Lactococci

Appl Biochem Biotechnol, 1998 Nov-Dec, 75(2-3), 295 - 306
Application of flow cytometry for rapid detection of Lactococcus garvieae; Endo H et al.; Flow cytometry (FCM) technique was applied to rapid determination of cell number of Lactococcus garvieae . An antiserum against L . garvieae was prepared and its immunological property was examined . The present antibody would recognize some epitopes of L . garvieae with a high specificity . The optimum conditions for the FCM assay were as follows: discriminate value, 60; dilution ratio of the antiserum, 1.0 x 10(4) . Calibration curve for L . garvieae cells was linear, in the range of 2.4 x 10(4)-1.5 x 10(7) cells/mL . The detection of L . garvieae in cell suspensions contaminated with Escherichia coli was carried out . A good correlation was observed in the range of 20-90% for the mixing ratio of L . garvieae . One FCM assay could be completed within 2 min, and the total assay time, including the preparation of bacterial sample, was within 3 h.

Res Microbiol, 1999 Apr, 150(3), 189 - 98
Cloning of branched chain amino acid biosynthesis genes and assays of alpha-acetolactate synthase activities in Leuconostoc mesenteroides subsp . cremoris; Cavin JF et al.; A genomic library from Leuconostoc mesenteroides subsp . cremoris (Lmc) in Escherichia coli was screened for alpha-acetolactate synthase (ALS) activity using a phenotypic test detecting the production of acetolactate or related C4 derivatives (diacetyl, acetoin or 2,3-butanediol) in the culture . Four recombinant E . coli clones, with plasmids containing overlapping DNA fragments and displaying anabolic ALS activity, were selected . This activity is encoded by an ilvB gene belonging to a putative operon which contains genes highly similar to the genes of the branched chain amino acid (BCAA) operon of Lactococcus lactis subsp . lactis . This putative BCAA operon is not functional as the ilvA gene is interrupted by a single mutation and the strain is auxotrophic for the three BCAAs . Only a very low anabolic ALS activity was present in cell-free extracts of Lmc and no transcript from the ilvB gene could be detected . Instability of ilvB expression in E . coli was the consequence of a frequent IS5 insertion sequence in this gene . Despite the detection of a high catabolic ALS activity in Lmc, no catabolic ALS activity gene could be found in the BCAA gene locus, indicating the presence of a catabolic als gene in the Lmc chromosome that could be absent or not expressed in the screened library.

Appl Environ Microbiol, 1999 May, 65(5), 2230 - 1
Excision of the conjugative transposon Tn916 in Lactococcus lactis; Marra D et al.; In Lactococcus lactis excision of Tn916 is limited by the concentration of integrase and is increased by providing more excisionase . However, even with increased excision of Tn916 in L . lactis, no conjugative transfer is detectable . This suggests that L . lactis is deficient in a host factor(s) required for conjugative transposition.

Appl Environ Microbiol, 1999 May, 65(5), 1891 - 9
Molecular characterization of a phage-encoded resistance system in Lactococcus lactis; McGrath S et al.; A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification . In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified . When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9 . The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication . Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats . Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed . UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

Mol Microbiol, 1999 Apr, 32(1), 75 - 87
Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis; Madsen SM et al.; In a previous study, we described the use of transposon Tn917-LTV1 for identification of environmentally regulated promoters in Lactococcus lactis . Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase . The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site . This fragment lacked the consensus -35 promoter region, but it contained an 'extended' -10 promoter region . When a 28 bp segment, containing the consensus -35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase . This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation . Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis . Analysis of total RNA from L . lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase . Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter . Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150- to 200-fold increase in the level of gene expression, without affecting the regulation . The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.

Eur J Biochem, 1999 Apr, 261(2), 524 - 32
Post-translational modification of nisin . The involvement of NisB in the dehydration process; Karakas Sen A et al.; The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis . As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide . These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide . In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33 . With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, {Trp30}nisin A and {Lys27,Lys31}nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50% . This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation . Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains . The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33 . The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule . The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.

Mol Microbiol, 1999 Mar, 31(5), 1523 - 35
Two operons that encode FNR-like proteins in Lactococcus lactis; Gostick DO et al.; Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products . Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR-like proteins (FlpA and FlpB) with the potential for mediating a dithiol-disulphide-dependent regulatory switch, were identified . Transcript analysis indicated that they are distal genes of two paralogous operons, orfX-orfY-flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively . The corresponding promoters were each associated with a potential FNR site (TTGAT----ATCAA) at positions +4.5 (flpA operon) and -42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated . The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases . No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two-dimensional gel electrophoresis): formate production was not affected . It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.

Lett Appl Microbiol, 1999 Mar, 28(3), 189 - 93
Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis; Montville TJ et al.; Nisin is a bacteriocin produced by many strains of Lactococcus lactis . This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium . Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco . smegmatis . Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment . Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability . the effect of nisin on the growth of Myco . smegmatis was both time- and concentration-dependent . Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco . smegmatis cells after several hours of treatment . These data suggest that nisin inhibits Myco . smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.

J Appl Microbiol, 1999 Mar, 86(3), 514 - 20
Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers; Mangin I et al.; Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy . It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas . The patterns from 58 isolates were analysed by UPGMA dendrograms . At a similarity level of 50%, four RAPD clusters were distinguished . Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis . The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3 . Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 191 - 5
Artificial promoters for metabolic optimization; Jensen PR et al.; In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts . In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides . From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities . Importantly, the range of promoter activities was covered in small steps of activity change . Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question . If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level . One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell .

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 115 - 22
A comparative analysis of the citrate permease P mRNA stability in Lactococcus lactis biovar diacetylactis and Escherichia coli; Drider D et al.; The role of ribonucleases in the control of gene expression remains unknown in lactic acid bacteria . In the present work, we analysed the expression of the citP gene, which encodes the lactococcal citrate permease P, through the stability of the citQRP messenger in both Lactococcus lactis biovar diacetylactis (L . diacetylactis) and Escherichia coli . The chemical half-life for citQRP mRNA observed in L . diacetylactis wild-type strain was abnormally long for bacteria . It was even longer than that detected in E . coli RNase E or RNase III mutant strains . A model of processing and fate of RNA species containing citP gene is presented.

Appl Environ Microbiol, 1999 Apr, 65(4), 1540 - 7
Identification of four phage resistance plasmids from Lactococcus lactis subsp . cremoris HO2; Forde A et al.; The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined . Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant . One of these isolates, Lactococcus lactis subsp . cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes . Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids . A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species) . pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule . pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.

Appl Environ Microbiol, 1999 Apr, 65(4), 1390 - 6
Role of calcium in activity and stability of the Lactococcus lactis cell envelope proteinase; Exterkate FA et al.; The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope . Different types of CEP can be distinguished on the basis of specificity and amino acid sequence . Removal of weakly bound Ca2+ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25 degrees C (pH 6.5) can be measured . The consequences of Ca2+ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity) . Autoproteolytic release of CEP from cells concerns this so-called "Ca-free" form only and occurs most efficiently in the case of the Wg2 CEP . The results of a study of the relationship between the Ca2+ concentration and the stability and activity of the cell-bound SK11 CEP at 25 degrees C suggested that binding of at least two Ca2+ ions occurred . Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca2+ dependence of the cell-bound CEP . The results are discussed in terms of predicted Ca2+ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site . We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.

Biotechnol Bioeng, 1999 May 5, 63(3), 356 - 62
Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction; Jensen NB et al.; An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented . The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity . In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching . A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels . The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components . The internal standard was recovered to an extent of 85-95% .

Gene, 1999 Mar 18, 229(1-2), 229 - 35
A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis; Khunajakr N et al.; Two regulatory genes (lcoR and lcoS) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (cat) . RT-PCR analysis indicates that lcoR and lcoS are organized within an operon, controlling the transcription of cat in a copper-inducible manner . The amino acid sequences deduced from lcoR and lcoS show homology to the response and sensor proteins of known two-component regulatory systems . Deletion within either lcoS or both genes inactivated the copper-dependent activity, suggesting the presence of no trans-acting lcoR and lcoS homologs in the lactococcal host chromosome . The transcription start site involved in copper induction was mapped by primer extension.

J Bacteriol, 1999 Apr, 181(7), 2075 - 83
Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria; Ingmer H et al.; In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains . The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families . The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-) . Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein . When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L . lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments . However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells . Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L . lactis, could be necessary for degrading proteins generated by certain types of stress.

J Bacteriol, 1999 Apr, 181(7), 2026 - 37
Regulation of expression of the Lactococcus lactis histidine operon; Delorme C et al.; In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis . It is transcribed from a unique promoter, localized 300 bp upstream of the first gene . The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria . Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium . In the absence of histidine, two transcripts are present . One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point . DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis . In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified . Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L . lactis . Analysis of the variable positions supports the folding model of the central part of the his leader region . Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine . An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism . The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon . The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively . Together, the two levels of control allow a 120-fold range of regulation of the L . lactis operon by histidine.

Mol Microbiol, 1998 Nov, 30(4), 789 - 98
Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA; Luesink EJ et al.; The Lactococcus lactis ccpA gene, encoding the global regulatory protein CcpA, was identified and characterized . Northern blot and primer extension analyses showed that the L . lactis ccpA gene is constitutively transcribed from a promoter that does not contain a cre sequence . Inactivation of the ccpA gene resulted in a twofold reduction in the growth rate compared with the wild type on glucose, sucrose and fructose, while growth on galactose was almost completely abolished . The observed growth defects could be complemented by the expression of either the L . lactis or the Bacillus subtilis ccpA gene . The disruption of the ccpA gene reduced the catabolite repression of the gal operon, which contains a cre site at the transcription start site and encodes enzymes involved in galactose catabolism . In contrast, CcpA activates the transcription of the cre-containing promoter of the las operon, encoding the glycolytic enzymes phosphofructokinase, pyruvate kinase and L-lactate dehydrogenase, because its transcription level was fourfold reduced in the ccpA mutant strain compared with the wild-type strain . The lower activities of pyruvate kinase and L-lactate dehydrogenase in the ccpA mutant strain resulted in the production of metabolites characteristic of a mixed-acid fermentation, whereas the fermentation pattern of the wild-type strain was essentially homolactic.

FEBS Lett, 1999 Feb 26, 445(2-3), 321 - 4
Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp . cremoris AM2; Bouchier PJ et al.; Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised . Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues . However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues . The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp . cremoris AM2 hydrolyse peptides containing proline residues.

Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1422 - 4
Crystallization and preliminary X-ray diffraction studies of 6-phosphogluconate dehydrogenase from Lactococcus lactis; Tetaud E et al.; 6-Phosphogluconate dehydrogenase is one of the seven enzymes involved in the pentose phosphate pathway . Crystals of a mammalian and a protozoan enzyme have been obtained previously and structures determined . It is reported here that a bacterial 6-phosphogluconate dehydrogenase, from Lactococcus lactis, has been purified and used in crystallization trials . Large prisms suitable for a detailed structural analysis have been obtained and characterized as orthorhombic, space group F222, with a = 70.4, b = 105.7, c = 474.6 A . Diffraction has been observed to 2.2 A resolution using synchrotron radiation . Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this enzyme.

J Biol Chem, 1999 Mar 26, 274(13), 8484 - 90
Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC . Involvement of LcnC in lactococcin a maturation; Franke CM et al.; Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway . These bacteriocins utilize a dedicated (type I) secretion system for externalization . The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD . LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems . This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA . By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm . As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.

Appl Microbiol Biotechnol, 1999 Jan, 51(1), 91 - 9
Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates; Corroler D et al.; A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized . As expected for environmental isolates, all strains had a Lactococcus lactis subsp . lactis phenotype . The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci . Two major clusters were identified containing the two subspecies lactis and cremoris . The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster . This RAPD classification was then compared with that of a traditional PCR assay using L . lactis species-specific primers corresponding to part of the histidine biosynthesis operon . The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris) . Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency.

Biochemistry, 1999 Mar 9, 38(10), 2899 - 908
The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis; Bjornberg O et al.; Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate . The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known . The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E . coli enzyme . Product inhibition showed that the E . coli enzyme, in contrast to the L . lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor . Trypsin readily cleaved the E . coli enzyme into two fragments of 182 and 154 residues, respectively . Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact . The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L . lactis enzyme, forms a loop where a cysteine residue is very critical for activity . In the corresponding position, the enzyme from E . coli has a serine residue . Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively . The S175C mutant was also defective with respect to substrate and product binding . Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed.

J Bacteriol, 1999 Mar, 181(6), 1924 - 6
Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis; Luesink EJ et al.; The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies . Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR . The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription.

Biopolymers, 1999 Jan, 49(1), 1 - 9
Isolation and physical characterization of an exocellular polysaccharide; Tuinier R et al.; The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp . cremoris strain NIZO B40 were investigated . Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes . Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water . Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide . Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13 . The number-averaged radius of gyration was found to be 86 +/- 2 nm . From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained . Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated . Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.

J Appl Microbiol, 1999 Feb, 86(2), 251 - 6
Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147-producing starter culture; McAuliffe O et al.; The efficacy of using a lacticin 3147-producing starter as a protective culture to improve the safety of cottage cheese was investigated . This involved the manufacture of cottage cheese using Lactococcus lactis DPC4268 (control) and L . lactis DPC4275, a bacteriocin-producing transconjugant strain derived from DPC4268 . A number of Listeria monocytogenes strains, including a number of industrial isolates, were assayed for their sensitivity to lacticin 3147 . These strains varied considerably with respect to their sensitivity to the bacteriocin . One of the more tolerant strains, Scott A, was used in the cottage cheese study; the cheese was subsequently inoculated with approximately 10(4) L . monocytogenes Scott A g-1 . The bacteriocin concentration in the curd was measured at 2560 AU ml-1, and bacteriocin activity could be detected throughout the 1 week storage period . In cottage cheese samples held at 4 degrees C, there was at least a 99.9% reduction in the numbers of L . monocytogenes Scott A in the bacteriocin-containing cheese within 5 d, whereas in the control cheeses, numbers remained essentially unchanged . At higher storage temperatures, the kill rate was more rapid . These results demonstrate the effectiveness of lacticin 3147 as an inhibitor of L . monocytogenes in a food system where post-manufacture contamination by this organism could be problematic.

Appl Environ Microbiol, 1999 Mar, 65(3), 1202 - 6
A general method for selection of alpha-acetolactate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation; Curic M et al.; The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the alpha-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV) . This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp . lactis biovar diacetylactis . Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains . Most dairy lactococcus strains are auxotrophic for the three amino acids . Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L . lactis NCDO2118 was constructed . Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype . By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained . These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald . Selected mutants lacking Ald were subsequently cured of pMC004 . Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting . The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade . Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms . These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L . lactis strains.

Appl Environ Microbiol, 1999 Mar, 65(3), 1005 - 8
Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia, and Australia; Eldar A et al.; Lactococcus garvieae (junior synonym, Enterococcus seriolicida) is a major pathogen of fish, producing fatal septicemia among fish species living in very diverse environments . The phenotypic traits of L . garvieae strains collected from three different continents (Asia, Europe, and Australia) indicated phenotypic heterogeneity . On the basis of the acidification of D-tagatose and sucrose, three biotypes were defined . DNA relatedness values and a specific PCR assay showed that all the biotypes belonged to the same genospecies, L . garvieae . All of the L . garvieae strains were serotyped as Lancefield group N . Ribotyping proved that one clone was found both in Japan, where it probably originated, and in Italy, where it was probably imported . PCR of environmental samples did not reveal the source of the contamination of the fish in Italy . Specific clones (ribotypes) were found in outbreaks in Spain and in Italy . The L . garvieae reference strain, isolated in the United Kingdom from a cow, belonged to a unique ribotype . L . garvieae is a rising zoonotic agent . The biotyping scheme, the ribotyping analysis, and the PCR assay described in this work allowed the proper identification of L . garvieae and the description of the origin and of the source of contamination of strains involved in outbreaks or in sporadic cases.

J Bacteriol, 1999 Mar, 181(5), 1451 - 7
Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH; Magni C et al.; Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH . The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells . The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism . The results clearly demonstrate that citrate metabolism in L . lactis is a secondary proton motive force-generating pathway . Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L . lactis in rich media . However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium . Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition . The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity . It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.

Mol Microbiol, 1999 Feb, 31(3), 845 - 58
Effects of metabolic flux on stress response pathways in Lactococcus lactis; Duwat P et al.; Studies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways . We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA-damaging agents but also to oxygen and heat . To further examine the stress response pathways in L . lactis, we isolated thermoresistant insertional mutants (Trm) of the recA strain . Eighteen independent trm mutations were identified and characterized . We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS), mRNA stability (pnpA) and in one uncharacterized gene (trmA) . All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell . Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild-type context . Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 163 - 6
Novel characteristic for distinguishing Lactococcus lactis subsp . lactis from subsp . cremoris; Nomura M et al.; Lactococcus lactis strains were examined for their ability to produce gamma-aminobutyric acid (GABA) . Results showed that strains of L . lactis subsp . lactis were able to produce this acid, whereas L . lactis subsp . cremoris were not . GABA production thus represents another effective characteristic for distinguishing L . lactis subsp . lactis from L . lactis subsp . cremoris.

J Biotechnol, 1999 Jan 22, 67(2-3), 135 - 49
Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis; Deng YM et al.; A plasmid-encoded phage abortive infection mechanism (AbiL) was identified from Lactococcus lactis biovar . diacetylactis LD10-1 . AbiL conferred complete resistance to the small isometric-headed phage phi 712 (936 species) and partial resistance to the prolate-headed phage phi c2 (c2 species) when introduced into L . lactis LM0230 . However, AbiL was not effective against the small isometric-headed phage ul36 (P335 species) . The AbiL determinant was sequenced and it consists of two open reading frames, abiLi and abiLii . Their encoded proteins did not share significant homology with any known proteins in the protein databases . Transcriptional analysis indicated that abiLi and abiLii are organized as a single operon . Deletion within abiLii abolished the phage resistance . The levels of four phi c2-specific transcripts, three within the early transcribed region and one within the late transcribed region, were examined by RT-PCR, no effect of AbiL on synthesis of these transcripts was detected, suggesting that AbiL may act at a point after the transcription of phi c2 in L . lactis.

FEMS Microbiol Lett, 1999 Feb 1, 171(1), 57 - 65
Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses; Kim WS et al.; Lactococcus lactis subspecies lactis (L . lactis ssp . lactis) and Lactococcus lactis subspecies cremoris (L . lactis ssp . cremoris) were investigated in respect to their response to acid, bile-salt and freezing stresses . First, the sublethal and lethal levels of each stress were determined for both subspecies . For acid stress, the levels were pH 4.5 and 2.5, respectively, for L . lactis ssp . lactis, and pH 5.0 and 3.0, respectively, for L . lactis ssp . cremoris . For bile-salt stress, the levels were 0.03 and 0.1%, respectively, for L . lactis ssp . lactis, and 0.01 and 0.04%, respectively, for L . lactis ssp . cremoris . For freezing stress, 10 degrees C was used as the sublethal temperature and -20 degrees C was used as the lethal temperature for both subspecies . To evaluate the effect of each stress at log phase, a log-phase culture was challenged directly with the appropriate lethal level (control culture) and a second log-phase culture was pre-exposed to the appropriate sublethal level prior to testing survival under normally lethal conditions (test culture) . Some, if not most, of the cells were killed in the control cultures for all three stresses . However, in the test cultures, the viability was significantly improved for all of the L . lactis ssp . lactis strains tested, but not for the L . lactis ssp . cremoris strains . It appears, therefore, that L . lactis ssp . lactis is capable of displaying adaptive response to stresses, whereas L . lactis ssp . cremoris seems to lack this phenotype or the response is much weaker in this subspecies . The effect of each stress on stationary-phase cultures was also investigated . Unlike the log-phase cultures, the stationary-phase cultures of both subspecies, challenged directly with the lethal levels, were highly resistant to each of the three stresses tested.

FEMS Microbiol Lett, 1999 Feb 1, 171(1), 43 - 8
Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains; Meghrous J et al.; From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification . The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity . By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification . A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

Mol Microbiol, 1999 Jan, 31(1), 79 - 87
ClpP participates in the degradation of misfolded protein in Lactococcus lactis; Frees D et al.; ClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms . In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases . We show here that the clpP gene from the Gram-positive bacterium Lactococcus lactis encodes a 22-kDa protein that is induced by low pH and by the t-RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl-containing peptides . Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram-positive bacteria . When we disrupted the L . lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild-type cells . Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl-containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress . Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.

Biochem J, 1999 Feb 15, 338 ( Pt 1), 55 - 60
6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme; Tetaud E et al.; A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1 . 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants . The cloned gene was then expressed to high levels in E . coli and the protein purified for kinetic analysis . The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5 . Sequence comparison of the L . lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly . A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan . In each case enzyme activity was lost, confirming an essential role for this residue on activity . A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis . This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude . A second 6-PGDH gene has been identified from the genome of B . subtilis . This second isoform contains an arginine (Arg-34) in this position, suggesting that B . subtilis has two 6-PGDHs with different coenzyme specificities.

