were supplemented with tetrazolium chloride or tetrazolium blue dyes . Comparisons of milk agar assays with M17 agar plates indicated that, out of 30 bacterial strains, 13 strains produced bacteriocins or inhibitory substances that were detectable on milk agar plates but not on M17 agar plates . Multiple-strain lactococcal cultures are used in milk fermentations . To identify suitable strains to combine for industrial use, component strains must be tested for compatibility . The procedure described allows optimization of compatibility . The assay of putative producer and sensitive indicator strains using milk agar plates (11% nonfat dry milk plus .8% agar and .02% tetrazolium chloride or tetrazolium blue) provides an important tool to prevent allopathic interactions in mixed cultures.

J Biol Chem, 1995 May 19, 270(20), 12226 - 34
Cloning, expression, sequence analysis, and site-directed mutagenesis of the Tn5306-encoded N5-(carboxyethyl)ornithine synthase from Lactococcus lactis K1; Donkersloot JA et al.; The gene (ceo) encoding N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) has been isolated from the sucrose-nisin transposon Tn5306 of Lactococcus lactis K1, sequenced, and expressed at high level in Escherichia coli . The cloned enzyme has allowed the synthesis of the novel N omega-carboxypropyl amino acids N5-(1-carboxypropyl)-L-ornithine and N6-(1-carboxypropyl)-L-lysine . Comparison of the deduced amino acid sequence of N5-(1-carboxyethyl)-L-ornithine synthase (M(r) = 35,323) to the functionally analogous octopine and nopaline synthases from crown gall tumors showed surprisingly little similarity . However, N5-(1-carboxyethyl)-L-ornithine synthase and yeast saccharopine dehydrogenase exhibit homology at their N and C termini, which suggests that these two proteins constitute a distinct branch of the amino acid dehydrogenase superfamily . A centrally located 9-amino acid segment (GSGNVAQGA) in N5-(1-carboxyethyl)-L-ornithine synthase is virtually identical with a sequence present in the beta alpha beta-fold of the nucleotide binding domain of several microbial NADPH-dependent glutamate dehydrogenases . A much longer sequence of approximately 80 residues has significant similarity to alanine dehydrogenase . Substitution of arginine 15 of N5-(1-carboxyethyl)-L-ornithine synthase by lysine resulted in loss of enzyme activity.

Gene, 1995 May 19, 157(1-2), 13 - 8
Restriction-modification systems in Lactococcus lactis; Nyengaard N et al.; Several restriction-modification (R-M) systems have been identified in Lactococcus lactis . Most of the systems have been plasmid encoded and function as phage-resistance mechanisms . At least five different type-II R-M systems, LlaAI, LlaBI, LlaCI, LlaDI and LlaEI, were identified in isolates from a mixed Cheddar starter culture . LlaAI and LlaBI recognized the DNA sequences 5'- decreases GATC-3' and 5'-C decreases TRYAG-3', respectively . The genes coding for the LlaAI and LlaBI R-M systems have been cloned and sequenced . The LlaAI R-M system had two genes coding for methyltransferases (MTases) and one gene coding for a restriction endonuclease (ENase) . The MTases showed high homology to the MTases from DpnII . The LlaBI R-M system had one gene coding for a MTase and one gene coding for an ENase.

J Bacteriol, 1995 May, 177(10), 2840 - 50
Physical and genetic map of the Lactococcus lactis subsp . cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp . lactis IL 1403 reveals a large genome inversion; Le Bourgeois P et al.; A physical and genetic map of the chromosome of the Lactococcus lactis subsp . cremoris reference strain MG1363 was established . The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments . The MG1363 chromosome appeared to be circular and 2,560 kb long . Seventy-seven chromosomal markers were located on the physical map by hybridization experiments . Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome . The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction . Comparison of the L . lactis subsp . cremoris MG1363 physical map with those of the two L . lactis subsp . lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism . At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.

Int J Food Microbiol, 1995 May, 25(3), 301 - 10
Behavior of Listeria monocytogenes in Mozzarella cheese in presence of Lactococcus lactis; Stecchini ML et al.; The behavior of Listeria monocytogenes (Scott A) on fully processed Italian Mozzarella cheese was examined in presence and in absence of bacteriocins produced by Lactococcus lactis ssp . lactis strains (DIP 15 and DIP 16) . These strains, isolated from raw milk, produced heat stable bacteriocins that were inactivated by pronase, alpha- chymotrypsin and proteinase K, but not by pepsin, trypsin and catalase . The addition of crude bacteriocins to the growing culture of Listeria monocytogenes resulted in a significant reduction in cell number at 5 degrees C, but not at 30 degrees C . Mozzarella cheese was inoculated with the Listeria culture to obtain an initial level of approximately 30 CFU/cm2 surface of Mozzarella and approximately 10(3) CFU/ml of the surrounding fluid and then packaged in bags containing the heat-treated neutralized-cultures of Lactococcus lactis ssp . lactis in skim milk (in Italy, Mozzarella is sold in small size pieces, individually packaged in bags containing some fluid) . Bags were stored at 5 degrees C up to 21 days . The presence of bacteriocins resulted in apparent death of Listeria monocytogenes after 24 h storage . After 7 days of storage, a revival of Listeria monocytogenes was observed, followed by an increase in number . However, for a storage period of 2-3 weeks the number of Listeria monocytogenes remained significantly below the number observed for Mozzarella cheese packaged in absence of the heat-treated cultures of Lactococcus lactis.

Appl Environ Microbiol, 1995 May, 61(5), 2023 - 6
Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp . lactis KR5; McLandsborough LA et al.; A 6.3-kb fragment from pBF61 in Lactococcus lactis subsp . lactis KR5 was cloned and found to confer an abortive phage infection (Abi+) phenotype exhibiting a reduction in efficiency of plating and plaque size for small isometric- and prolate-headed bacteriophages sk1 and c2, respectively, and to produce a 10-fold decrease in c2 phage burst size . Phage adsorption was not significantly reduced . An open reading frame of 1,098 bp was sequenced and designated abiD . Tn5 mutagenesis confirmed that abiD was required for the Abi+ phenotype.

Plasmid, 1995 May, 33(3), 218 - 25
Novel insertion sequence-like element IS982 in lactococci; Yu W et al.; A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp . cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster . The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats . IS982 contained an open reading frame encoding a putative transposase of 296 amino acids . An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp . cremoris Wg2 with 17-bp perfect inverted repeats . Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20 . IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Yeast, 1995 Apr 30, 11(5), 455 - 8
Sequence analysis of a 5.6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28; Baur S et al.; The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28 . Gene disruption reveals both putative genes as non-essential . ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Surl protein of S . cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product.

Microbiology, 1995 Apr, 141 ( Pt 4), 1027 - 36
Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes; Cancilla MR et al.; The gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA . Plasmids containing the L . lactis gene were able to complement a gap mutant of Escherichia coli . The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36,043 . The codon usage in gap and four other glycolytic genes from L . lactis showed a high degree of bias, when compared with 84 other chromosomal genes . Northern blot analysis of total L . lactis RNA showed that gap hybridized strongly with a 1.3 kb transcript . The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon . These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L . lactis gap is expressed on a monocistronic transcript . Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes . The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function . The 3' end of a clpA homologue was identified in the sequence upstream of ORF156 . The location of gap on the L . lactis DL11 chromosome map was determined to be between map coordinates 0.530 and 0.660.

Appl Environ Microbiol, 1995 Apr, 61(4), 1627 - 9
Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp . lactis; Steidler L et al.; Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system . The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium . Sequence analysis revealed correct processing at the first amino acid of the mature protein . A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source.

J Appl Bacteriol, 1995 Apr, 78(4), 341 - 8
Locating nisin-producing Lactococcus lactis in a fermented meat system; Stringer SC et al.; Antibody-linked probes were used to locate nisin in a fermented meat system . Free nisin or nisin bound to susceptible cells or food components was not detected . Colonies of nisin-producing Lactococcus lactis were stained at all times during growth . The position of nisin-producing L . lactis colonies was noted and compared with the location of spoilage organisms or the distribution of areas with a fermented meat appearance . No relationship between the distribution of starter culture and the location of spoilage organisms or areas of fermentation was observed . In addition to the presence of L . lactis, a rapidly fermentable sugar was also required to obtain a fermented appearance and to reduce the levels of spoilage organisms.

Int J Food Microbiol, 1995 Apr, 25(2), 153 - 8
Enhancement of antigen-specific antibody production by extracellular slime products from slime-forming Lactococcus lactis subspecies cremoris SBT 0495 in mice; Nakajima H et al.; The effect of extracellular slime products (ESP) produced by Lactococcus lactis subspecies cremoris SBT 0495 on antigen specific antibody production was studied in mice . ESP contained 48.5% protein, 15.4% neutral sugar, and 1.1% of phosphorus . The optimum dose of ESP was between 100 to 500 micrograms per mouse . ESP administered intraperitoneally (200 micrograms per mouse) enhanced the production of specific antibody in mice . These results indicate that ESP may act as an adjuvant.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 105 - 9
Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon; MacCormick CA et al.; A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism . L . lactis MG5267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion . The lacF gene was deleted from this strain by a double cross-over homologous recombination event . The lacF-deficient strain produced a Lac- phenotype on indicator agar . A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF-deficient strain resulting in a Lac+ phenotype . This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2244 - 8
A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis; Biswas I et al.; Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected . It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD . Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro . A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria . The properties of this sequence in L . lactis correspond to those of a Chi site . Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Mol Gen Genet, 1995 Mar 10, 246(5), 590 - 9
Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression; Lopez de Felipe F et al.; The transport of citrate in Lactococcus lactis biovar diacetylactis is mediated by the citrate permease P . This polypeptide is encoded by the citP gene carried by plasmid pCIT264 . In this report, we characterize the citP transcript, identify a cluster of two genes cotranscribed with citP and describe their post-transcriptional regulation . The transcriptional promoter is located 1500 nucleotides upstream of the citP gene and the transcriptional terminator is positioned next to the 3'-end of this gene . The DNA sequence was determined of the region upstream of the citP gene, including the promoter . Two partially overlapping open reading frames, citQ and citR were identified, which could encode polypeptides of 3.9 and 13 kDa respectively . These two genes, together with citP, constitute the cit cluster . Moreover, an IS-like element located between the cit promoter and the citQ open reading frame was identified . This element includes an open reading frame ORF1, which could encode a 33 kDa polypeptide . A translational fusion between the citP and a cat reporter gene showed that translation of citR and citP is coupled, and regulated by CitR . The cit mRNA was subjected to specific cleavage after addition of rifampicin to the bacterial cultures . We propose that expression of the cit cluster is controlled at the post-transcriptional level by mRNA processing at a putative complex secondary structure and by translational repression mediated by CitR.

Appl Environ Microbiol, 1995 Mar, 61(3), 1082 - 9
Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3; Siegers K et al.; The lantibiotic nisin is produced by several strains of Lactococcus lactis . The complete gene cluster for nisin biosynthesis in L . lactis 6F3 comprises 15 kb of DNA . As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK . Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK . Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified . One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L . lactis 6F3 have now been identified . Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins . Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin . However, in comparison with the wild-type strain, these mutants were more sensitive to nisin . This indicates that besides nisI the newly identified genes are also involved in immunity to nisin . The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.