Appl Environ Microbiol, 1999 Feb, 65(2), 665 - 73
Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp . lactis; Stuart MR et al.; Two strains of Lactococcus lactis subsp . lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation . Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion . Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium . By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L . lactis ML3, whereas the serine concentration increased by 97% during the same period . When no lactose was added, the concentrations of these amino acids remained constant . Similar trends were observed for L . lactis 11454 . Without lactose or arginine, L . lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels . However, L . lactis 11454 without lactose or arginine remained culturable for at least 14 days . These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion . Additionally, these data indicate that amino acids other than arginine facilitate the survival of L . lactis during carbohydrate starvation.

Appl Environ Microbiol, 1999 Feb, 65(2), 752 - 8
Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis; Brondsted L et al.; Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp . cremoris (B . Christiansen, L . Brondsted, F . K . Vogensen, and K . Hammer, J . Bacteriol . 178:5164-5173, 1996) . In this work, a further analysis of the phage-encoded elements involved in integration was performed . Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L . lactis subsp . cremoris chromosome . Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out . We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L . lactis subsp . cremoris when the orf1 gene is donated in trans . The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages . Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1 . By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L . lactis was constructed . Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed . Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest . By transformation of L . lactis subsp . cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

Appl Environ Microbiol, 1999 Feb, 65(2), 686 - 93
LlaFI, a type III restriction and modification system in Lactococcus lactis; Su P et al.; We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis . LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L . lactis LL42-1 . Sequencing revealed two adjacent open reading frames (ORFs) . One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide . The two ORFs appear to be organized in an operon . A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits . The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases . Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable . An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein . The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence . LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes . ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it . To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

Int J Food Microbiol, 1998 Dec 8, 45(2), 85 - 92
Characterization of lactococci isolated from minimally processed fresh fruit and vegetables; Kelly WJ et al.; Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus lactis subsp . lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the 16S rRNA gene . Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic DNA . Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products during the period they are intended to be eaten . Although these plant strains showed many similarities to strains of L . lactis used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental origin . Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose . Most of the bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin activity, or that produced a different bacteriocin with a narrow spectrum of activity . One strain produced nisin but was unable to ferment sucrose, properties that have been generally regarded as linked . These strains may have uses as biopreservatives for minimally processed plant products.

Syst Appl Microbiol, 1998 Dec, 21(4), 530 - 8
Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD); Tailliez P et al.; Lactococcus lactis strains are widely used in industrial dairy fermentations . Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations . DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups . However, results showed discrepancies between the two methods of classification . We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications . Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose . Our analysis revealed three major RAPD groups, designated G1, G2 and G3 . G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics . Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one . The taxonomic structure within L . lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous . We hypothesize that a subfamily of the subsp . lactis group gave rise to the cremoris subspecies.

J Bacteriol, 1999 Feb, 181(3), 764 - 71
Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr; Luesink EJ et al.; The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene . A promoter sequence was identified upstream of the ptsHI operon, and the transcription start site was mapped by primer extension . The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing ptsH and a second of 2.0 kb containing both ptsH and ptsI . Disruption of the ptsH and ptsI genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in L . lactis . Complementation of the ptsH and ptsI mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype . The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine . The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon . Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and L-lactate dehydrogenase genes.

Biochemistry, 1999 Jan 19, 38(3), 1002 - 8
Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP; Putman M et al.; The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter . LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force . The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes . Surprisingly, dodecyl maltoside did not modulate the activity of LmrP . Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine . Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins . These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

J Dairy Sci, 1998 Dec, 81(12), 3109 - 16
Isolation and identification of some major water-soluble peptides in Feta cheese; Michaelidou A et al.; Peptides were isolated from the water-soluble fraction of Feta cheese by reversed-phase HPLC in three successive steps . Peptide sequencing was performed by automatic Edman degradation . Most of the peptides originated from alpha s1-casein (CN), especially from the N-terminal half of the molecule . Two peptides originated from the C-terminal domain of beta-CN . Only one peptide, which was rich in histidine, originated from kappa-CN . beta-Lactoglobulin and alpha-lactalbumin were also identified in the water extract of Feta cheese . The origin of most of these peptides could be explained on the basis of known specificities of chymosin and lactococcal cell-wall proteinases.

Appl Environ Microbiol, 1999 Jan, 65(1), 330 - 5
Expression, regulation, and mode of action of the AbiG abortive infection system of lactococcus lactis subsp . cremoris UC653
O'Connor L, Tangney M, Fitzgerald GF.
The abortive infection system AbiG is encoded by the lactococcal plasmid pCI750 . The abiG locus (consisting of two genes, abiGi and abiGii) was examined by Northern blot analysis, revealing two transcripts of approximately 2.8 and 1.5 kb which were homologous to the two gene-specific probes . A transcriptional start site was mapped upstream of abiGi, and it appeared that the two genes were cotranscribed, resulting in the 2.8-kb transcript . The smaller transcript may be the result of independent transcription of abiGii within abiGi or of the presence of a weak terminator within abiGii . The locus was shown to be constitutively expressed . Evidence is presented for the possible existence of a second Abi mechanism on pCI750 . Examination of phage sk1 RNA synthesis demonstrated that both the subcloned AbiG and, to a greater extent, pCI750 inhibited this process . pCI750 also severely inhibited synthesis of both early and late phage c2 transcripts, while the presence of the subclone resulted in a reduction in late transcript synthesis only.

Appl Environ Microbiol, 1999 Jan, 65(1), 291 - 3
Influence of osmolarity and the presence of an osmoprotectant on lactococcus lactis growth and bacteriocin production
Uguen P, Hamelin J, Le Pennec JP, Blanco C.
Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium . Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.

Lett Appl Microbiol, 1998 Dec, 27(6), 345 - 8
Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish; Nykanen A et al.; The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms . Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only . The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.

J Appl Microbiol, 1998 Dec, 85(6), 999 - 1005
Characterization of lactococci isolated from milk produced in the Camembert region of Normandy; Desmasures N et al.; Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level . Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples . Most strains fermented lactose (97%) and showed proteinase activity (76%) . Isolates were characterized by RAPD technique and rRNA gene restriction analysis . More L . lactis strains with the lactis genotype were found in the first area, while L . lactis strains with the cremoris genotype predominated in the second area . RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp . lactis genotype . For L . lactis with the subsp . cremoris genotype, the second method gave a better result but there was poor discrimination between strains . Plasmid profiles were determined . Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found . Six groups of strains were determined, five of which were specific for the area of origin . It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

J Bacteriol, 1999 Jan, 181(1), 338 - 40
Exopolysaccharide biosynthesis in Lactococcus lactis NIZO B40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone; van Kranenburg R et al.; We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit . EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose . Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.

Nucleic Acids Res, 1999 Jan 15, 27(2), 608 - 15
AP site structural determinants for Fpg specific recognition; Castaing B et al.; The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments . We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM) . The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes . The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively) . These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue . High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand . This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.

Biochemistry, 1998 Nov 24, 37(47), 16671 - 9
Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis; Detmers FJ et al.; To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins . The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins . beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp . To study the specificity of Opp and the kinetics of peptide uptake in L . lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC) . These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested . Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration . Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.

Appl Environ Microbiol, 1998 Dec, 64(12), 4842 - 5
Use of a genetically enhanced, pediocin-producing starter culture, lactococcus lactis subsp . lactis MM217, To control listeria monocytogenes in cheddar cheese
Buyong N, Kok J, Luchansky JB.
Cheddar cheese was prepared with Lactococcus lactis subsp . lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1 . About 75 liters of pasteurized milk (containing ca . 3.6% fat) was inoculated with strain MM217 (ca . 10(6) CFU per ml) and a mixture of three Listeria monocytogenes strains (ca . 10(3) CFU per ml) . The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8 degreesC for 6 months . In control cheese made with the isogenic, non-pediocin-producing starter culture L . lactis subsp . lactis MM210, the counts of the pathogen increased to about 10(7) CFU per g after 2 weeks of ripening and then gradually decreased to about 10(3) CFU per g after 6 months . In the experimental cheese made with strain MM217, the counts of L . monocytogenes decreased to 10(2) CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months . The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months . No pediocin activity (<200 AU per g) was detected in the control cheese . Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar . Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L . monocytogenes.

Appl Environ Microbiol, 1998 Dec, 64(12), 4748 - 56
AbiQ, an abortive infection mechanism from Lactococcus lactis; Emond E et al.; Lactococcus lactis W-37 is highly resistant to phage infection . The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L . lactis LM0230 . In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L . lactis W-37 . This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages . pSRQ900 did not confer any resistance to phages of the P335 species . Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism . The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment . Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids . A frameshift mutation within abiQ completely abolished the resistant phenotype . The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein . AbiQ has no homology to known or deduced proteins in the databases . DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ . However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form . The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.

Appl Environ Microbiol, 1998 Dec, 64(12), 4736 - 42
Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis; Leenhouts K et al.; Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses . The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P . shermanii . The levels of expression of the pip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter in L . lactis or the inducible tac promoter in E . coli . Stable replication of the rolling-circle replicating (rcr) plasmid, used to express pip in L . lactis, could only be obtained by providing the repA gene in trans . Upon the integration of pip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose . The multicopy integrants demonstrated a high degree of stability in the presence of glucose . This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes in L . lactis.

Appl Environ Microbiol, 1998 Dec, 64(12), 4729 - 35
An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area; Corroler D et al.; The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied . Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy . All of the strains analyzed had a Lactococcus lactis subsp . lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L . lactis subsp . lactis or L . lactis subsp . cremoris . The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon . The geographic distribution of each subspecies of L . lactis was determined; 80% of the Bocage Falaisien strains were members of L . lactis subsp . lactis, and 30.5% of the Bessin strains were members of L . lactis subsp . lactis . A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level . The coefficient of similarity for 117 of the 139 strains identified as members of L . lactis subsp . cremoris was as high as 66% . The L . lactis subsp . lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%) . Reference strains of L . lactis subsp . lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different . As determined by the RAPD profiles, some L . lactis subsp . lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter) . Therefore, the typicality of L . lactis subsp . lactis strains was linked to the farm of origin rather than the area . These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Appl Environ Microbiol, 1998 Dec, 64(12), 4670 - 5
Use of 13C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci; Gao S et al.; Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor . However, the catabolism of methionine by cheese-related microorganisms has not been well characterized . Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase . To differentiate between these two pathways and to determine the possible distribution between the pathways, 13C nuclear magnetic resonance (NMR) performed with uniformly enriched {13C}methionine was utilized . The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined . Only the aminotransferase-initiated pathway was observed . The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively . Production of methanethiol was not observed in any of the 13C NMR studies . Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol . The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid . These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.