Int J Food Microbiol, 1995 Mar, 25(1), 83 - 94
Induction of thermotolerance by chemical agents in Lactococcus lactis subsp . lactis IL1403; Boutibonnes P et al.; Like in other organisms tested to date, adapted cells of Lactococcus lactis subsp . lactis IL1403 pretreated at 42 degrees C for 30 min develop a thermotolerant state, i.e . an increased ability to survive subsequent exposure to a lethal challenge temperature (52 degrees C for 15 or 30 min) . In different cellular systems, chemicals as diverse as divalent metal salts, natural or synthetic compounds trigger the development of thermotolerance . Yet, in L . lactis subsp . lactis IL1403, among the 17 chemicals tested, only four induced this transient increased tolerance to heat: cadmium chloride, mercury chloride, sodium azide and beta-mercaptoethanol . Intriguingly, none of these four compounds induced the synthesis of three major heat shock proteins (DnaK, GroEL and hsp104-analogue), which are believed to be responsible for thermotolerance in most organisms . It is suggested that: (i) the lesions produced by these various 'proteotoxic' agents are fundamentally different from those produced by heat; (ii) heat shock protein synthesis and transient induced tolerance to heat are not tightly correlated phenomena in L . lactis subsp . lactis as they are in Escherichia coli and some other organisms.

Plasmid, 1995 Mar, 33(2), 91 - 9
Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in Lactococcus lactis; Meijer WJ et al.; The effects of the single-strand origins (SSOs) of the broad-host-range streptococcal plasmid pMV158 on (i) the conversion of its single-stranded (ss) DNA replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed . pMV158 is distinguished from most other plasmids that replicate by the rolling-circle mechanism by the presence of two single-strand origins of replication, palA and palU . In this paper the results obtained with Lactococcus lactis are presented; complementary studies with Bacillus subtilis are presented in the accompanying paper (Meijer et al., 1995) . In the presence of both SSOs, no ss plasmid DNA was observed in L . lactis . The removal of either palA or palU resulted in the appearance of low amounts of ssDNA . High amounts of ssDNA were detected, however, when both SSOs were deleted . The results indicated that both SSOs were active, albeit that palU was the most effective of the two . In the presence of both SSOs, the plasmid was stably maintained in L . lactis under nonselective growth conditions . Also, the derivatives containing only one of the two SSOs were maintained rather stably . In contrast, the derivative devoid of both SSOs was poorly maintained . It was concluded that, in the absence of a functional SSO, the generation of large amounts of ssDNA drastically reduces the maintenance of pMV158 in L . lactis . The results also showed that the presence of the plasmid-located mob gene, required for conjugative mobilization, was involved neither in the accumulation of ssDNA nor in the maintenance of pMV158.

Plasmid, 1995 Mar, 33(2), 79 - 89
Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in different Bacillus subtilis strains; Meijer WJ et al.; The effects of the single-strand origins (SSOs) of plasmid pMV158 on (i) the conversion of its single-stranded (ss) replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed . The rolling-circle plasmid pMV158, which replicates via ssDNA intermediates, contains two single-strand origins (SSOs) of replication, palA and palU . In this paper the results obtained with Bacillus subtilis are described; complementary studies with Lactococcus lactis are presented in the accompanying paper (Meijer et al., 1995) . While in L . lactis both SSOs are functional as ssDNA conversion signal, only palU appeared to be active B . subtilis . Similar to the situation in L . lactis, the accumulation of large amounts of ssDNA resulted in a severe decrease in plasmid maintenance in B . subtilis . In the latter bacterium large amounts of ssDNA were only accumulated, however, when plasmids lacking a functional SSO were propagated in RecA mutant strains . In wild-type RecA strains these plasmids accumulated only modest amounts of ssDNA and they were maintained at fairly stable levels . The results suggest that in B . subtilis a RecA-mediated alternative pathway exists for the conversion of ssDNA which can improve plasmid maintenance . In addition to ssDNA accumulation and the antagonizing role of RecA therein, two other plasmid regions were shown to affect pMV158 maintenance in B . subtilis . One was the mob gene region, which had a negative effect on plasmid maintenance, and the other the palA type SSO . Although palA was not functional as an ssDNA conversion signal in B . subtilis, its presence had a positive effect on pMV158 maintenance.

Mol Microbiol, 1995 Mar, 15(5), 839 - 47
Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector; Dickely F et al.; Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant . The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined . One suppressor is an ochre suppressor with an altered tRNA(gln) and the other an amber suppressor with an altered tRNA(ser) . The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L . lactis MG1363 . A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker . Selection, following electroporation of a suppressible purine auxotroph, can be done on purine-free medium . The pepN gene from L . lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.

Biochim Biophys Acta, 1995 Feb 23, 1243(2), 209 - 15
An aminopeptidase P from Lactococcus lactis with original specificity; Mars I et al.; An aminopeptidase P (E.C . 3.4.11.9) that cleaves the Arg-1-Pro-2 bond of bradykinin has been isolated for the first time from Lactococcus lactis . The peptidase was purified to homogeneity in a 3-step procedure and characterized . It is a monomeric metalloenzyme with a 43 kDa molecular mass, activated by Mn2+ and inhibited by DTT . It differs from the majority of aminopeptidases P already described by displaying a specificity for X-Pro-Pro N-terminal and probably an extended binding site that could accommodate amino acid residues beyond the P'2 position of the substrate.

Gene, 1995 Feb 3, 153(1), 25 - 31
Competence for genetic transformation in Streptococcus pneumoniae: organization of a regulatory locus with homology to two lactococcin A secretion genes; Hui FM et al.; Regulation of competence for genetic transformation in Streptococcus pneumoniae involves the comAB locus and a small extracellular protein, the competence factor (CF) . The comA or comB mutations block both spontaneous competence induction and elaboration of CF, yet permit induction of competence by added CF and subsequent transformation at normal levels . Sequence and genetic studies showed that the com locus comprised the comA and comB genes, encoding 77- and 50-kDa proteins, respectively, and demonstrated that they were closely flanked by genes and DNA not required for competence regulation . In-frame deletion of comA demonstrated that the translation product of this gene is required for normal competence regulation; deletion-replacement mutations showed that no com gene lay in the 0.2-kb gap between comB and purC or within 2.5 kb upstream from comA . Strong sequence similarities (51-59% identities) showed that ComA and the proteins, PdcD and LcnC, which act in the secretion of pediocin A-1 and lactococcin A, respectively, form a subfamily within the large ABC-transporter protein family . ComB was found to be homologous to a single known protein, LcnD, required for secretion of the peptide antibiotic lactococcin A . Thus, the comAB locus displays homology to two lactococcin A secretion genes, but is devoid of additional linked com genes . The results suggest that the mechanism for CF production is similar to that for the small peptide bacteriocins, lactococcin A and pediocin A-1, but that its genetic organization is unusual in being split into at least two separate operons.

Microbiology, 1995 Feb, 141 ( Pt 2), 411 - 7
Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein; Duwat P et al.; The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp . cremoris ML3 has been cloned, characterized and sequenced . The fpg-L gene is composed of 819 bp encoding a protein of 31.3 kDa (Fpg-L) . The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identity with the Escherichia coli Fpg protein (Fpg-E) . Polyclonal antibodies against Fpg-E react with the Fpg-L protein . The Fpg-L protein was purified to apparent homogeneity from the overproducing E . coli strain BH410 hosting plasmid pVE1064, which carries fpg-L under the control of the E . coli lac promoter . In its active form, Fpg-L is a 30 kDa monomeric enzyme with a measured isoelectric point of 9.0 . It contains one zinc per molecule and has a zinc finger motif localized at the carboxyterminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH) . The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites . Furthermore, the expression of the fpg-L gene in fpg and mutY mutants of E . coli suppresses their spontaneous GC-->TA mutator phenotype . The similarity of the activity of the two Fpg proteins and its conversation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.

J Appl Bacteriol, 1995 Feb, 78(2), 109 - 15
Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages; Rodriguez JM et al.; A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production . Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria . BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics . The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin . This was confirmed by PCR analysis of their genomic DNA . Nucleotide sequencing revealed that they produced nisin A . The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L . lactis strains may be more widespread in meat products than previously thought . Nisin produced by L . lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography . The purification procedure was simple, rapid and reproducible.

Protein Eng, 1995 Feb, 8(2), 117 - 25
Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin; Siezen RJ et al.; A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin . This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin . Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP . Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin . A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin . Specific acidic residues in the S1 subsite of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the P1 Arg residue of precursor nisin after which cleavage occurs . The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins . Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP . An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Feb, 61(2), 788 - 92
Detection and characterization of lactose-utilizing Lactococcus spp . in natural ecosystems; Klijn N et al.; The presence of lactose-utilizing Lactococcus species in nondairy environments was studied by using identification methods based on PCR amplification and (sub)species-specific probes derived from 16S rRNA sequences . Environmental isolates from samples taken on cattle farms and in the waste flow of a cheese production plant were first identified to the genus level, using a Lactococcus genus-specific probe . Isolates which showed a positive signal with this probe were further identified to the (sub)species level . Lactococcus lactis isolates were also characterized at the phenotypic level for the ability to hydrolyze arginine, to ferment citrate, and to produce proteases and bacteriocins . With specific PCR amplifications, the presence of sequences related to citP, coding for citrate permease; prtP, coding for protease; and nisA or nisZ, the structural genes for production of nisin A or nisin Z, respectively, was verified . By these methods, it was possible to isolate lactococci from various environmental sources, such as soil, effluent water, and the skin of cattle . The strains of L . lactis isolated differed in a number of properties, such as the ability to hydrolyze arginine or the absence of citP-related sequences, from those found in industrial starter cultures . The results indicate that the majority of the industrially produced lactococci do not survive outside the dairy environment, although natural niches are available . However, from those niches strains with the potential to be developed into novel starter cultures may be isolated.

Appl Environ Microbiol, 1995 Feb, 61(2), 561 - 6
Cloning and molecular analysis of the dihydrofolate reductase gene from Lactococcus lactis; Leszczynska K et al.; The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis . Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR) . (i) It fully complements the fol "null" mutation in E . coli . (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes . (iii) Overexpression of this open reading frame in E . coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity . In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration . By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined . Results of these experiments indicate that in L . lactis the dhfr gene represents part of a larger transcription unit.

J Biol Chem, 1995 Jan 27, 270(4), 1569 - 74
Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis; Kunji ER et al.; In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP) . The fate of these peptides, i.e . extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed . Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein) . The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy . The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants . When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains . Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system . These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L . lactis.

Mol Gen Genet, 1995 Jan 6, 246(1), 119 - 27
Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis; Griffin HG et al.; Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA . An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined . This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism . TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids . The aroA gene from L . lactis has been shown to complement an E . coli mutant strain deficient in this gene . The arrangement of genes involved in aromatic amino acid biosynthesis in L . lactis appears to differ from that in other organisms.

Chin J Biotechnol, 1995, 11(4), 281 - 4
Cloning of a gene encoding the precursor of nisin by PCR; Huan L et al.; The structural gene for the precursor of nisin was synthesized by polymerase chain reaction and then cloned in pUC18 . The nucleotide sequence of the cloned precursor nisin gene was determined by dideoxy termination method . The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains.

J Tongji Med Univ, 1995, 15(4), 193 - 7
NisP is related to nisin precursor processing and possibly to immunity in Lactococcus lactis; Ye SY et al.; In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene . The mutant strain secreted fully modified nisin with the N-terminal leader still attached . The presence of the leader was confirmed by N-terminal sequencing of the purified precursor . The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells . Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity . The results show that NisP is needed fro precursor processing and for development of high immunity of nisin.

Dev Biol Stand, 1995, 85, 561 - 7
Molecular biology of lactococcal bacteriophage c2; Jarvis AW et al.; The 22163 bp genome of the lytic prolate-headed lactococcal phage c2 was fully sequenced . Mapping of restriction sites and RNA transcripts demonstrated the presence of early and late genes . Early and late promoters were identified . The early region contained 21 ORFs, with predicted protein products of 4.4-32.9 kDA, all reading right to left . Significant similarity was found between a putative protein encoded by an early region ORF and the erf (essential recombination function) gene product of Salmonella phage P22 . The late genes for which a function has been identified, all of which read from left to right, included a possible holin gene, and genes encoding three major and six minor phage structural proteins . Analysis of the cohesive termini revealed complementary, non-symmetrical, 9-base single-stranded 3' extended DNAs . The exploitation of phage sequence data and analysis of phage genomes to find ways of inhibiting phage replication is discussed.

Dev Biol Stand, 1995, 85, 535 - 41
Specificity of insertion of Tn1545 transposon family in Lactococcus lactis subsp . lactis; Renault P et al.; A collection of Lactococcus lactis subsp . lactis strains carrying a derivative of Tn1545 inserted in the chromosome was generated . Some 34 insertions were cloned in Escherichia coli and the flanking DNA sequences determined . Insertions are distributed non randomly and several hot spots were observed . The hot spots do not have sequence features which would distinguish them from other insertion sites, suggesting that they may have special structural properties . Insertions in open reading frames occurred at a far lower frequency than expected for random transposition and were most often located near potential terminator and promoter sequences . Different mutations in the extremities of the transposon do not affect the specificity of insertion . We suggest that target specificity is mainly due to the properties of the intergrase itself.

Dev Biol Stand, 1995, 85, 469 - 80
Polysaccharide expression in Lactococci; Dierksen KP et al.; Lactococcus lactis produces polysaccharide under defined environmental conditions . Three approaches are being used to identify regulators of polysaccharide synthesis in the organism . i) Two new lactococcal vectors, each of which contains a promoterless reporter gene, have been developed . They are being used to select for chromosomal insertions that affect expression of polysaccharide ii) Genetic complementation with lactococcal genomic libraries identified two classes of lactococcal genomic activities, both of which affect capsule expression in Escherichia coli iii) Lon, a highly conserved protease, is a negative regulator of polysaccharide synthesis in E . coli . Antiserum specific to Lon protease reacted with a lactococcal protein similar in size to E . coli Lon . This reaction was strongest in lactococcal strains which could not be induced to express polysaccharide.

Dev Biol Stand, 1995, 85, 455 - 67
recA gene involvement in oxidative and thermal stress in Lactococcus lactis; Duwat P et al.; The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction . There is gathering evidence that recA also affects expression of genes associated with different types of stress . We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain . recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair . Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals . Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C) . Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain . In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature . HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli . We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.

Dev Biol Stand, 1995, 85, 431 - 41
Metabolic operons in Lactococci; Renault P et al.; The genes for the biosynthesis of histidine, tryptophan and branched-chain amino acids (ilv for isoleucine, leucine and valine) are clustered in large operons . In addition to genes encoding the pathway enzymes, the his and the ilv operons contain 4 and 3 other genes, respectively . The functions of two of these, orf3 and aldB are regulatory . The second gene of the his operon, orf3, encodes a protein homologous to the histidyl-tRNA synthetases . It is involved in transcription attenuation upstream of the his operon . This regulation is related to a new class of attenuation mechanisms controlling the expression of most tRNA synthetase genes and a few metabolic operons in Gram-positive bacteria . Gene aldB, the penultimate gene of the ilv operon, encodes acetolactate decarboxylase . This enzyme transforms acetolactate (AL), the first intermediate of leucine and valine biosynthesis, into acetoin . AL decarboxylase is positively controlled by the availability of leucine and possibly valine in the cell . This is the key enzyme for a new class of regulatory mechanisms, a metabolic shunt which guides the AL flux towards synthesis of amino acids or a secondary metabolite.

Dev Biol Stand, 1995, 85, 411 - 22
Genomic organization of Lactococci; Davidson BE et al.; The genus Lactococcus contains four species of which L . lactis is the most thoroughly studied . Its genome is A+T-rich and consists of a circular chromosome of 2.0 to 2.7 Mbp, a wide range of plasmids, and frequently one or more prophages . Insertion sequence elements are commonly present in both the chromosome and the plasmids, while one conjugative transposon of 68 kbp has been described . Genetic maps of the chromosomes of a number of different L . lactis strains have been determined and found to differ quite significantly . For example, the maps of two L . lactis subsp . cremoris strains, MG1363 and FG2, have an inversion of approximately 40% of the chromosome when compared with the maps of two L . lactis subsp . lactis strains, DL11 and IL1403 . Other differences indicative of smaller scale translocations and inversions are also found . Chromosomal rearrangements have also been induced in the laboratory by incubation of L . lactis cultures with infecting lytic phage and by treatment with mutagens . These results are indicative of a great deal of plasticity in the lactococcal genome.

Appl Environ Microbiol, 1995 Jan, 61(1), 226 - 33
A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth; Verheul A et al.; Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein . This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A . Hagting, E . R . S . Kunji, K . J . Leenhouts, B . Poolman, and W . N . Konings, J . Biol . Chem . 269:11391-11399, 1994) . The peptide permease of L . monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides . No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell . Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L . monocytogenes cells grown in brain heart infusion broth or defined medium . The di- and tripeptide permease can supply L . monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods . Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L . monocytogenes are discussed.

FEMS Microbiol Lett, 1995 Jan 1, 125(1), 37 - 43
Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme; Denayrolles M et al.; Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into L-lactic acid . This direct decarboxylation is catalysed by the malolactic enzyme . Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme . Heterologous expression of mleS in Saccharomyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast . mleS gene was cloned in a yeast multicopy vector under a strong promoter . Malolactic activity was present in crude extracts of recombinant yeasts . Malic acid degradation was tested during alcoholic fermentation in synthetic media and must . Yeasts expressing the mleS gene actually produced L-lactate from L-malate; nevertheless malate degradation was far from complete.

J Bacteriol, 1995 Jan, 177(2), 283 - 9
Two Lactococcus lactis genes, including lacX, cooperate to trigger an SOS response in a recA-negative background; Huang XF et al.; A 4.3-kb EcoRI fragment from a Lactococcus lactis genomic library alleviates the methyl methanesulfonate, mitomycin C, and UV sensitivities of an Escherichia coli recA mutant (M . Novel, X . F . Huang, and G . Novel, FEMS Microbiol . Lett . 72:309-314, 1990) . It complements recA1 and delta recA mutations but not recA13 . Three proteins (with molecular masses of 20, 35, and 23 kDa) were produced from this fragment in a T7-directed system, and three corresponding genes were detected by DNA sequencing, namely, ISS1CH;lacX, which is the distal gene of the lac operon; and a third open reading frame, named lacN, which encodes 211 amino acids . Mutations produced in either lacX or in lacN resulted in the loss of the resistance to DNA-damaging agents . Thus, these two genes appeared to be involved in this activity . Introduction of pUCB214 carrying the 4.3-kb fragment into a lexA+ delta recA306 sfiA::lacZ strain resulted in UV-inducible synthesis of beta-galactosidase . A uvrA strain or a lexA (Ind-) strain containing pUCB214 did not support any DNA repair . However, a lexA (Def-) strain carrying pUCB214 could partly repair UV damage . We discuss possible targets for LacX and LacN products, and we speculate that LacX and LacN may constitute a two-component regulatory system that is able to respond to SOS signals, and then to act in the SOS response, bypassing the RecA-activated function.

Biosci Biotechnol Biochem, 1995 Jan, 59(1), 73 - 7
Identification and molecular analysis of Lactococcus lactis rpoD operon; Araya-Kojima T et al.; The complete nucleotide sequence of an open reading frame (ORF) preceding the Lactococcus lactis rpoD gene is reported . It was suggested that this ORF encodes Lactococcus lactis DNA primase and that the L . lactis rpoD operon consists of only two genes . Northern hybridization analysis showed that i) there are four mRNAs transcribing the rpoD gene (from upstream, M1-M4), ii) only the 3.7-kb transcript M1 includes the entire rpoD operon, iii) the shortest transcript M4 exists at both logarithmic and stationary phases of growth while the other three transcripts appear only at logarithmic phase, and iv) no apparent induction at the transcriptional level but transient repression of M1 was found when the growth temperature was shifted from 30 degrees C to 42 degrees C . 5'-ends of all four mRNAs were identified by primer extension analyses.

Curr Microbiol, 1995 Jan, 30(1), 33 - 7
Segregational stability and copy number of the theta-type lactococcal replicon Rep22 in Lactococcus; Frere J et al.; Rep22 is the replication region of the lactococcal theta replicating pUCL22 plasmid . The copy number of Rep22-based plasmids in Lactococcus was determined by using a chromosomal DNA fragment from Lactococcus lactis subsp . lactis MMS368 as reference . Segregational behavior appeared to be linked to copy number and therefore indicated random distribution of copies to daughter cells . Nevertheless, an active partitioning system was detected in the parental plasmid pUCL22 . A pUCL22 138-bp DNA restriction fragment bearing a perfect 18-bp inverted repeat was involved in the improvement of Rep22-based plasmid segregational stability during discontinuous exponential growth.

Plasmid, 1995 Jan, 33(1), 71 - 7
Use of continuous culture for the selection of plasmids with improved segregational stability; Seegers JF et al.; In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described . The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched . We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors . The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties . The first class involved mutations in the pAM beta 1-derived selection plasmid . These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable . The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids . We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance . The site-specific resolution function of pAM beta 1 was used as an example of the latter functions . We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

Anal Biochem, 1995 Jan 1, 224(1), 245 - 9
Preparation of bacterial X-prolyl dipeptidyl aminopeptidase and its stabilization by organic cosolvents; Chich JF et al.; To obtain large amounts of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp lactis (PepX, E.C . 3.4.14.5), PepX was purified from a commercial L . lactis cell extract . The enzyme was purified in only three steps and the last one was performed by HPLC on a C4 reverse-phase column using acetonitrile as an eluent . Despite its high molecular mass (175 kDa), the enzyme was recovered with a good activity yield (75%) . Advantages and drawbacks of this technique compared to the classical ones are discussed . The stability of the enzyme in aqueous solutions and in the presence of 10 water-miscible solvents was also investigated . PepX was found to be stabilized by dimethyl sulfoxide, triglyme, and glycerol.