Mol Gen Genet, 1998 Oct, 260(1), 38 - 47
In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2; Grohmann E et al.; The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism . Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01 . We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae . In the latter host, we have defined in vivo the nick site introduced by the RepX protein . Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S . pneumoniae . Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1 . Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements . Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place.

Dis Aquat Organ, 1998 Oct 8, 34(2), 93 - 101
Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality in Macrobrachium rosenbergii in Taiwan; Cheng W et al.; A Gram-positive, ovoid, diplococoid bacterium tentatively identified as Enterococcus-like was isolated from diseased Macrobrachium rosenbergii in Taiwanese aquaculture ponds . The diseased prawns displayed poor growth, anorexia, inactivity, opaque and whitish musculature, and mortality . In histological preparations, melanized hemocytic granulomas were seen in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature . Five isolates of diplococci were collected from diseased prawns at 4 farms and these were evaluated for 93 characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics . The results indicated that the isolates belonged to a single species . They grew in 0.5 to 6.0% NaCl, at 10 to 40 degrees C, at pH 9.6 and on bile esculin medium, gave positive pyrrolidonylarylamidase, arginine dehydrolase and Voges-Proskauer tests, were resistant to bacitracin and SXT, and were CAMP-negative and non-hemolytic on sheep blood agar . These findings indicated an Enterococcus-like bacterium closely related to Enterococcus seriolicida (recently reduced to synonymy with Lactococcus garvieae) . Experimental injection of 3 x 10(5) cells of strain KM002 of this Enterococcus-like bacterium into the ventral sinus of the prawn cephalothorax caused 100% mortality in 11 d, and induced muscular necrosis and hepatopancreatitis, gross signs and histopathology similar to those observed in the naturally infected prawns . It was concluded that this Enterococcus-like bacterium was the etiological agent associated with mortality of the farmed, diseased prawns.

Diagn Microbiol Infect Dis, 1998 Oct, 32(2), 127 - 30
Lactococcus garvieae endocarditis: report of a case and review of the literature; Fefer JJ et al.; Bacterial endocarditis due to Lactococcus garvieae is extremely unusual, and may actually be underreported due to its morphologic and biochemical similarities with enterococci . Only three cases have been reported in the medical literature, and all involved prosthetic valves . We report a case of native valve bacterial endocarditis caused by L . garvieae.

J Bacteriol, 1998 Nov, 180(22), 5947 - 53
Autolysis of Lactococcus lactis is influenced by proteolysis; Buist G et al.; The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP . Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L . lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI) . On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity . Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac . Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities . Autolysis of L . lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase . No autolysis was observed when the various proteinases were expressed in an L . lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells . The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.

J Bacteriol, 1998 Nov, 180(22), 5860 - 5
A Saccharomyces cerevisiae mutant lacking a K+/H+ exchanger; Ramirez J et al.; The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K+/H+ antiporter (873 amino acids) in Saccharomyces cerevisiae was disrupted by homologous recombination . The core protein is similar to the putative Na+/H+ antiporters from Enterococcus hirae (NAPA gene) and Lactococcus lactis (LLUPP gene) and the putative K+/H+ exchanger from Escherichia coli (KEFC gene) . Disruption of the KHA1 gene resulted in an increased K+ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used . Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant . Kinetic studies of 86Rb+ uptake showed the same saturable system for wild-type and disruptant strains . Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity . We speculate that higher K+ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.

J Bacteriol, 1998 Nov, 180(22), 5844 - 54
Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes; Twomey DP et al.; The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp . lactis biovar diacetylactis KR2 was determined . This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM . The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3' . In addition, M . LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M . Sau3AI . Both of these proteins shared notable homology throughout their putative target recognition domains . Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M . LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites . The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine . The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats . A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982 . Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

J Biotechnol, 1998 Aug 27, 63(3), 229 - 33
Procedure for quantifiable assessment of nutritional parameters influencing nisin production by Lactococcus lactis subsp . lactis; Chandrapati S et al.; A modified rapid plate assay procedure was developed, that allowed quantifiable measurement of nisin production by Lactococcus lactis growing directly on agar media . Using this direct plate assay, several nutritional parameters were assessed for their influence on nisin production (as distinct from their influence on growth) by L . lactis subsp . lactis ATCC 11454 growing on standard M17 based media over 3 and 6 h incubation periods . Glucose was found to be the optimal carbon source tested, with glycerol having the greatest suppressive effect . The addition of salts suppressed nisin production on a per cell basis, except MnCl2 . This direct plate method proved to be a good pilot assay for rapidly and quantifiably investigating the initial effects of different parameters on nisin production by L . lactis, prior to conducting more intensive broth batch culture assays . The data obtained in this study indicate that certain nutritional parameters can impose a repressive effect on nisin production . Elucidation of how these parameters control the amount of nisin produced will provide further insight into the regulation of nisin biosynthesis in L . lactis.

Microbiology, 1998 Oct, 144 ( Pt 10), 2885 - 93
Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus lactis MG1363; Wouters JA et al.; A family of genes encoding cold-shock proteins, named cspA, cspB, cspC, cspD and cspE, was cloned and sequenced from Lactococcus lactis MG1363 . The genes cspA and cspB and the genes cspC and cspD are located in tandem repeats, an organization of csp genes that has never been encountered before . The five genes encode small (7.1-7.6 kDa) proteins with high mutual sequence identities (up to 85%) and high identities (about 45-65%) with the major cold-shock proteins from Escherichia coli (CspA) and Bacillus subtilis (CspB) . Northern-blot analysis revealed single transcripts of about 300 nucleotides for each csp gene and showed that cspA, cspB, cspC and cspD mRNA levels were strongly increased upon cold shock to 10 degrees C (about 10-, 40-, 10- and 30-fold compared to 30 degrees C, respectively), whereas the cspE mRNA level was not increased . The expression of the cold-induced csp genes was highest in the 6-8 h lag phase after cold shock . A differential expression in time, in which cspA and cspC were maximally expressed at 2 h and cspB and cspD at 4 h after cold shock, was observed . The -35 and -10 regions of the five promoters were identified and transcriptional start sites were mapped in each case by primer extension at different temperatures which confirmed that regulation takes place at the transcriptional level . Significant differences were observed between the 5'-untranslated leader regions of the four cold-induced csp genes and the corresponding region of the non-cold-induced cspE gene.

Microbiology, 1998 Oct, 144 ( Pt 10), 2845 - 54
Heterologous expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway; Biet F et al.; Two different N-terminal extensions have been identified within class II bacteriocin precursors . The first one is a two-glycine-type leader peptide associated with a dedicated ATP-binding cassette transporter . The second is a signal peptide which directs the bacteriocin precursor to the general secretion machinery . Mesentericin Y105 is a class II anti-Listeria bacteriocin produced by Leuconostoc mesenteroides Y105 via a dedicated transport system (DTS) . To investigate heterologous expression systems capable of producing mesentericin Y105 in various hosts, two different secretion vectors were constructed . One of them, containing the mesentericin Y105 structural gene fused to the segment encoding the divergicin A signal peptide, was introduced into Escherichia coli, Leuconostoc subsp . and Lactococcus subsp . In E . coli, mesentericin Y105 production was linked to a putative periplasmic toxicity . To take advantage of this secretion system, the mesentericin Y105 precursor was also produced in E . coli . It was demonstrated that this pre-bacteriocin exhibited some antagonistic activity against Listeria . To allow for a comparison between the two different transport systems, mesentericin Y105 production using the vector containing the mesentericin Y105 structural gene and its DTS transporter operon was examined . The production of mesentericin Y105 was monitored by a new fast purification method followed by MS analysis . It was shown that, in Leuconostoc, the production of mesentericin Y105 is enhanced via the DTS compared to the general secretion pathway.

Appl Environ Microbiol, 1998 Nov, 64(11), 4618 - 22
Design of a phage-insensitive lactococcal dairy starter via sequential transfer of naturally occurring conjugative plasmids
O'Sullivan D, Coffey A, Fitzgerald GF, Hill C, Ross RP.
The plasmid-free Lactococcus lactis subsp . cremoris MG1614 is highly phage sensitive and lacks lactose fermenting ability (Lac) and primary casein degrading ability (Prt) . Food grade gene transfer systems were used to sequentially superimpose different phage defense systems on this background, resulting in a gradual increase in resistance to bacteriophage in the derivatives . pLP712, encoding Lac and Prt, was then transferred to one of these hosts, into which plasmids encoding adsorption inhibition, restriction modification, and abortive infection had already been introduced . This resulted in a phage-resistant strain which was successfully used as a single-strain starter for cheddar cheese manufacture under industrial conditions.

Appl Environ Microbiol, 1998 Nov, 64(11), 4507 - 12
Humic acid reduction by propionibacterium freudenreichii and other fermenting bacteria
Benz M, Schink B, Brune A.
Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation . Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction . Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids . Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P . freudenreichii even oxidized propionate to acetate under these conditions . When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts . These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction . This information needs to be considered in future studies of electron flow in soils and sediments.

Appl Environ Microbiol, 1998 Nov, 64(11), 4142 - 8
Analysis of the bacteriolytic enzymes of the autolytic lactococcus lactis subsp . cremoris strain AM2 by renaturing polyacrylamide gel electrophoresis: identification of a prophage-encoded enzyme
Lepeuple AS, Van Gemert E, Chapot-Chartier MP.
Lactococcus lactis subsp . cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P . Chapot-Chartier, C . Deniel, M . Rousseau, L . Vassal, and J.-C . Gripon, Int . Dairy J . 4:251-269, 1994) . We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L . lactis autoclaved cells . Several lytic activities were detected in L . lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L . lactis cell substrate . This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G . Buist, J . Kok, K . J . Leenhouts, M . Dabrowska, G . Venema, and A . J . Haandrickman, J . Bacteriol . 177:1554-1563, 1995), which has a similar molecular mass . The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds . L . lactis AM2 is lysogenic and mitomycin C inducible . Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded . It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages . A prophage-cured derivative of L . lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2 . After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture . These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.

Appl Environ Microbiol, 1998 Nov, 64(11), 4591 - 5
Genetic characterization of pepP, which encodes an aminopeptidase P whose deficiency does not affect Lactococcus lactis growth in milk, unlike deficiency of the X-prolyl dipeptidyl aminopeptidase; Matos J et al.; We sequenced the pepP gene of Lactococcus lactis, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition . PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.