DNA Seq, 1995, 5(4), 203 - 18
The codon usage of the nisZ operon in Lactococcus lactis N8 suggests a non-lactococcal origin of the conjugative nisin-sucrose transposon; Immonen T et al.; An 11.6 kb area downstream from the structural gene of nisin Z in the conjugative nisin-sucrose transposon of Lactococcus lactis subsp . lactis N8 was cloned and sequenced . Analysis of the sequence revealed eight open reading frames, nisZBTClPRK, followed by a putative rho-independent terminator (delta G degrees = -4.7 kcal/mol) . The C-terminal hydrophilic domain of the NisK protein is homologous to the C-termini of several histidine kinases of bacterial two-component regulator systems, such as SpaK from Bacillus subtilis and KdpD and RcsC of Escherichia coli . The nisin Z biosynthetic genes were highly similar with the genes of the nisin A operons having, however, a 0-3% difference in the amino acid sequences of the individual proteins . The codon usage of eleven genes within the same conjugative transposon was calculated and found to be strikingly different from that of other lactococcal genes . This, together with the low GC-content (32%) compared to the 38% (G+C) of the lactococcal chromosome in general strongly suggests a non-lactococcal origin of this transposon.

Microbiology, 1995 Jan, 141 ( Pt 1), 229 - 38
The Lactococcus lactis triosephosphate isomerase gene, tpi, is monocistronic; Cancilla MR et al.; Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity . Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells . In solution, triosephosphate isomerase (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) of noncovalently linked subunits . The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation . This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR . The probe was used to isolate a molecular clone of tpi from a lambda GEM11 library of L . lactis LM0230 DNA . The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit M(r) of 26,802 after removal of the NH2-terminal methionine . Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene . Northern analysis of L . lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi . The 5' end of the transcript was determined by primer extension analysis to be a G located 65 bp upstream from the tpi start codon . These transcript analyses indicated that in L . lactis, tpi is expressed on a monocistronic transcript . Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme . The location of tpi on the L . lactis DL11 chromosome map was determined to be between map coordinates 1.818 and 1.978.

J Bacteriol, 1995 Jan, 177(1), 134 - 43
In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030; O'Sullivan DJ et al.; The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis . The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located approximately 5 kb upstream from the abiA gene, encoding abortive phage resistance . In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs) . Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit . The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins . Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with MerB, in three regions that coincided with the GTP-binding motifs in both proteins . To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2 . The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L . lactis NCK203 than the low-copy-number parental plasmid, pTR2030 . A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3 . Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype . ORF4 had no discernible effect on in vivo restriction . A frameshift mutation in llaIM proved lethal to L . lactis NCK203, implying that the restriction component was active without the modification subunit . These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.

J Biol Chem, 1994 Dec 23, 269(51), 32070 - 6
Biochemical and genetic characterization of PepF, an oligopeptidase from Lactococcus lactis; Monnet V et al.; Lactococcus lactis possesses a complex proteolytic system which is essential for its growth in milk . We characterized one of the peptidases of this system, oligopeptidase PepF, together with its structural gene . PepF hydrolyzed peptides containing between 7 and 17 amino acids with a rather wide specificity . It was purified to homogeneity . The N-terminal sequences of PepF and of peptides resulting from tryptic digestion of PepF were determined and used to design degenerate oligonucleotides which served to amplify a DNA fragment internal to pepF . This fragment was used as a probe to screen a lactococcal genomic library in Escherichia coli and to clone the entire gene pepF . The gene coded for a 70 kDa protein and was located on a 55-kilobase lactose-protease plasmid . A motif His-Glu-X-X-His, characteristic of metallopeptidases was evidenced . Two regions of PepF were found similar, first to a stretch of 43 amino acids around the zinc-binding site of several other peptidases, second to a stretch of 33 amino acids well conserved among creatine and arginine kinases . Preliminary results suggest the presence of a second copy of pepF.

Microbiology, 1994 Dec, 140 ( Pt 12), 3421 - 9
Regulation of 2-deoxyglucose phosphate accumulation in Lactococcus lactis vesicles by metabolite-activated, ATP-dependent phosphorylation of serine-46 in HPr of the phosphotransferase system; Ye JJ et al.; Lactococcus lactis takes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase . Uptake of 2DG into L . lactis vesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP) . 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles . Intravesicular but not extravesicular wild-type HPr of Bacillus subtilis alone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DG-P . Intravesicular H15A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites . Intravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites . The S46D mutant HPr protein promoted regulation, even in the absence of a metabolite . The Vmax but not the Km value for 2DG uptake was affected . Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr . The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L . lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1994 Dec, 60(12), 4413 - 20
Genetic analysis of regions of the Lactococcus lactis subsp . lactis plasmid pRS01 involved in conjugative transfer; Mills DA et al.; The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp . lactis ML3 plasmid pRS01 were localized by insertional mutagenesis . Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency . Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer . Tra3 corresponds closely to a region previously identified (D . G . Anderson and L . L . McKay, J . Bacteriol . 158:954-962, 1984) . Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype . Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3 . When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.

J Bacteriol, 1994 Nov, 176(22), 6957 - 64
Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis; Bolhuis H et al.; Three mutants of Lactococcus lactis subsp . lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively . These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G . The three multidrug-resistant strains showed an increased rate of energy-dependent ethidium and daunomycin efflux compared with that of the wild-type strain . This suggests that resistance to these toxic compounds is at least partly due to active efflux . Efflux of ethidium from the EthR strain could occur against a 37-fold inwardly directed concentration gradient . In all strains, ethidium efflux was inhibited by reserpine, a well-known inhibitor of P-glycoprotein . Ionophores which selectively dissipate the membrane potential or the pH gradient across the membrane inhibited ethidium and daunomycin efflux in the wild-type strain, corresponding with a proton motive force-driven efflux system . The ethidium efflux system in the EthR strain, on the other hand, was inhibited by ortho-vanadate and not upon dissipation of the proton motive force, which suggests the involvement of ATP in the energization of transport . The partial inhibition of ethidium efflux by ortho-vanadate and nigericin in the DauR and RhoR strains suggest that a proton motive force-dependent and an ATP-dependent system are expressed simultaneously . This is the first report of an ATP-dependent transport system in prokaryotes which confers multidrug resistance to the organism.

J Bacteriol, 1994 Nov, 176(21), 6754 - 8
Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp . lactis and Escherichia coli; Bojovic B et al.; Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp . lactis NP4510 by using promoter-probe vector pGKV210 . N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L . lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression . Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine) . A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis . These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L . lactis and E . coli . The data suggest that cat expression is dependent on the secondary structure of the cat mRNA . New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.

J Bacteriol, 1994 Nov, 176(21), 6457 - 63
Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis; Martinussen J et al.; Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms . The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp . cremoris MG1363 by complementation of an Escherichia coli mutant . The gene was sequenced, and the putative amino acid sequence was deduced . The promoter was mapped by both primer extension and analysis of beta-galactosidase expressed from strains carrying fusion between upp promoter fragments and the lacLM gene . The results showed that the upp gene was expressed from its own promoter . After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event . This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains . The phenotype of the uracil phosphoribosyltransferase-deficient strain was established . Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil . Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance to high concentrations of 5-fluorouracil.

Microbiology, 1994 Nov, 140 ( Pt 11), 3061 - 9
Sequence and organization of the lactococcal prolate-headed bIL67 phage genome; Schouler C et al.; bIL67 is a broad-host-range prolate-headed phage that is active against Lactococcus cells . The complete phage genome sequence of 22195 bp was established . Thirty-seven open reading frames (ORFs) organized in two clusters were identified . Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs . These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.

Microbiology, 1994 Nov, 140 ( Pt 11), 3053 - 60
Mosaic structure of large regions of the Lactococcus lactis subsp . cremoris chromosome; Delorme C et al.; Lactococcus lactis subsp . lactis and Lactococcus lactis subsp . cremoris are closely related phenotypically and genetically . Here we report that certain regions of their chromosomes diverge considerably more than others . Conserved regions differ by less than 20%, whilst variable regions differ by more than 60% . This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L . lactis subsp . cremoris chromosome the G+C content and the codon bias are not typical for lactococci . Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.

Mol Microbiol, 1994 Nov, 14(3), 521 - 32
Mode of action of LciA, the lactococcin A immunity protein; Venema K et al.; Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A . One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L . lactis producer cell . LciA was present in the cytosolic, the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A . The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein . It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A . Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA . The epitope in LciA was localized between amino acid residues 60 and 80 . As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell . The immunity protein contains a putative alpha-amphiphilic helix from residue 29 to 47 . A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell . Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.

Mol Gen Genet, 1994 Oct 28, 245(2), 160 - 6
Sequencing and analysis of the cos region of the lactococcal bacteriophage c2; Lubbers MW et al.; The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3' extended DNAs, with the following sequence: 5'-GTTAGGCTT-3' 3'-CAATCCGAA-5' . DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found . Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features . All of the bacteriophages from gram-positive hosts had 3' extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5' extended DNA termini . All bacteriophages had a region of dyad symmetry close to the cohesive termini . A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.

FEMS Microbiol Lett, 1994 Oct 1, 122(3), 289 - 95
Comparative analysis of gene expression in Streptococcus pneumoniae and Lactococcus lactis; Lopez de Felipe F et al.; The pFL10 plasmid vector for translational fusions was constructed . pFL10 is based in the promiscuous pLS1 replicon and contains the pC194 cat gene deprived of its transcriptional promoter and Shine-Dalgarno (SD) sequence . Three promoters (Pcit, PpolA and PtetL) from Gram-positive bacteria, inserted in pFL10, were tested for their ability to drive transcription in Lactococcus lactis and Streptococcus pneumoniae . These promoters were coupled to the SD sequence of the lactococcal citP gene fused to the cat gene . Determination of the 5' ends of the three mRNAs by primer extension revealed the same start sites in both bacterial systems . However, it was observed a differential efficiency of promoter utilization by the RNA polymerases from the two hosts . The transcriptional behavior correlates with expression of the cat gene measured by determinations of chloramphenicol acetyltransferase (CAT) activity . Substitution of the SD of citP by that of the T7 phi 10 gene rendered a similar decrease of the CAT production in both bacterial systems.

Yeast, 1994 Oct, 10(10), 1389 - 94
Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA-protein family; Guelin E et al.; A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature-sensitive respiratory-deficient mutant . DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA-protein family (ATPases associated with diverse cellular activities) . The members of this family are involved in very different biological processes . Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post-translational processing of PBP3, a protein necessary for septation during cell division . This newly sequenced gene, which we have designated AFG3 for ATPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively.

Biotechnology (N Y), 1994 Oct, 12(10), 1017 - 23
A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli; Gram H et al.; We describe a novel approach to the production in E . coli of a peptide fragment derived from the human parathyroid hormone (hPTH) . The first 38 amino acids of hPTH were fused at the amino terminus to a derivative of the bacteriophage T4-encoded gp55 protein, and were expressed in the E . coli cytoplasm in inclusion bodies at levels exceeding 50% of the total cell protein . Solubilization and subsequent incubation of the inclusion bodies in dilute hydrochloric acid facilitated the cleavage of an acid-labile aspartyl-prolyl peptide bond engineered into the fusion protein, thus releasing the hormone fragment directly from the inclusion body preparation . The amino-terminal prolyl-prolyl dipeptide-extension was subsequently removed by treatment with Lactococcus lactis dipeptidyl peptidase IV which was overexpressed in E . coli and purified to near homogeneity from the cytosol of the recombinant bacteria . In pilot-scale fermentations, more than 80 mg of pure hPTH(1-38) were isolated per liter of bacterial culture, with an overall yield of 35% . This process is suitable for scale-up, is cost effective, and by employing recombinant dipeptidyl peptidase IV, should be widely and directly applicable to the manufacturing of peptides of pharmaceutical interest.

FEBS Lett, 1994 Sep 26, 352(2), 180 - 4
Confirmation of the existence of a third family among peptidyl-prolyl cis/trans isomerases . Amino acid sequence and recombinant production of parvulin; Rahfeld JU et al.; In addition to the major cyclophilin-like peptidyl-prolyl cis/trans isomerases (PPIases) of Escherichia coli an enzyme of very low relative molecular mass (10.1 kDa) was discovered in this organism which gave first indication of the existence of a novel family in this enzyme class {1994, FEBS Lett . 343, 65-69} . In the present report we describe the chemically determined amino acid sequence of four peptides derived from the 10.1 kDa protein by the treatment with either cyanogen bromide or endoproteinase Lys-C . Together with a continuous run of 75 amino acids starting N-terminally, the sequence of the mature enzyme, 92 residues in length, was elucidated . Cloning and determination of the primary structure of a DNA fragment encoding this enzyme were also performed . Overexpression of the enzyme by using multicopies of plasmid pSEP38 in E . coli and detecting an enhanced PPIase activity attributed to the 10.1 kDa enzyme provided additional proof that the 92 amino acid protein was a PPIase . The enzyme was called parvulin (lat.: parvulus, very small) . Homology analyses indicated that several parvulin-like proteins could be found in the database screened . To further elucidate the functional role of PPIases it might be of some importance that homologous proteins like the PrtM protein of Lactococcus lactis and the PrsA lipoprotein of Bacillus subtilis are known to be involved in the protein export and maturation machinery of the bacteria.

Biochem J, 1994 Sep 15, 302 ( Pt 3), 957 - 63
Prevention of C-terminal autoprocessing of Lactococcus lactis SK11 cell-envelope proteinase by engineering of an essential surface loop; Bruinenberg PG et al.; The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has various inserts, situated in external loops of the catalytic domain, compared with the related subtilisins . Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis . We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4, 7 and 16 serine residues) . Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity . The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substrates . The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic specificity or altered accessibility of the processing site . The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the breakdown of casein substrates.

Biochim Biophys Acta, 1994 Sep 13, 1219(1), 141 - 4
A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis; Nilsson D et al.; A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced . Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L . lactis . Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).

J Bacteriol, 1994 Sep, 176(17), 5304 - 11
Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp . lactis and their regulation in maltose- and glucose-utilizing cells; Qian N et al.; Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp . lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM) . beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared . The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8 . The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively . The expression of both PGM enzymes was investigated under different growth conditions . The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium . When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly . This suggests that synthesis of beta-PGM is repressed by glucose in the medium . Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.

Microbiology, 1994 Sep, 140 ( Pt 9), 2251 - 61
Temporal transcription map of the Lactococcus lactis bacteriophage sk1; Chandry PS et al.; Bacteriophage sk1 is a small isometric-headed lytic phage that infects Lactococcus lactis . The phage has a linear double-stranded DNA genome of 28 kbp, with cohesive ends . RNA was prepared from phage-infected L . lactis cells harvested at various intervals after infection, and the RNA molecules were resolved by electrophoresis . Northern blots of these gels were hybridized with sk1 DNA probes and the results obtained from these experiments, together with the results of primer extension analyses, enabled a transcription map of the phage genome to be prepared . Three classes of phage transcripts, designated as early, middle or late based on their time of appearance, were detected . Seven partially overlapping early transcripts were detected; these were transcribed from a 10 kbp region of the phage . The nine middle transcripts were derived from a 2 kbp region, limited by cos at one end and the start of the early transcripts at the other . The early and middle transcripts were transcribed divergently from a region mapping at 26 kbp on the sk1 physical map . The four late transcripts were derived from a 16 kbp region of the phage limited at one end by cos . The late transcripts were transcribed in the opposite direction to the early transcripts and three of the late transcripts terminated in the same region of the phage genome as three of the early transcripts.

Appl Environ Microbiol, 1994 Sep, 60(9), 3063 - 73
Controlled expression and structural organization of a Lactococcus lactis bacteriophage lysin encoded by two overlapping genes; Shearman CA et al.; The phi vML3 bacteriophage lysin is specific for lactococci and could be used to promote enzyme release during cheese manufacture . The level of lysin expression from the cloned gene using its own upstream sequences is very low . Expression in Escherichia coli by using a synthetic hybrid lysin gene and a series of BAL 31 deletions of the original cloned DNA fragment suggested that the start of the gene had previously been incorrectly assigned . Reevaluation of homology between the lysin and Bacillus subtilis PZA protein 15 led to the identification of a new potential ribosome binding site (RBS) . A 0.72-kb PCR-generated fragment including this RBS and the complete lysin gene was expressed and inducibly controlled . The translational start of the lysin gene was identified as an isoleucine codon, and this may lead to a low translation rate . During the analysis of the BAL 31 deletion fragments, two proteins of 20 and 8 kDa were shown to be expressed from the originally defined lysin gene . The DNA sequence has a second open reading frame with a good RBS and two potential start methionines . The smaller lysin protein was isolated, and the N terminus was sequenced, confirming that one methionine codon acted as the start of a second gene . The larger lysin protein has homology with lysozymes . The smaller lysin protein has some features resembling those of a holin . The possible roles of these two proteins in lysis of lactococci are discussed.

FEBS Lett, 1994 Aug 29, 351(1), 95 - 9
Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp . lactis NCDO 2118; Phalip V et al.; alpha-Acetolactate decarboxylase from Lactococcus lactis subsp . lactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable . The purification was carried out from E . coli in which the enzyme was expressed 36-fold higher . The specific activity was 24-fold enhanced after purification . The main characteristics of alpha-acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine . The enzyme is composed of six identical subunits of 26,500 Da.

Mol Gen Genet, 1994 Aug 15, 244(4), 374 - 82
Genetic analysis of the minimal replicon of the Lactococcus lactis subsp . lactis biovar diacetylactis citrate plasmid; Pedersen ML et al.; Using a combination of mutagenesis with the transposon gamma delta and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp . lactis biovar diacetylactis citrate plasmid have been identified . An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential . This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid . A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication . A region containing two 10 bp direct repeats and three tandem repeats of a 22 bp sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication . A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon . Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.

J Appl Bacteriol, 1994 Aug, 77(2), 229 - 36
Characteristics of Vagococcus salmoninarum isolated from diseased salmonid fish; Schmidtke LM et al.; Isolates of the salmonid pathogen Vagococcus salmoninarum were recovered from Atlantic salmon, rainbow trout and brown trout with peritonitis . The phenotypes of these isolates and the type strain of Vag . salmoninarum NCFB 2777 were determined by morphological, biochemical and physiological tests and whole cell protein profiles by SDS-PAGE . There was a high level of phenetic similarity between the salmonid isolates and the type strain . The species forms short Gram-positive rods, hydrolyses L-pyrrolidonyl-beta-naphthylamide, is alpha-haemolytic on sheep's blood agar, grows at pH 9.6 and 10 degrees C but not at 40 degrees C or in 6.5% NaCl and is catalase-negative; a Lancefield group N antigen is not present . Vagococcus salmoninarum can be distinguished phenetically from similar fish pathogens including Carnobacterium piscicola, Enterococcus seriolicida and Lactococcus piscium.

Biotechnol Appl Biochem, 1994 Aug, 20 ( Pt 1), 131 - 40
Xaa-Pro-dipeptidyl-aminopeptidase from Lactococcus lactis catalyses kinetically controlled synthesis of peptide bonds involving proline; Yoshpe-Besancon I et al.; Xaa-Pro-dipeptidyl-aminopeptidase (EC 3.4.14.5) from Lactococcus lactis (PepX) was used, for the first time, as a catalyst in kinetically controlled synthesis of peptide bonds involving proline . PepX had amidase and esterase activities in addition to peptidase activity . Thus amide and ester derivatives of X-Pro peptides could be employed as acyl donors . PepX showed a broad specificity for the residue in position P'1, accepting a large variety of amino acid amides, esters, peptides as well as free amino acids as nucleophiles . This also indicated that it was not necessary to protect the C-terminus of the nucleophile . The major factors controlling yield, e.g . pH, an excess of nucleophile, ionic strength and type of carboxyl protecting and activating groups, were evaluated . Under optimum reaction conditions (pH 8.5, high excess of nucleophile over acyl donor and moderate ionic strength) the selectivity of the reaction ranged from 5 to 99% depending on the structure of the nucleophile and the acyl donor . Our work contributes to the elucidatation of the mechanism of aminolysis reactions catalysed by an aminopeptidase.

Appl Microbiol Biotechnol, 1994 Aug, 41(6), 644 - 51
Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp . cremoris AM1 on bovine kappa-casein; Visser S et al.; The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp . cremoris AM1 in its action on bovine kappa-casein was studied . A 4-h digest (pH 6.2, 15 degrees C) of kappa-casein was made with the purified proteinase . The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques . Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry . On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components . The HMM products formed were the fragments 1-160, 1-151, 1-95 and 1-79 of kappa-casein, whereas the main LMM products found were the 161-169 and 152-160 fragments . The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds . Two minor LMM products were identified as the fragments 96-104 and 103-106, indicating additional cleavage at positions 102-103, 104-105 and 106-107 of the sequence . Also several peptide bonds within the 161-169 sequence were found to be subject to secondary cleavage by the proteinase . From electrophoretic and identification data it is concluded that the lactococcal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1994 Aug, 7(8), 991 - 6
Evidence for a large dispensable segment in the subtilisin-like catalytic domain of the Lactococcus lactis cell-envelope proteinase; Bruinenberg PG et al.; The Lactococcus lactis SK11 cell-envelope proteinase contains various inserts, located in external loops of the catalytic domain compared with related subtilisins . In this study, protein engineering was employed to determine the function of the largest loop insertion (residues 238-388) relative to the subtilisin structure . By site-directed mutagenesis we have deleted the fragment of the proteinase gene encoding these 151 residues and analyzed the mutant delta 238-388 proteinase for activity, (auto)processing and cleavage specificity . This extra segment is found to be inessential for activity, and its removal does not inhibit folding as the mutant proteinase is still active . In addition, the N- and C-terminal autoprocessing of the delta 238-388 proteinase appears to be unchanged . However, removal of residues 238-388 altered substantially the caseinolytic specificity of the enzyme, indicating that this extra segment influences substrate specificity . Residues 238-388 were shown to contain a specific epitope for a monoclonal antibody.

FEMS Microbiol Lett, 1994 Jul 15, 120(3), 249 - 56
The immune response to Lactococcus lactis: implications for its use as a vaccine delivery vehicle; Norton PM et al.; The development of natural antibodies to Lactococcus lactis in three inbred strains of mice and the effect of inoculating L . lactis into these mice has been investigated . All animals developed detectable levels of natural (systemic and secretory) anti-lactococcal antibodies . Systemic anti-lactococcal antibodies were principally IgG in young mice . An increase in anti-lactococcal IgM occurred in all older animals . Inoculation of L . lactis strains MG1820 or MG1363 by the oral and parenteral routes induced systemic anti-lactococcal antibody responses in a dose-dependent fashion . The relative immunogenicity of individual bacterial proteins varied according to mouse strain and route of inoculation.