Appl Environ Microbiol, 1998 Nov, 64(11), 4321 - 7
Cloning and expression of the Lactococcus lactis purDEK genes, required for growth in milk; Nilsson D et al.; An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram-positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609 . The genes encode enzymes in the de novo pathway of purine nucleotides . The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium . Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which encode beta-galactosidase . Deletion of a region 79 bp upstream of the transcription start point reduced the promoter activity 33-fold when incubated in a purine-free medium and to values below the detection limit when incubated in a purine-containing medium . No secondary transcription start points were mapped in or close to this region, indicating that a putative activator site and not a promoter was deleted or partly destroyed.

Appl Environ Microbiol, 1998 Nov, 64(11), 4255 - 9
Monoclonal antibodies raised against native major capsid proteins of lactococcal c2-like bacteriophages; Chibani Azaiez SR et al.; Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice . Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay . Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA . Immunoelectron microscopy showed that both MAbs recognized only phage head proteins . They did not react with any denatured phage proteins in Western blot assays . However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa . Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes . These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.

Mol Microbiol, 1998 Aug, 29(4), 1029 - 38
Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147; Dougherty BA et al.; The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp . lactis DPC3147, has been determined . Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy) . Sixty-four open reading frames (ORFs) were identified . Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements . The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid . The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general . The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.

Res Microbiol, 1997 Sep-Oct, 148(7), 573 - 83
Comparative analysis of the genomic DNA terminal regions of the lactococcal bacteriophages from species c2; Perrin R et al.; In an attempt to compare the cos intergenic region and bordering ORFs from Lactococcus lactis bacteriophages of the species c2, the nucleotide sequence of a 2479-bp fragment containing the cos site of phage P001 DNA was determined and compared with the corresponding regions of phages c2, bIL67 and P6 (partial sequence), which belong to species c2 . This comparative analysis revealed that some characteristic features of the cos intergenic region are conserved in all members of species c2 . Some of them are specific to species c2, as is the case for a GC-rich repeat in phase with the double helix that is located close to cos . One conserved motif seems to be more general, as it is found in all the cos regions of L . lactis bacteriophages that have been sequenced . It consists in a 4-nt indirect repeat TCAN/NACT located in a 15-bp fragment containing cos . This motif may be related to terminase specificity, as most of the cos asymmetric cleavages identified up to now are located within, or at the border of, these indirectly repeated sequences . Finally, some of the conserved DNA motifs of the species c2 cos-intergenic region seem to be even more general, as they are homologous to the lambda-R sites known to be involved in the maturation and the encapsidation of phage lambda DNA . Our comparative analysis also showed that within c2 phage DNAs, large blocks of sequences, i.e . the intergenic cos region and ORF/17 on the one hand, and ORF/16 on the other hand, evolved as distinct entities, probably by block recombination between phage DNAs of the same species.

J Bacteriol, 1998 Oct, 180(20), 5285 - 90
Characterization of multiple regions involved in replication and mobilization of plasmid pNZ4000 coding for exopolysaccharide production in Lactococcus lactis; van Kranenburg R et al.; We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40 . The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2, repB3, and repB4) that belong to the lactococcal theta replicon family . Subcloning of each replicon individually showed that all are functional and compatible in L . lactis . Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L . lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid . Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats . Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality . One oriT sequence was followed by a mobA gene, coding for a trans-acting protein, which increased the frequency of conjugative transfer 100-fold . The predicted MobA protein and the oriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and nic site of the oriT from the Escherichia coli plasmid R64 . The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1574 - 80
Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions; Amachi S et al.; A mutant of Lactococcus lactis subsp . lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity . The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions . At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain . The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured . The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain . At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0 while the level of increase in the mutant was only 1.6 . Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1 beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene . On the other hand, at pH 4.5 the transcriptional level and the amount of F1 beta subunit were found to be significantly higher in both strains than those at pH 7.0 . From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression . It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

Syst Appl Microbiol, 1998 Mar, 21(1), 28 - 32
Bacillus subtilis develops competence for uptake of plasmid DNA when growing in milk products; Zenz KI et al.; Transformation with plasmid DNA of naturally competent cells of Bacillus subtilis 168 in milk products was studied . Plasmid pMG36enpr, a broad host-range lactococcal vector carrying an erythromycin resistance and the B . subtilis npr gene encoding neutral protease, was taken up by B . subtilis cells grown in UHT chocolate milk . Under these conditions competence was optimal during transition from exponential to stationary growth phase, resulting in 9 x 10(1) transformants per 0.01 microgram DNA . No manipulation of the cells was necessary for competence to develop . When cells were pregrown in synthetic medium, higher transformation rates were obtained in assays, where the subsequent transformation experiments were either done in chocolate milk diluted 1:1 (v/v) with synthetic growth medium (up to 8 x 10(2) transformants) or in undiluted chocolate milk (1 x 10(2) transformants) . The number of transformants was reduced to 4 x 10 (1), when diluted milk or flavored milks were used . No transformants were obtained in diluted yoghurt . Controls, in which both the preculturing and the transformation assays were done in synthetic medium, gave the maximum number of transformants (4 x 10(3) transformants per 0.01 microgram DNA).

FEMS Microbiol Lett, 1998 Sep 1, 166(1), 15 - 20
Two methods for the genetic differentiation of Lactococcus lactis ssp . lactis and cremoris based on differences in the 16S rRNA gene sequence; Ward LJ et al.; The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp . lactis and ssp . cremoris differ by 9-10 bp (depending on strain), within the first 200 bp of the sequence . These differences were used to develop two methods of genetically differentiating lactis and cremoris strains . Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene . A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences . The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence . Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris . This method was used to examine lactococci isolated from raw milk . Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.

J Bacteriol, 1998 Sep, 180(18), 4893 - 902
Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO2054; Vaughan EE et al.; The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes . Initially the beta-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment . Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements . The gene order is galK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, and lacA encodes a galactoside acetyltransferase . The galT and galE genes of L . lactis LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L . lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054 . Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units . The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless beta-glucuronidase gene (gusA) from E . coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon . The greater similarity of the lacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L . lactis NCDO2054 have been recently acquired . Thus, the lacA-lacZ genes appear to have engaged the promoters of the gal operon in order to direct and control their expression.

J Bacteriol, 1998 Sep, 180(18), 4834 - 42
A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences; Daveran-Mingot ML et al.; Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Lactococcus lactis subsp . cremoris strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome . To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized . Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS905 element in each junction . Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase . Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS905 element . The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the oriC-terC symmetry of the chromosome and the local genetic environment.

Cell, 1998 Aug 21, 94(4), 451 - 62
Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination; Cousineau B et al.; The mobile group II intron of Lactococcus lactis, Ll.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems . Here, we show that Ll.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L . lactis . Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homology and without coconversion of exon markers . These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis . The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles . Thus, Ll.LtrB has many features of retrotransposons, with practical and evolutionary implications.

Appl Environ Microbiol, 1998 Sep, 64(9), 3159 - 65
Modeling of the competitive growth of Listeria monocytogenes and Lactococcus lactis in vegetable broth; Breidt F et al.; Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria . We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures . In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH . In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium . Predictions of the model indicate that pH is the primary factor that limits the growth of L . monocytogenes in competition with a strain of L . lactis which does not produce the bacteriocin nisin . The model also predicts the values of parameters that affect the growth and death of the competing populations . Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.

Lett Appl Microbiol, 1998 Jul, 27(1), 45 - 8
Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods; Sunen E; Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied . The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms . The main smoke components were identified and quantified by gas chromatography/mass spectrometry . The most effective condensate was S2 . All strains except Salmonella enteritidis were inhibited by S2 with an MIC < 0.5-1.5% . Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L . inocua, Brochothrix thermosphacta and Lactococcus lactis ssp lactis with an MIC of < 0.2-0.8% . The condensate L3 inhibited effectively V . vulnificus, B . subtilis, L . innocua and Staph . aureus . L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms . Vibrio vulnificus was the most susceptible micro-organism to test compounds . The antimicrobial activity of smoke preparations was related to the concentration of phenols.

J Bacteriol, 1998 Sep, 180(17), 4487 - 96
Transcription analysis of the prolate-headed lactococcal bacteriophage c2; Lubbers MW et al.; A detailed transcription map of the prolate-headed lactococcal phage c2 has been constructed . Transcription of about one-third of the genome, encoding 22 open reading frames, began within the first 2 min of infection and produced at least 12 overlapping transcripts that persisted until lysis occurred at 30 min after initiation of infection . The remaining two-thirds of the genome, encoding 17 open reading frames, was divergently transcribed, beginning between 4 and 6 min after initiation of infection, and resulted in at least 18 overlapping transcripts that persisted until lysis . Five very strong, simultaneously active, and probably unregulated early promoters and a single positively regulated late promoter were identified . The late promoter had an extended -10 sequence, had a significant basal level of activity in the uninduced state, and was induced to high activity by a phage gene product . The complex overlapping pattern of transcripts resulted from the action of the multiple early promoters, inefficient termination of transcription, and (possibly) processing of a late precursor transcript(s) . Phage proteins were not required for these processes, and the host RNA polymerase was probably used for both early and late transcription.

J Bacteriol, 1998 Sep, 180(17), 4380 - 6
The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically; Martinussen J et al.; The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase . The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit . Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine . By mutant analysis, L . lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways . Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway . The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism . Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.

Microbiology, 1998 Aug, 144 ( Pt 8), 2203 - 15
Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region; Madsen PL et al.; Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments . The latent period was found to be 65 min and the burst size 40 +/- 10 . The eight early transcripts, all mapping in a 13 kb region adjacent to the attachment site of TP901-1, were present at maximal levels 10 min after infection . The four middle transcripts, observed at maximal levels 30 min after infection, are all located within a 2 kb region at the distal end of the early transcripts . The late class of transcripts were detected 40 min after infection and the amounts of these transcripts increased with time . The late transcripts were localized to the 13 kb region adjacent to the 2 kb middle transcribed region . The sequence of almost 4 kb of the early region was determined, allowing a detailed transcriptional map for the early region of which in total 6.4 kb was sequenced . Sequence analysis of the early region revealed two closely positioned but divergently orientated promoters, PL and PR, in accordance with the orientation of the ORFs and the transcriptional map . Nine ORFs were found, and similarities to a phage repressor, a single-stranded DNA-binding protein, a topoisomerase, a Cro-like protein and two other phage proteins of unknown function were detected . The gene arrangement in the early transcribed region of TP901-1 thus consists of two transcriptional units: one from PR containing four genes, of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle . ORFs with homology to proteins involved in DNA replication were identified on the latter transcriptional unit.