Appl Environ Microbiol, 1994 Jul, 60(7), 2324 - 9
Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363; van de Guchte M et al.; The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented . In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified . The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp . cremoris UC509 were determined . A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L . lactis subsp . cremoris MG1363 . This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.

J Bacteriol, 1994 Jul, 176(13), 3975 - 82
Two different dihydroorotate dehydrogenases in Lactococcus lactis; Andersen PS et al.; The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms . The general pathway consists of six enzymatic steps . In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated . The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined . One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis . It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors . We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes . Only the double mutant is pyrimidine auxotrophic.

Protein Sci, 1994 Jul, 3(7), 1114 - 6
Expression, purification, and characterization of thymidylate synthase from Lactococcus lactis; Greene PJ et al.; The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli . The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose . Six grams of cell pellet yielded 140 mg of homogeneous TS . TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L . lactis TS . By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences . With the large amounts of L . lactis TS now available, studies can be pursued to understand the structure-function relationships of this enzyme compared to other TSs and to confirm the presumed roles of the compensatory changes predicted in the homology model.

Gene, 1994 Jun 24, 144(1), 93 - 5
Identification of the putative repressor-encoding gene cI of the temperate lactococcal bacteriophage Tuc2009; van de Guchte M et al.; The putative repressor-encoding gene cI of the temperate lactococcal bacteriophage Tuc2009 was cloned and sequenced . In the inferred amino-acid sequence, two domains can be recognized, one of which shows homology to DNA-binding domains of various regulatory proteins, while the other is thought to be involved in oligomerisation.

Biochemistry, 1994 Jun 7, 33(22), 6911 - 7
Purification and characterization of colicin V from Escherichia coli culture supernatants; Fath MJ et al.; The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC) . Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa . The protein identified remains biologically active after purification and SDS-PAGE . A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues . The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC . N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids . The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase . The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria . Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide . The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

J Bacteriol, 1994 Jun, 176(11), 3393 - 6
Characterization of IS905, a new multicopy insertion sequence identified in lactococci; Dodd HM et al.; IS905 is a multicopy insertion sequence identified in Lactococcus lactis . It is 1313 bp long, bounded by 28-bp imperfect inverted repeats, and encodes a putative transposase of 391 amino acids . One end of IS905 contains sequences that are potentially promoter active . It displays sequence homology to the IS256 class of elements.

Microbiology, 1994 Jun, 140 ( Pt 6), 1301 - 5
Cloning, sequencing and comparison of three lactococcal L-lactate dehydrogenase genes; Swindell SR et al.; The conversion of pyruvate to lactate is a key feature of lactococcal strains . The enzyme which facilitates this conversion, L-lactate dehydrogenase (LDH), and the gene which encodes it (Idh), are therefore of great significance . This paper presents the cloning and DNA sequence analysis of three further lactococcal genes which are of key importance in the genetic manipulation of commercial starter strains . The Idh gene from Lactococcus lactis subsp . Lactis biovar diacetylactis BU2-60 has been isolated from a lambda library and sequenced . The Idh gene from L . lactis subsp . cremoris NCDO 762 and that from L . lactis subsp . Lactis IL1403 have been amplified by the polymerase chain reaction (PCR) and sequenced . These DNA sequences and deduced amino acid sequences have been compared with those from L . lactis subsp . Lactis MG1363 . The LDHs from L . lactis subsp . Lactis MG1363 and L . lactis subsp . cremoris NCDO 762 are 99.4% homologous . The LDHs from L . lactis subsp . lactis MG1363 and L . lactis subsp . Lactis IL1403 are 96.4% homologous . The LDHs from L . lactis subsp . lactis IL1403 and L . lactis subsp . lactis biovar diacetylactis BU2-60 are 99.9% homologous . Our results provide further evidence that L . lactis subsp . Lactis MG1363 and other L . lactis subsp . Lactis NCDO 712 derived strains should be reclassified as Lactococcus lactis subsp . cremoris.

Microbiology, 1994 Jun, 140 ( Pt 6), 1291 - 300
The majority of lactococcal plasmids carry a highly related replicon; Seegers JF et al.; DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons . pWVO2, pWVO4 and pWVO5, which co-exist in Lactococcus lactis subsp . cremoris Wg2, and pIL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids . They contain a gene encoding a highly conserved RepB protein (60-80% amino acid identity between pWVO2, pWVO4 and pWVO5), which is essential for replication . When supplied in trans, repB of pWVO2 complemented a repB deficiency of pWVO5 . Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an A/T-rich region . The similarity with pWVO2, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication . Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.

Appl Environ Microbiol, 1994 Jun, 60(6), 1875 - 83
Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins; Arendt EK et al.; Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp . cremoris UC509 . The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy . The unit genome size is approximately 39 kb . A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins . lys, S, mp1, and mp2 were further characterized . lys and S are partially overlapping and appear to be part of one operon . The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7 . The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.

Appl Environ Microbiol, 1994 Jun, 60(6), 1798 - 804
Distribution and evolution of nisin-sucrose elements in Lactococcus lactis; Rauch PJ et al.; The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied . Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L . lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene . Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated . Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively . Representative conjugative transposons of these classes include Tn5276 (class I) from L . lactis NIZO R5 and Tn5278 (class II) from L . lactis ILC11 . The class II transposon found in L . lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition . Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position . The class III element from L . lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation . The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain . A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.

Gene, 1994 May 3, 142(1), 91 - 6
Cloning and sequencing of the Lactococcus lactis subsp . lactis dnaK gene using a PCR-based approach; Barril JS et al.; The coding region for the dnaK gene from Lactococcus lactis subsp . lactis LM0230 was isolated and sequenced . An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK . This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene . The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions . The inverse PCR products were then cloned and partially sequenced . The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein . Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.

J Bacteriol, 1994 May, 176(10), 2854 - 61
Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant; Mierau I et al.; The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp . cremoris (formerly subsp . lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced . The tripeptidase of L . lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences . L . lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E . coli strain by liberation of Leu from a tripeptide . Using a two-step integration-excision system, a pepT-negative mutant of L . lactis was constructed . No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.

Appl Environ Microbiol, 1994 May, 60(5), 1652 - 7
Cloning, expression, and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from lactococcus lactis subsp . lactis; Rince A et al.; The partial nucleotide sequence of a Lactococcus lactis subsp . lactis ADRIA 85LO30 bacteriocin-producing operon was determined . The first two open reading frames of the operon are necessary to get bacteriocin expression in L . lactis IL1403R.

J Appl Bacteriol, 1994 May, 76(5), 431 - 41
Genetic studies of lactococcal bacteriophages--taxonomic differentiations and DNA analysis: evidence for 3' cohesive ends; Ermel G et al.; Twenty-four bacteriophages of Lactococcus lactis subsp . lactis and L . lactis subsp . cremoris were classified . Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies . These criteria showed that prolate phages are quite homogeneous . In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions . Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52 . These two families were clearly defined . The unique phage which does not fit in either group probably belongs to a third one which is much less represented . No obvious relationships between these criteria and the lytic spectra were detected . Evidence of the presence of cohesive ends in phage genomes is also presented in this study . A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.

Mol Microbiol, 1994 May, 12(4), 655 - 63
The Lactococcus lactis sex-factor aggregation gene cluA; Godon JJ et al.; A gene, cluA, was cloned from the chromosomally located sex factor of Lactococcus lactis MG1363 . Sequence analysis revealed significant homology with previously described aggregation proteins in Enterococcus and Streptococcus species . The possibility that cluA was an equivalent protein involved in cell aggregation between donor and recipient bacteria during lactococcal conjugation was confirmed by its expression under the control of a heterologous promoter in L . lactis . Analysis of the homology between the CluA protein and the related proteins of Enterococcus and Streptococcus allowed a common structure for these proteins to be postulated . This consisted of five domains . Functionally conserved domains I and V act respectively as a secretory leader and C-terminal membrane anchor . Domains II and IV are conserved at the amino acid level and probably have common structural roles whereas domain III is variable and may control binding specificity.

Lett Appl Microbiol, 1994 May, 18(5), 292 - 3
Rapid isolation and purification of lactococcal bacteriophage DNA without the use of caesium chloride gradients; Brown JC et al.; Intact bacteriophage of Lactococcus lactis were recovered from small volumes of lysate by centrifugation at 15,000 g without precipitation with polyethylene glycol and sodium chloride, or ultracentrifugation in a caesium chloride gradient . DNA was then extracted and purified by standard protocols . This DNA was readily digested with restriction endonucleases and used successfully in hybridization experiments.

J Biotechnol, 1994 Apr 30, 34(1), 87 - 95
Influence of dilution rate and cell immobilization on plasmid stability during continuous cultures of recombinant strains of Lactococcus lactis subsp . lactis; D'Angio C et al.; The influence of dilution rate and cell immobilization on plasmid stability in recombinant strains of Lactococcus lactis subsp . lactis was investigated during continuous cultures . The studied strains, L . lactis IL2682 and IL2683, contained plasmids pIL9 (Lac+), pIL205 (CmR) and plasmids pIL252 (low copy number) and pIL253 (high copy number), respectively, that conferred resistance to erythromycin . Plasmid pIL205 was remarkably stable . Dilution rate did not affect the rate of loss of plasmids pIL252 and pIL253 significantly . Nevertheless, the loss of plasmid pIL253 was apparent after a further 21 generations when the dilution rate was decreased from 0.70 h-1 to 0.55 h-1 . Cell immobilization in beads of kappa-carrageenan/locust bean gum improved plasmid stability by factors of 4.5 for pIL253 and 6.5 for pIL252 . Thus, 10% of cells containing plasmids pIL252 or pIL253 were still present after 370 or 540 generations, respectively, compared with 50 or 210 generations in free cell cultures.

J Biol Chem, 1994 Apr 22, 269(16), 11837 - 44
Inhibition of the phosphoenolpyruvate:lactose phosphotransferase system and activation of a cytoplasmic sugar-phosphate phosphatase in Lactococcus lactis by ATP-dependent metabolite-activated phosphorylation of serine 46 in the phosphocarrier protein HPr; Ye JJ et al.; Lactococcus lactis takes up lactose and the nonmetabolizable lactose analogue, thiomethyl-beta-galactoside (TMG), via the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which couples sugar transport to sugar phosphorylation . Earlier studies had shown that TMG-phosphate, previously accumulated in L . lactis cells, is rapidly dephosphorylated in the cytoplasm and effluxes from the cells upon addition of glucose and that glucose inhibits further uptake of TMG . We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeable metabolites into the vesicles . Uptake of TMG was dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate at low concentrations or extravesicular phosphoenolpyruvate at high concentrations . TMG uptake into osmotically shocked vesicles was only weakly inhibited, and expulsion of preaccumulated TMG was only slightly stimulated upon addition of glucose . Intravesicular (but not extravesicular) wild-type HPr of Bacillus subtilis completely restored the regulatory behavior observed in vivo when glucose was present in the external medium . Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-diphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro . Intravesicular mutant HPr(S46A) protein could not promote regulation of lactose permease activity when electroporated into the vesicles regardless of the presence or absence of glucose or the various phosphorylated metabolites, but the HPr(S46D) mutant protein promoted regulation, even in the absence of glucose or a metabolite, and HPr(H15A) was more effective than the wild-type protein in promoting regulation . Intravesicular wild-type and H15A HPrs, but not the S46A or S46D mutant proteins, were found to be phosphorylated by ATP under the conditions which promoted TMG efflux . In toluenized vesicles, the conditions which promoted TMG efflux also promoted TMG-P hydrolysis . These results establish for the first time that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates the expulsion of intracellular sugar-phosphate as well as the uptake of sugar via the PTS in L . lactis.

J Biol Chem, 1994 Apr 15, 269(15), 11391 - 9
The di- and tripeptide transport protein of Lactococcus lactis . A new type of bacterial peptide transporter; Hagting A et al.; Lactococcus lactis takes up di- and tripeptides via a proton motive force-dependent carrier protein . The gene (dtpT) encoding the di-tripeptide transport protein of L . lactis was cloned by complementation of a dipeptide transport-deficient and proline auxotrophic Escherichia coli strain . Functional expression of the dipeptide transport gene was demonstrated by uptake studies of alanyl-{14C}glutamate and other peptides in E . coli cells . The di-tripeptide transport protein catalyzes proton motive force-driven peptide uptake and dipeptide exchange activity . The nucleotide sequence of dtpT was determined and the translated sequence corresponds with a protein of 463 amino acid residues . Hydropathy profiling indicates that the protein could form 12 membrane-spanning segments with the amino and carboxyl termini at the outer surface of the membrane . A secondary structure model is presented which is substantiated by analysis of DtpT-PhoA fusion constructs . Amino acid sequence comparisons showed no significant homology with other bacterial peptide transport systems nor with any other known protein . Flanking regions of the di-tripeptide transport gene were used to delete dtpT from the chromosome of L . lactis . Genetic and biochemical characterization of this mutant indicates that DtpT is the only transport protein in L . lactis for hydrophilic di- and tripeptides.

J Bacteriol, 1994 Apr, 176(8), 2165 - 71
Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276; Rauch PJ et al.; The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L . lactis strains . Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision . The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons . The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein . Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process . Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.

Appl Environ Microbiol, 1994 Apr, 60(4), 1390 - 4
Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp . lactis biovar diacetylactis; Marugg JD et al.; The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp . lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis . The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations . This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als) . Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids . The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes . The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

Microbiology, 1994 Apr, 140 ( Pt 4), 923 - 30
Purification and characterization of an endopeptidase from Lactococcus lactis subsp . cremoris SK11; Pritchard GG et al.; An endopeptidase has been purified from Lactococcus lactis subsp . cremoris SK11 . The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and phosphoramidon but relatively insensitive to EDTA . It is not significantly inhibited by the thiol enzyme inhibitor p-chloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl fluoride . The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis . The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond . It thus has a specificity very similar to that of thermolysin . Two of the oligopeptides produced during the early stages of beta-casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis . Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme . These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic system.

FEMS Microbiol Lett, 1994 Mar 15, 117(1), 29 - 33
Application of the ligase chain reaction to the detection of nisinA and nisinZ genes in Lactococcus lactis ssp . lactis; Ward LJ et al.; This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis . The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained . This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.

Appl Environ Microbiol, 1994 Mar, 60(3), 814 - 25
Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3; Engelke G et al.; The biosynthetic genes of the nisin-producing strain Lactococcus lactis 6F3 are organized in an operon-like structure starting with the structural gene nisA followed by the genes nisB, nisT, and nisC, which are probably involved in chemical modification and secretion of the prepeptide (G . Engelke, Z . Gutowski-Eckel, M . Hammelmann, and K.-D . Entian, Appl . Environ . Microbiol . 58:3730-3743, 1992) . Subcloning of an adjacent 5-kb downstream region revealed additional genes involved in nisin biosynthesis . The gene nisI, which encodes a lipoprotein, causes increased immunity after its transformation into nisin-sensitive L . lactis MG1614 . It is followed by the gene nisP, coding for a subtilisin-like serine protease possibly involved in processing of the secreted leader peptide . Adjacent to the 3' end of nisP the genes nisR and nisK were identified, coding for a regulatory protein and a histidine kinase, showing marked similarities to members of the OmpR/EnvZ-like subgroup of two-component regulatory systems . The deduced amino acid sequences of nisR and nisK exhibit marked similarities to SpaR and SpaK, which were recently identified as the response regulator and the corresponding histidine kinase of subtilin biosynthesis . By using antibodies directed against the nisin prepeptide and the NisB protein, respectively, we could show that nisin biosynthesis is regulated by the expression of its structural and biosynthetic genes . Prenisin expression starts in the exponential growth phase and precedes that of the NisB protein by approximately 30 min . Both proteins are expressed to a maximum in the stationary growth phase.

Appl Environ Microbiol, 1994 Mar, 60(3), 801 - 6
Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains; Reid JR et al.; The cell envelope-associated proteinases from Lactococcus lactis subsp . cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis . The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases . Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth . The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule . The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion) . Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1994 Mar, 176(5), 1514 - 6
Nucleotide metabolism in Lactococcus lactis: salvage pathways of exogenous pyrimidines; Martinussen J et al.; By measuring enzyme activities in crude extracts and studying the effect of toxic analogs (5-fluoropyrimidines) on cell growth, the metabolism of pyrimidines in Lactococcus lactis was analyzed . Pathways by which uracil, uridine, deoxyuridine, cytidine, and deoxycytidine are metabolized in L . lactis were established . They are similar to those found in Escherichia coli except that lactococci are unable to utilize cytosine.

J Microencapsul, 1994 Mar-Apr, 11(2), 189 - 95
Microencapsulation of Lactococcus lactis subsp . cremoris; Larisch BC et al.; Lactococcus lactis subsp . cremoris was microencapsulated within alginate/poly-L-lysine (alg/PLL), nylon or crosslinked polyethyleneimine (PEI) membranes . Toxic effects were observed with solvents and reagents used in nylon and PEI membrane formation . Alg/PLL encapsulation resulted in viable and active cell preparations which acidified milk at a rate proportional to the cell concentration, but at rates less than that of free cell preparations . At 4 x 10(8) colony-forming units (cfu/ml milk), encapsulated cells took 17 per cent longer than free lactococci to reduce the pH of milk to 5.5 . Similar activities of free and micro-encapsulated cells may be attained at higher cell concentrations (10(9) cfu/ml milk) . The rate of lactic acid production was approximately 2 mmol/h at an encapsulated cell concentration of 4 x 10(8) cfu/ml.

J Biol Chem, 1994 Feb 4, 269(5), 3555 - 62
Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis; van der Meer JR et al.; Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process . Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences . We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence . Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide . Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4-->Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it . The Ala-4-->Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin . These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide . Several mutations, i.e . Pro-2-->Gly,Pro-2-->Val, Asp-7-->Ala,Lys-9-->Leu,Ser-10-->Ala/Ser-12-->Ala and Val-11-->Asp/Val-13-->Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues . When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e . Phe-18-->Leu,Leu-16-->Lys,Asp-15-->Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors . This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.

FEMS Microbiol Lett, 1994 Feb 1, 116(1), 79 - 86
Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes; Denayrolles M et al.; Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria . Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction . This specific probe was used to isolate two contiguous fragments covering the gene as a whole . The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids . The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms . Expression of the mleS gene in Escherichia coli results in malolactic activity.

J Bacteriol, 1994 Feb, 176(4), 1069 - 76
Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration; Christiansen B et al.; The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp . cremoris 901-1, was characterized . The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism . The pac region was localized on the 38.4-kb phage genome . TP901-1 belongs to the class of P335 phages (V . Braun, S . Hertwig, H . Neve, A . Geis, and M . Teuber, J . Gen . Microbiol . 135:2551-2560, 1989) . Evidence is presented that the phages TP936-1 (V . Braun, S . Hertwig, H . Neve, A . Geis, and M . Teuber, J . Gen . Microbiol . 135:2551-2560, 1989) and C3-T1 (A . W . Jarvis, V . R . Parker, and M . B . Bianchin, Can . J . Microbiol . 38:398-404, 1992) are very closely related to or are identical to TP901-1 . The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2 . Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome . Only one chromosomal attB site was found in 20 independent lysogens . The attP region of TP901-1 and the attL and attR regions were cloned and sequenced . The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA . This result was further verified by sequencing of the attB region obtained by PCR . An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP . This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci.

Gene, 1994 Jan 28, 138(1-2), 123 - 6
Analysis of the cos region of the Lactococcus lactis bacteriophage sk1; Chandry PS et al.; The location, structure and nature of the cos site of the Lactococcus lactis bacteriophage sk1 was determined using a Taq DNA polymerase runoff sequencing technique . The cos site contains a single-stranded 3' overhang of 11 nucleotides . The region surrounding cos contains several features which may be involved in the binding and catalytic action of a phage terminase . These include four putative terminase-binding sites which show some homology to lambda R-sites, an 11-bp direct repeat, a 10-bp inverted repeat, a string of eight consecutive C residues and six copies of the pentanucleotide, AATCT . The spacing between adjacent copies of the pentanucleotides would place them on the same side of the DNA helix.

Plasmid, 1994 Jan, 31(1), 106 - 10
A variant of the staphylococcal chloramphenicol resistance plasmid pC194 with enhanced ability to transform Lactococcus lactis subsp . lactis; von Wright A et al.; In our attempts to transform Lactococcus lactis subsp . lactis with pC194, a staphylococcal chloramphenicol resistance plasmid, only a few transformants could be obtained and only when relatively large amounts of plasmid DNA were used . However, when pC194 DNA from lactococcal transformants was introduced back to Staphylococcus aureus and reisolated, it could be retransformed into L . lactis at substantially higher frequencies . It was concluded that pC194 had undergone mutation expanding its host range . By exchanging DNA fragments between the original pC194 and the variant transforming L . lactis (named pVS41), the mutation could tentatively be located within the 1.1-kbp AccI-HaeIII fragment . Comparison of the DNA sequences in the vicinity of the replication plus-origin revealed the formation of an "opal" stop codon (TGA) apparently interrupting the synthesis of the putative protein C for which no function has been described . Cloning this mutation within a 194 bp AccI-MspI fragment on pC194 made this plasmid able to transform L . lactis . Whether the mutation somehow affects functions controlling plasmid host-specificity, or whether the extended host range reflects, for example, mutational inactivation of some lactococcal restriction site, cannot yet be stated on the basis of these data.

Ciba Found Symp, 1994, 186, 27 - 42; discussion 42-53
Gene-encoded antibiotics made in bacteria; Sahl HG; Production of antimicrobial peptides and proteins is very common among bacteria and a variety of such substances has been described . In general Gram-negative bacteria produce protein bacteriocins (e.g . colicins) with narrow action spectra based on receptor-mediated activity . They produce comparatively few peptides, such as the post-translationally modified microcin B17 . In contrast Gram-positive bacteria tend to produce peptide bacteriocins smaller than 10 kDa and of wider activity spectra . These show particular potential for application . They can be divided into unmodified peptides (e.g . lactococcins, lactacins, pediocins) and lanthionine-containing peptides (lantibiotics, e.g . nisin, epidermin, Pep5) . The unmodified peptides are mostly hydrophobic or amphiphilic and act by disturbing the function of the cytoplasmic membrane . They are synthesized as prepeptides with a characteristic N-terminal leader peptide . In some cases genes for immunity peptides were found in close proximity to structural genes; furthermore, two-component response regulators seem to be involved in the regulation of their synthesis . The biosynthetic genes for lantibiotics are also organized in operons . Lantibiotic gene clusters include genes encoding the unique enzymes which dehydrate serine and threonine and form the characteristic thioether-bridged lanthionines . Three types of lantibiotics are currently distinguished on the basis of structural features and functional aspects: type A, which include elongated, amphiphilic, pore-forming peptides (e.g . nisin); type B, which are of globular shape and inhibit phospholipases (e.g . duramycins); and type C (e.g . actagardine) with intermediate features which act by inhibiting bacterial cell wall biosynthesis.