Microbiology, 1998 Aug, 144 ( Pt 8), 2103 - 11
Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region; Frenkiel H et al.; The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis . This leader presents striking similarity to 'T-box' leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA . A region of the L . lactis leader transcript also contains a series of (G/U) AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription . A screen was developed for the isolation of regulatory mutants affected in the leader region . All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis . All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.

J Appl Microbiol, 1998 Jun, 84(6), 1099 - 103
Detection of specific bacteriocin-producing lactic acid bacteria by colony hybridization; Martinez MI et al.; A colony hybridization method for detecting lactic acid bacteria encoding specific bacteriocins was developed . Specific PCR-generated probes were used to detect colonies of pediocin PA-1, lactococcin A, enterocin AS-48, nisin A and lacticin 481 producing strains . The probes were shown to be sensitive and specific for sequences belonging to the structural genes of the respective bacteriocins.

J Food Prot, 1998 Aug, 61(8), 1007 - 12
Selective hydrolysis of milk proteins to facilitate the elimination of the ABBOS epitope of bovine serum albumin and other immunoreactive epitopes; Alting AC et al.; Milk proteins are hydrolyzed to prevent immunological reactions, but immunoreactive epitopes, including the ABBOS epitope of bovine serum albumin (BSA), can still be detected in commercially available milk protein hydrolysates . We used lactococcal cell-envelope proteinase (CEP) for the hydrolysis of the individual milk proteins and of mixtures thereof, or for the hydrolysis of sodium caseinate (contaminated with whey proteins) . CEP exclusively degraded casein, leaving the four major whey proteins intact . This property facilitated the removal of the intact whey proteins from the casein fragments by ultrafiltration . Depending on the molecular mass of the whey protein to be removed, membranes with cutoff values between 3 and 30 kDa were used, resulting in casein hydrolysates free of protein fragments with cross-reactive whey-protein-specific IgE (immunoglobulin E) or ABBOS antibody-binding sites . Even the casein itself was degraded in such a way by CEP that cross-reactive casein-specific IgE antibody-binding sites could be eliminated . The product could find application in infant formulas for therapeutic and preventive treatment of children with cow's milk allergy; in addition, the preventive use of such formulas in children genetically susceptible to the development of insulin-dependent diabetes mellitus (IDDM) should be considered if a relationship between the consumption of BSA and IDDM were to become more apparent . The method is also applicable for preparing casein-free whey protein preparations.

Mol Microbiol, 1998 Jul, 29(1), 61 - 74
tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon; van de Guchte M et al.; The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination . We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation . In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination . These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA . Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon . For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA . In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA . Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript . Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Cryobiology, 1998 Aug, 37(1), 86 - 91
Effect of cold shock on protein synthesis and on cryotolerance of cells frozen for long periods in Lactococcus lactis; Kim WS et al.; Aspects of the cold-shock response in Lactococcus lactis subsp . lactis LL41-1 were investigated . First, it was determined whether new proteins were synthesized in response to cold shock . Cell-free extracts were prepared from a cold-shocked (exposed to 10 degreesC for 5 h) culture (cfe-cs) and from a non-cold-shocked (held at 30 degreesC continuously) culture (cfe-non), and were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis . A protein of approximately 6.3 kDa was present in the cfe-cs and appeared not to be present in the cfe-non . No other changes were evident . Second, the effect of cold shock on cryotolerance of cells that have been frozen at -20 degreesC for up to 1 year was examined . Without the cold-shock treatment prior to freezing the cell viability following freezing for 1 day was 34%, 14 days 32%, 182 days 7%, and 364 days 0.2% . However, with the cold shock treatment it was 83%, 82%, 12%, and 0.8%, respectively . It appears that cold shock significantly improves cryotolerance of the cells for short periods of freezing, but the protective effect was less marked following longer storage periods .

Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 31 - 6
The ABC family of multidrug transporters in microorganisms; van Veen HW et al.; Multidrug transporters are membrane proteins that are able to expel a broad range of toxic molecules from the cell . In humans, the overexpression of the multidrug resistance P-glycoprotein (Pgp) and the multidrug resistance-associated protein MRP1 (MRP) is a principal cause of resistance of cancers to chemotherapy . These multidrug transporters belong to the ATP-binding cassette (ABC) family of transport proteins that utilize the energy of ATP hydrolysis for activity . In microorganisms, multidrug transporters play an important role in conferring antibiotic resistance on pathogens . In the last decade, homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, Candida albicans, Saccharomyces cerevisiae and, more recently, in Lactococcus lactis . In this review, we will summarize the current state of knowledge on three major aspects of microbial ABC-type multidrug transporters: (i) the functional and structural similarities among these proteins in prokaryotic and eukaryotic cells, (ii) the molecular mechanism of these transporters, and (iii) their potential physiological role.

J Dairy Sci, 1998 Jun, 81(6), 1486 - 91
Production of gamma-aminobutyric acid by cheese starters during cheese ripening; Nomura M et al.; Nine mixed-strain starters were examined for their abilities to produce gamma-aminobutyric acid . Six commercial starters were found to produce gamma-aminobutyric acid in a skim milk culture . The bacterium that produced gamma-aminobutyric acid was isolated from the mixed-strain starters, identified as citrate-utilizing Lactococcus lactis ssp . lactis (formerly L . lactis ssp . lactis biovar diacetylactis) and designated as strain 01-7 . A cell extract showed glutamate decarboxylase activity, for which the optimum pH was 4.7 . In pH-controlled cultivation, gamma-aminobutyric acid was generated at pH 5.0 but not above pH 5.5 . Cheeses were prepared experimentally using strain 01-7 to determine the relationship between the pH values and the production of gamma-aminobutyric acid during cheese ripening . gamma-Aminobutyric acid increased linearly in the experimental cheeses as the pH of the cheese decreased . Based on these results, gamma-aminobutyric acid was concluded to be produced by the cheese starters during ripening.

J Bacteriol, 1998 Aug, 180(15), 3907 - 16
A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis; Kilstrup M et al.; A purR::pGh9:ISS1 mutant of Lactococcus lactis was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxotrophs . After determination of the nucleotide sequence and deduction of the purR reading frame, the PurR product was found to be highly similar to the purR-encoded repressor from Bacillus subtilis . The wild-type purR gene complemented the purine auxotrophy of a purR::ISS1 mutant, and it was shown that the purR::ISS1 mutation lowered the level of transcription from the purine-regulated L . lactis purD promoter . In a parallel study on the regulation of purC and purD expression in L . lactis (M . Kilstrup, S . G . Jessing, S . B . Wichmand-Jorgensen, M . Madsen, and D . Nilsson, J . Bacteriol . 180:3900-3906, 1998), we identified regions (PurBox sequences: AWWWCCGAACWWT) upstream of the promoters with a central G residue at exactly position -76 relative to the transcriptional start site . The PurBox sequences were found to be required for high-level promoter activity and purine regulation . We identified a PurBox sequence overlapping the -35 region of the L . lactis purR promoter and found, by studies of a purR-lacLM fusion plasmid, that purR is autoregulated . Because of the high degree of similarity of the PurR proteins from B . subtilis and L . lactis, we looked for PurBox sequences in the promoter regions of the PurR-regulated genes in B . subtilis and identified a perfectly matching PurBox sequence in the purA promoter region and slightly degenerate PurBox-like sequences in the promoter regions for the pur operon and the purR gene . Interestingly, the PurBox in the pur operon of B . subtilis is located almost identically, with respect to the promoter, to the PurBox sequences located in front of purC and purD in L . lactis . We present a hypothesis to explain how an ancestral PurR protein in B . subtilis could have evolved from an activator of the pur operon into a repressor which regulates transcription initiation from the same pur promoter by using the same PurR binding site and a similar response toward its effectors.

J Bacteriol, 1998 Aug, 180(15), 3900 - 6
Activation control of pur gene expression in Lactococcus lactis: proposal for a consensus activator binding sequence based on deletion analysis and site-directed mutagenesis of purC and purD promoter regions; Kilstrup M et al.; A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between -79 and -70 nucleotides upstream from the transcriptional start sites . Both promoters have well-defined -10 regions but lack sequences resembling -35 regions for sigma70 promoters . Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation . Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene . A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions . By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the purC and purD promoters . Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important . All results support the idea that purC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

J Bacteriol, 1998 Aug, 180(15), 3873 - 81
Induced levels of heat shock proteins in a dnaK mutant of Lactococcus lactis; Koch B et al.; The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes . In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK . The minor deletion of 65 amino acids in the dnaKDelta2 mutant resulted in a slight temperature-sensitive phenotype . BK6, containing the larger deletion of 174 amino acids (dnaKDelta1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response . The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L . lactis . For Bacillus subtilis, it has been found (A . Mogk, G . Homuth, C . Scholz, L . Kim, F . X . Schmid, and W . Schumann, EMBO J . 16:4579-4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins . The present data from L . lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.

J Bacteriol, 1998 Aug, 180(15), 3804 - 8
Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase; Lopez de Felipe F et al.; NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L . lactis nisA promoter . This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions . Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism . The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced . At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0 . Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values . Pyruvate was mostly converted to acetoin or diacetyl via alpha-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase . The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide . Under these conditions, lactate production was completely absent . Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor . These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ ratios in the cells.

FEMS Microbiol Lett, 1998 Jul 15, 164(2), 419 - 26
Involvement of antisense RNA in replication control of the lactococcal plasmid pND324; Duan K et al.; pND324 belongs to a family of closely related theta-type plasmids from Lactococcus lactis . An antisense RNA, termed countertranscript (ctRNA), was identified which is complementary to the leader sequence of the mRNA that encodes RepB, a protein essential for plasmid replication . When the synthesis of ctRNA was abolished by site-directed mutagenesis within its promoter region, the mutant replicon showed a 1.8-fold increase in copy number . Similar ctRNA promoter sequences are readily identifiable in 12 other published lactococcal theta-type plasmids, suggesting that they all encode a similar ctRNA-mediated regulatory mechanism.

Gene, 1998 May 28, 212(1), 5 - 11
A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species; Dinsmore PK et al.; The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species . Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein . The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis . Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively . The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263 . Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested . Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested . Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity . Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained . Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important . Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity . This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Mol Biotechnol, 1998 Apr, 9(2), 127 - 39
Inducible gene expression systems in Lactococcus lactis; Djordjevic GM et al.; Lactococcus lactis is industrially important microorganism used in many dairy fermentations . Numerous genes and gene expression signals from this organism have now been identified and characterized . Recently, several naturally occurring, inducible gene-expression systems have also been described in L . lactis . The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes . Novel gene-expression systems in Lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.

Protein Sci, 1998 Jun, 7(6), 1269 - 79
The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function; Rowland P et al.; Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides . A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate . The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme . The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop . In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues . The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop . The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71 . Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate . Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling . The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.

Appl Environ Microbiol, 1998 Jul, 64(7), 2721 - 2
Use of luciferase genes as biosensors to study bacterial physiology in the digestive tract; Corthier G et al.; A method based on the use of the bacterial luciferase genes was developed in order to investigate Lactococcus lactis gene expression in the mouse digestive tract . Germfree mice were monoassociated with different strains containing transcriptional fusions of promoters with the luciferase genes . Our results demonstrate that this method is readily applicable to the study of promoter strength and physiology of bacteria in the digestive tract.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2485 - 9
Glutamate Biosynthesis in Lactococcus lactis subsp . lactis NCDO 2118
Lapujade P, Cocaign-Bousquet M, Loubiere P.
Unlike other lactic acid bacteria, Lactococcus lactis subsp . lactis NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family . Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h-1) compared to that in the reference medium containing glutamate (0.16 h-1) . The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected . As in most anaerobic bacteria, no alpha-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of alpha-ketoglutarate . The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway . As regards the synthesis of glutamate from alpha-ketoglutarate, no glutamate dehydrogenase was detected . While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured . The conversion of alpha-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor . All of the enzymes assayed were shown to be constitutive.

Appl Environ Microbiol, 1998 Jul, 64(7), 2424 - 31
Cloning and characterization of the lactococcal plasmid-encoded type II restriction/modification system, LlaDII; Madsen A et al.; The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp . cremoris W39 . A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L . lactis shuttle vector pCI3340 conferred to L . lactis LM2301 and L . lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2 . The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site . It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI . Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region . The endonuclease gene of 543 bp preceded the methylase gene of 954 bp . The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp . strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases . The genetic organizations of the LlaDII and Bsp6I R/M systems are identical . Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences . Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.

Mol Microbiol, 1998 May, 28(4), 823 - 34
Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC; Bidnenko E et al.; The function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined . Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E . coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs) . The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation . DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids . The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures . Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orfs (orf2 and orf3) are needed for nucleolytic activity . The orf3 product has amino acid homology with the E . coli RuvC Holliday junction resolvase . By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s) . We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.

Wei Sheng Wu Xue Bao, 1996 Aug, 36(4), 269 - 75
{Studies on purification and some properties of nisin from Lactococcus lactis subsp . lactis Al2}; Chen X et al.; Nisin from Lactococcus lactis subsp . lactis AL2 was extracted with n-propanol from NaCl-saturated culture and purified by ion-exchange chromotography on CM-Sephadex C-25 . Nisin was purified 1.63 fold with a yield of 41.7% . The molecular weight of nisin was determined by SDS-PAGE to be about 3500 . Nisin activity was stable at low pH and sensitive to digestion by a-chymotrypsin . Nisin is capable of inhibiting a broad range of gram-positive bacteria . In contrast, the gram-negative bacteria, yeasts, molds and Nip+ L . lactis subsp . lactis ATCC11454 were not inhibited.

Infect Immun, 1998 Jul, 66(7), 3183 - 9
Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine; Steidler L et al.; Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes . To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L . lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6 . When mice were immunized intranasally with various different expression strains of L . lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC . This adjuvant effect was lost when the recombinant strains of L . lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci . These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.

J Appl Microbiol, 1998 Feb, 84(2), 176 - 84
Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence; Bouksaim M et al.; A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp . lactis biovar . diacetylactis UL 719 . A dot immunoblot assay was then developed to detect nisin Z in milk and whey . As few as 1.5 10(-1) international units per ml (IU ml-1), corresponding to 0.003 microgram ml-1 of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence . When milk and whey samples were tested, approximately 0.155 microgram ml-1 (7.9 IU ml-1) of nisin Z was detected . The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.

Lett Appl Microbiol, 1998 Apr, 26(4), 293 - 6
Using specific polyclonal antibodies to study the malolactic enzyme from Leuconostoc oenos and other lactic acid bacteria; Labarre C et al.; Specific polyclonal antibodies directed against the malolactic enzyme of Leuconostoc oenos were obtained . Despite the homologies between the malolactic enzymes from Leuc . oenos and Lactococcus lactis, no immunological relationship was detected with the L . lactis malolactic enzyme, suggesting differences in their structural organization . The use of the antiserum also demonstrated that the problem of heterologous expression occurring in the recombinant Escherichia coli strain (Labarre et al . 1996a) resulted in a low synthesis of the malolactic enzyme from Leuc . oenos . Moreover, a small amount of the protein was found to be peripherally associated to the membrane of Leuc . oenos.

Lett Appl Microbiol, 1998 Apr, 26(4), 270 - 4
Biogenic amine production by wild lactococcal and leuconostoc strains; Gonzalez de Llano D et al.; Two qualitative and one quantitative HPLC methods were evaluated for the detection of biogenic amine producers among wild dairy lactococcal and leuconostoc strains . High tyramine producers ranging from 370 to 807 mg l-1 were detected by qualitative methods and confirmed by HPLC analysis . Tyramine levels detected throughout the incubation time depended on the concentration of the amino acid precursor available and no tyramine production was observed when strains were grown in milk . However, increasing amounts of tyramine were detected in cultures grown in milk supplemented with different concentrations of tyrosine . Qualitative methods failed to detect weak producers so that tryptamine production (< 7 mg l-1) could only be determined by HPLC . None of the tested strains was able to produce histamine . Simultaneous production of different amines was observed by HPLC although no colour change was observed in the specific decarboxylase media . Thus, it was concluded that the amine forming ability should be taken into account when selecting starters for milk fermentations . Qualitative methods could be used as a first screening step to eliminate the highest amine producers while the quantitative methods would detect any producing strain.

Yakugaku Zasshi, 1998 Apr, 118(4), 150 - 7
{Biological response modifier activity of Lactococcus lactis 332}; Maeda M et al.; The biological response modifier (BRM) activity of a preparation of heat killed cells of Lactococcus lactis 332 which can produce large amount of lactate in culture, was investigated in C3H/He and ICR mice . Intraperitoneal injection of Lactococcus lactis 332 caused an accumulation of neutrophils and macrophages in the peritoneal cavity of the mice, similarly to BRMs used clinically . As a parameter of the activation of macrophages, the effect of the Lactococcus lactis 332 preparation on the production of tumor necrosis factor (TNF) was examined . The Lactococcus lactis 332 preparation was revealed to have both priming and triggering activities for the production of TNF . The TNF level in the sera reached about 1000 IU/ml in mice 2 h after the triggering injection . This preparation also stimulated peritoneal macrophages to produce TNF in vitro . Intratumoral injection of the Lactococcus lactis 332 preparation regressed MM46 tumor cells in C3H/He mice . These results suggest that the Lactococcus lactis 332 preparation is a biological response modifier (BRM) with various activities on phagocytes similarly to a streptococcal antitumor agent, OK432, used clinically.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 25 - 9
Determination of the recognition sequence of the type II restriction endonuclease, LlaCI, from Lactococcus lactis W15; Josephsen J et al.; A new type II restriction endonuclease, called LlaCI, was partially purified from Lactococcus lactis subsp . cremoris W15 . The characterisation of the LlaCI endonuclease showed it to be an isoschizomer of HindIII, recognising the sequence 5-'A decreases AGCTT-3' . The cleavage site is indicated by the arrow.

Int J Food Microbiol, 1998 May 5, 41(1), 73 - 80
Production of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp . lactis biovar . diacetylactis, isolated from Zimbabwean naturally fermented milk; Narvhus JA et al.; Malty compound-producing Lactococcus lactis subsp . lactis biovar . diacetylactis strain INF-DM1, originally isolated from naturally fermented milk in Zimbabwe was used to prepare fermented milk from ordinary milk, milk enriched with 2.5% (w/v) skimmed milk powder and by 2.5% (w/v) increase in dry matter by ultrafiltration . Inoculated milk was incubated at 22, 30 and 37 degrees C . Analyses were made after 0, 9, 18 and 24 h incubation and also after 24 h incubation followed by storage for one week at 4 degrees C . Samples were analysed for volatile compounds including malty compounds and for organic acids, pH and log cfu/g . All samples were also judged for sensory attributes . Products made from enriched milks showed increased viscosity which was most marked in ultrafiltrated milk incubated at 30 and 37 degrees C . The levels of certain compounds (lactic acid, citrate and diacetyl) were significantly affected by milk type . Incubation temperature had a significant effect on starter growth rate and the rate of production and amount of the malty compounds, lactate, diacetyl, ethanol, acetoin and acetaldehyde . 3-Methyl butanal concentrations were above the taste threshold level of 0.06 ppm in almost all products, including stored products . Although initial growth rate was fastest at 37 degrees C, an uncoupling of acid production and growth was observed after 9 h incubation, suggesting that this is above the optimum temperature for this strain . In addition, products incubated at 37 degrees C showed a tendency to whey separation, indicating that this temperature is also too high to give optimum product quality . All products attained good scores in sensory analysis provided that fermentation was complete . Variation in the levels of malty compounds during the fermentation had no significant effect on the sensory score for total flavour.

Biol Chem, 1998 Apr-May, 379(4-5), 443 - 9
Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp . cremoris W15; Madsen A et al.; The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L . lactis subsp . cremoris W15 . The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat) . Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L . lactis LM2301 and L . lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively . Increased plasmid copy number enhanced the level of phage restriction . Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation . IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR . The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction . The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon . It shows 24% identity to the HindIII endonuclease.

J Bacteriol, 1998 Jun, 180(12), 3174 - 80
Multiple transcriptional control of the Lactococcus lactis trp operon; Raya R et al.; The Lactococcus lactis trpEGDCFBA operon is preceded by a noncoding leader region . Transcriptional studies of the trp operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region) . These transcripts most likely result from initiation at the unique Ptrp promoter, transcription termination at either T1 (upstream of the trp operon) or T2 (downstream of the trp operon), and/or processing . Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly . The tryptophan-dependent transcription control is exerted through transcription antitermination.