Gene, 1993 Dec 22, 136(1-2), 371 - 2
LlaAI and LlaBI, two type-II restriction endonucleases from Lactococcus lactis subsp . cremoris W9 and W56 recognizing, respectively, 5'-/GATC-3' and 5'-C/TRYAG-3'; Nyengaard N et al.; Two type-II restriction endonucleases have been purified from Lactococcus lactis subsp . cremoris W9 and W56, the strains isolated from a mixed Cheddar starter . Their characterization showed that LlaAI was an isoschizomer of MboI from Morexella bovis with the cleaving sequence, 5'/GATC-3', being sensitive to methylation of the adenine residue; LlaBI was an isoschizomer to SfcI from Streptococcus faceium with the cleaving sequence, 5'-C/TRYAG-3' . Both LlaAI and LlaBI restriction-modification (R-M) systems are encoded by the plasmids, respectively, pFW094 and pJW563, protecting the harboring strain against phage attack.

J Bacteriol, 1993 Dec, 175(23), 7594 - 603
Genetic analysis of the marine manganese-oxidizing Bacillus sp . strain SG-1: protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation; van Waasbergen LG et al.; Mature spores of the marine Bacillus sp . strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide . Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved . To further study this phenomenon, methods of genetic analysis were developed for SG-1 . By a modified protoplast transformation procedure, SG-1 was transformed (approximately 100 transformants per micrograms of DNA) with several different plasmids of gram-positive origin . Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1 . Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10(-3) . Each mutant was found to be the result of a single insertion event . Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated . Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions . The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants . These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.

J Bacteriol, 1993 Dec, 175(23), 7523 - 32
Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis; Tynkkynen S et al.; The nucleotide sequence of a chromosomal DNA fragment of Lactococcus lactis subsp . lactis SSL135, previously implicated in peptide utilization, has been determined . The genes oppDFBCA, encoding the oligopeptide transport system (Opp), and that encoding the endopeptidase PepO were located on this 8.9-kb DNA fragment . The oppDFBCA and pepO genes are probably organized in an operon . Analysis of the deduced amino acid sequences of the genes indicated that the oligopeptide transport system consists of two ATP-binding proteins OppD and OppF, two integral membrane proteins OppB and OppC, and a substrate-binding protein OppA . On the basis of the homology of OppF and OppD of L . lactis with other ABC (ATP-binding cassette) transporter proteins, the L . lactis Opp system can be classified as a member of this group . Two integration mutants, one defective in OppA and the other defective in PepO, were constructed . Growth of these mutants in a chemically defined medium with oligopeptides showed that the transport system, but not the endopeptidase, is essential for the utilization of peptides longer than three residues . Uptake of the pentapeptide Leu-enkephalin in glycolyzing lactococcal cells was followed by rapid hydrolysis of the peptide intracellularly . Importantly, extracellular hydrolysis of Leu-enkephalin is not observed . The OppA-deficient mutant was unable to transport Leu-enkephalin . Growth experiments with pasteurized milk revealed that transport of oligopeptides forms an essential part of the proteolytic system in lactococci.

Int J Food Microbiol, 1993 Dec, 20(4), 199 - 210
Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various gram-positive bacteria including Listeria sp; Stoffels G et al.; This paper describes a simple purification method for the purification of carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola UI49 . The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83 . The protocol consists of only two purification steps, XAD chromatography and cation exchange chromatography . It is quick, easy, and can be used for large scale purification of these lantibiotics . The bactericidal activity of carnocin UI49 against carnobacteria, lactococci and Listeria was compared with that of nisin Z . The carnobacteria showed similar sensitivity towards carnocin UI49 and nisin . The nisin producing L . lactis strains were very sensitive towards carnocin UI49, while the non-producing L . lactis strains were more sensitive to nisin . The Listeria strains were weakly sensitive to carnocin UI49, lower concentrations of nisin were needed to inhibit growth.

Mol Microbiol, 1993 Dec, 10(5), 1113 - 24
Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22; Frere J et al.; pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp . lactis CNRZ270 . The nucleotide sequence of its replication region Rep22 contains a non-transcribed region, the replication origin, followed by a gene encoding a putative 388-amino-acid protein named Rep22A . The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L . lactis . We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated . We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L . lactis, and is representative of a widely distributed replicon family, members of which could be co-resident . We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co-existence of numerous related replicons in lactococcal strains.

Protein Eng, 1993 Nov, 6(8), 927 - 37
Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis; Siezen RJ et al.; The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase . Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties . The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23) . Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged . The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site . Several mutant SK11 proteinases have novel properties not previously encountered in natural variants . Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity . Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase . Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Appl Environ Microbiol, 1993 Nov, 59(11), 3954 - 9
A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis; Wells JM et al.; The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase . The vectors incorporated different lactococcal secretion leaders and translation initiation sequences . When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium . However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane . The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain . This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions . Our results indicate that L . lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.

Appl Environ Microbiol, 1993 Nov, 59(11), 3640 - 7
Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region; Exterkate FA et al.; The biochemical and genetical diversity of the subtilisin-like cell envelope proteinase (CEP) among Lactococcus lactis strains was investigated . The specificities of the proteinases of 16 strains toward the important cheese peptide alpha s1-casein fragment 1 to 23 and toward two differently charged chromophoric peptides have been determined . On the basis of the results, these strains could be classified into seven groups . The contribution to the specificity of specific residues in the large C-terminal segment, which differentiates this proteinase from most other members of the subtilisin family, was established with hybrid proteinases, even in the case of the small substrates . These remote residues and the subtilisin-like substrate-binding region are therefore assumed to be spatially close to each other and together constitute most of the binding region of CEP . DNA sequence analysis of fragments of the gene (prtP) encoding segments of the proteinase which contain the relevant residues of the substrate-binding region shows that among the strains studied, this binding region is the most negatively charged in the CEP group represented by strain HP and the positively charged in the CEP group represented by strains AM1 and SK11 . Consequently, these two proteinase groups show the most divergent specificities . Each of the proteinases of the other groups shows a different intermediate specificity which in part is the reflection of an intermediate charge in the binding region . However, the results suggest that amino acid residues outside the segments known to be part of the CEP-binding region also contribute to specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1993 Nov 1, 113(3), 315 - 9
Insertion sequence analysis of protoplast fused strains of Lactococcus lactis ssp . cremoris; Ward LJ et al.; The use of insertion sequence probes for the analysis of fusants obtained following protoplast fusion is described . Hybridization of both total and plasmid DNA from parent and fusant strains with probes to IS904 and ISS1 showed that of the four protoplast fusions examined, three appeared to involve a rearrangement of genetic material while in the fourth the fusant appeared similar to one of the parental strains . This method of analysis provides more information about the changes induced by protoplast fusion than that obtained by monitoring the acquisition or loss of individual characteristics.

Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 348 - 55
Construction of first-generation lactococcal integrative cloning vectors; McIntyre DA et al.; Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp . lactis LM0230, first-generation lactococcal integrative cloning vectors were developed . Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs . Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L . lactis and L . lactis subsp . cremoris . Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (eryr) gene obtained from L . lactis IL1837 . Integration of the eryr gene into the L . lactis LM0230 chromosome was achieved by a Campbell-like recombination . The nisin (Nis)-resistance (nisr) gene from L . lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L . lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nisr fragment flanked by the cloned chromosomal DNA . Transformants grown in the absence of either Em or Nis for > 200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes . Further studies involving the Nis-resistant (NisR) transformant suggested that the integrated nisr gene may be amplifying within the host chromosome.

Appl Environ Microbiol, 1993 Nov, 59(11), 3941 - 5
Isolation of Lactococcus lactis subsp . cremoris from nature by colony hybridization with rRNA probes; Salama MS et al.; Lactococcus lactis subsp . cremoris is widely used in the manufacture of fermented milk products . Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful . Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L . lactis subsp . cremoris from related strains . These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures . A total of 170 strains of L . lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L . lactis . Fifty-nine of these isolates also hybridized to L . lactis subsp . cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L . lactis subsp . cremoris-specific probe had the L . lactis subsp . cremoris phenotype.

FEBS Lett, 1993 Oct 11, 332(1-2), 74 - 80
Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis; Ansanay V et al.; Many lactic acid bacteria can carry out malolactic fermentation . This secondary fermentation is mediated by the NAD- and Mn(2+)-dependent malolactic enzyme, which catalyses the decarboxylation of L-malate to L-lactate . The gene we call mleS, coding for malolactic enzyme, was isolated from Lactococcus lactis . The mleS gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 59 kDa . The amino acid sequence of the predicted MleS gene product is homologous to the sequences of different malic enzymes . Bacterial and yeast cells expressing the malolactic gene convert L-malate to L-lactate.

Naturwissenschaften, 1993 Oct, 80(10), 454 - 60
{Lantibiotics, a class of ribosomally synthesized peptide antibiotics}; Entian KD et al.; Lantibiotics are defined as peptide antibiotics containing the unusual amino acids mesolanthionine, 3-methyllanthionine, dehydroalanine, and dehydrobutyrine . They are synthesized by some gram-positive bacteria . Their inhibitory effect on certain other gram-positive bacteria is explained by detergent-like damage of cytoplasmic membranes . Prominent members of the lantibiotics are nisin of Lactococcus lactis, which can be used as a food preservative, subtilin of Bacillus subtilis, which is similar to nisin, and epidermin of Staphylococcus epidermidis, which is considered in the treatment of acne . Lantibiotics are ribosomally synthesized as prepeptides, which are posttranslationally modified . Genes probably encoding these biosynthetic enzymes and regulatory factors have been identified adjacent to the structural genes of the lantibiotics subtilin, nisin, and epidermin.

Mol Microbiol, 1993 Oct, 10(2), 319 - 27
Theta replication of the lactococcal plasmid pWVO2; Kiewiet R et al.; pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp . cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli . Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism . The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present . By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism . This is the first proof for the existence of theta-replicating plasmids in lactococci . The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons . It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times . Further upstream is another 10 bp direct repeat present in an A/T-rich sequence . This structural organization resembles that of several iteron-containing theta-type plasmids from E . coli . Derivatives of pWVO2 were stably maintained in L . lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.

FEMS Microbiol Lett, 1993 Sep 15, 112(3), 313 - 8
Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp . lactis CNRZ 481; Piard JC et al.; The lacticin 481-producer (Lct+), L . lactis subsp . lactis (L . lactis) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb . Novobiocin treatment of L . lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid . Conjugal transfer of the lacticin 481 structural gene (lct) into the plasmid free strain L . lactis IL1441 yielded Lct+ transconjugants at a 10(-4) frequency, which carried a plasmid with an apparent size of 120-130 kb . Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L . lactis 481 and on the 120-130 kb plasmid in the transconjugants . The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.

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