J Bacteriol, 1998 Jun, 180(12), 3049 - 55
Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli; Arnau J et al.; The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized . A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L . lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically . Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence . Analysis of adhE expression in L . lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose . Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions . With the L . lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

Appl Microbiol Biotechnol, 1998 Apr, 49(4), 417 - 23
Construction of a food-grade multiple-copy integration system for Lactococcus lactis; Leenhouts K et al.; A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis . The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region . The system includes two L . lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors . These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background . Single-crossover integration of the plasmids in L . lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar . The amplifications were stable under selective growth conditions . In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation . One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth . These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

J Bacteriol, 1998 Jun, 180(11), 2950 - 7
D-Alanylcardiolipin, a major component of the unique lipid pattern of Vagococcus fluvialis; Fischer W et al.; Motile group N streptococci, classified as Vagococcus fluvialis, have been isolated from cows' udders, human and animal feces, river water, and seawater . They possess an unusual membrane lipid and fatty acid pattern . We isolated and characterized 13 polar lipids, 8 of them also found in other gram-positive bacteria: mono- and dihexosyldiacylglycerol, an acylated and a glycerophosphate-substituted derivative of the latter, cardiolipin, phosphatidylglycerol, D-alanylphosphatidylglycerol, and L-lysylphosphatidylglycerol . Besides them, we characterized two rare compounds, bis(acylglycero)phosphate and alpha-D-glucopyranosylcardiolipin, and two compounds so far not detected in nature, D-alanylbis(acylglycero)phosphate and D-alanylcardiolipin . The concomitant occurrence of four aminoacyl phospholipids in one organism is another unique finding . Substituted cardiolipins represent a novel lipid class: in vagococci, D-alanylcardiolipin is a major membrane lipid component, contributing 11 and 26 mol% of total lipids in the exponential and stationary phases of growth, respectively . The vagococcal lipids contain even-numbered straight-chain saturated and cis-monounsaturated fatty acids, but the cis-monoenic acids belong to the omega-9 series and not the omega-7 series, found in enterococci, lactococci, and streptococci.

Mol Gen Genet, 1998 Apr, 258(1-2), 9 - 15
RNA processing is involved in the post-transcriptional control of the citQRP operon from Lactococcus lactis biovar diacetylactis; Drider D et al.; The importance of Lactococcus lactis biovar diacetylactis (L . diacetylactis) in the dairy industry is due to its ability to produce aroma compounds, such as acetoin and diacetyl, from citrate . The first step in citrate utilization is its uptake by the cells . In L . diacetylactis, the citrate transport system is encoded by the citQRP operon . We have previously proposed that expression of citQRP operon is regulated at the post-transcriptional level . In this paper, we show that the cit mRNA is processed at a complex secondary structure in L . diacetylactis and Escherichia coli . This secondary structure includes the 5'-terminal two-thirds of citQ and the overlap between citQ and citR . Primer-extension analysis revealed that the major cleavage sites are located upstream of citR and within citQ . In an attempt to identify the enzyme(s) responsible for this cleavage, we have analyzed this processing in E . coli mutants deficient in endoribonucleases . A comparative analysis of cit mRNA degradation was performed in RNase E and RNase III mutants and in wild-type strains using Northern blot hybridization . This analysis revealed that the cit transcript is degraded into several breakdown products, which are significantly stabilized in the mutant lacking RNase III . Our results indicate that the complex secondary structure has a critical role in the control of the expression of cit mRNA . A model for processing is discussed.

Microbiology, 1998 May, 144 ( Pt 5), 1145 - 56
Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers; Ze-Ze L et al.; A physical map of the chromosome of Oenococcus oeni PSU-1 was constructed . This represents the first map for a strain of this species . A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb . Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O . oeni strain GM . Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS1165-like elements and rrn loci were located on the physical map . Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified . The two ribosomal operons have been precisely located and their transcription direction determined.

Appl Environ Microbiol, 1998 Jun, 64(6), 2111 - 6
Influence of reduced water activity on lactose metabolism by lactococcus lactis subsp . cremoris At different pH values
Liu S, Asmundson RV, Gopal PK, Holland R, Crow VL.
The influence of reduced water activity (aw) on lactose metabolism by Lactococcus lactis subsp . cremoris 2254 and 2272 was studied at different pH values . In control incubations (aw, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered as L-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5 . However, the levels of recovery decreased to approximately 50% at all pH values tested when the aw was 0.88 (with glycerol as the humectant) . When growing cells in broth controlled at pH 6.3 were used, a reduction in the aw from 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered as L-(+)-lactate from 100 to 71% . Low levels of L-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at aw values of 0.99 and 0 . 87 and in young cheese curds . The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate . Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used . Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants . Decreasing either the aw or the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect . The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions . A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-aw and low-pH stress conditions.

Appl Environ Microbiol, 1998 Jun, 64(6), 2287 - 90
Evaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens; Ryan MP et al.; Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp . lactis DPC3147 which is bactericidal against a range of mastitis-causing streptococci and staphylococci . In this study, both lacticin 3147 and the lantibiotic nisin were separately incorporated into an intramammary teat seal product . The seal containing lacticin 3147 exhibited excellent antimicrobial activity and might form the basis of an improved treatment for the prevention of mastitis in dry cows.

Appl Environ Microbiol, 1998 Jun, 64(6), 1991 - 6
The contribution of caseins to the amino acid supply for Lactococcus lactis depends on the type of cell envelope proteinase; Flambard B et al.; The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type) . Two genetically engineered strains of L . lactis that differed only in the type of proteinase were grown in chemically defined media containing alphas1-, beta-, and kappa-caseins (alone or in combination) as the sources of amino acids . Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase . Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation . This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L . lactis in milk (S . Helinck, J . Richard, and V . Juillard, Appl . Environ . Microbiol . 63:2124-2130, 1997) as a function of the type of proteinase . Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined . The results were in agreement only in the case of the PI-type proteinase . Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

Mol Gen Genet, 1998 Apr, 257(6), 681 - 5
Identification of a sodium chloride-regulated promoter in Lactococcus lactis by single-copy chromosomal fusion with a reporter gene; Sanders JW et al.; An integration vector, pORI13, was developed to screen in Lactococcus lactis for expression signals induced by changes in the environment and to assay transcriptional activity of genes in single copy . The plasmid carries a promoterless Escherichia coli lacZ gene preceded by a start codon, a lactococcal ribosome binding site, and a multiple cloning site . Chromosomal Sau3AI fragments of L . lactis MG1363 DNA were cloned in pORI13 using a RepA+ E . coli as host . The resulting bank of plasmids was used for Campbell-type integration into the chromosome of L . lactis MG1363 . The relatively large size of the chromosomal fragments used increases the chance of retaining complete genes in the targeted region . Screening of integrants in the presence of 0.3 M NaCl resulted in the isolation of a clone (NS3) in which expression of lacZ was dependent on the concentration of chloride ions.

J Clin Microbiol, 1998 Apr, 36(4), 983 - 5
Identification of Lactococcus garvieae by PCR; Zlotkin A et al.; Lactococcus garvieae junior synonym Enterococcus seriolicida) is an emerging zoonotic agent isolated from economically important fish (rainbow trout and yellowtail), from cattle, and from humans . Clindamycin susceptibility is the only phenotypic test which can differentiate L . garvieae from Lactococcus lactis, another emerging agent in humans . A PCR assay for the identification of L . garvieae was developed and resulted in an amplified fragment of 1,100 bp in size . The PCR assay was shown to be specific to L . garvieae . The PCR assay was positive for all the L . garvieae strains tested, which originated from three different continents (Asia, Australia, and Europe) . The PCR assay was negative for the phenotypically similar L . lactis and for all the other fish pathogens tested, including Streptococcus iniae and Aeromonas salmonicida . The PCR assay was applied to plasma obtained from diseased animals and was found sensitive enough to detect bacteria from 1 microl of plasma . The PCR assay that was developed is the only practical test besides the clindamycin test which can specifically identify the zoonotic agent L . garvieae and which can differentiate it from L . lactis.

FEMS Microbiol Lett, 1998 May 1, 162(1), 111 - 5
A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis; Urbach E et al.; A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis . The method distinguishes closely related strains and discriminates among some with identical ldh sequences . The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G + C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G + C Gram-positive species.

Mol Microbiol, 1998 Apr, 28(1), 169 - 78
Combinational variation of restriction modification specificities in Lactococcus lactis; Schouler C et al.; Three genes coding for a type I R-M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403 . In addition, plasmids were found that encode only the HsdS subunit that directs R-M specificity . The presence of these plasmids in IL1403 conferred a new R-M phenotype on the host, indicating that the plasmid-encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits . Such combinational variation of type I R-M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.

Int J Food Microbiol, 1998 Jan 6, 39(1-2), 129 - 32
Inhibitory activity of a nisin-producing starter culture on Listeria innocua in raw ewes milk Manchego cheese; Rodriguez E et al.; The inhibitory activity of nisin-producing Lactococcus lactis subsp . lactis ESI 515 on the survival of Listeria innocua during ripening of raw ewes milk Manchego cheese was investigated . After 60 days of ripening, counts of L . innocua in cheese were 4.08 log units lower than the control when Lc . lactis subsp . lactis ESI 515 was used as a single-strain starter . Nisin activity was detected in cheeses manufactured with Lc . lactis subsp . lactis ESI 515 throughout the ripening period.

Appl Environ Microbiol, 1998 May, 64(5), 1950 - 3
Lysis of Lactococcus lactis subsp . cremoris SK110 and its nisin-immune transconjugant in relation to flavor development in cheese; Meijer W et al.; To develop a nisin-producing cheese starter, Lactococcus lactis subsp . cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production . Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter . The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.

Appl Environ Microbiol, 1998 May, 64(5), 1673 - 9
A deficiency in aspartate biosynthesis in Lactococcus lactis subsp . lactis C2 causes slow milk coagulation; Wang H et al.; A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp . lactis C2, designated L . lactis KB4, was identified . Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype . When the amino acid requirements of L . lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid . This aspartic acid requirement could also be met by aspartate-containing peptides . The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4 . KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide . The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.

Appl Environ Microbiol, 1998 May, 64(5), 1594 - 600
Mechanism of the citrate transporters in carbohydrate and citrate cometabolism in Lactococcus and Leuconostoc species; Bandell M et al.; Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C . Marty-Teysset, C . Posthuma, J . S . Lolkema, P . Schmitt, C . Divies, and W . N . Konings, J . Bacteriol . 178:2178-2185, 1996) . Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue . Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis . The cloned gene was expressed in a Lactococcus lactis Cit- strain, and the gene product was functionally characterized in membrane vesicles . Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange . These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis . Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms . The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.

Mol Microbiol, 1998 Mar, 27(6), 1107 - 18
Reconstruction of the proteolytic pathway for use of beta-casein by Lactococcus lactis; Kunji ER et al.; Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids . For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell . We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures . This approach revealed in detail how L . lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet . Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo . To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds . The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues . The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 165 - 72
Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates; Sechi LA et al.; Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci . We tested several enterococcal ATCC strains and found that only E . hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence . Moreover, we wanted to investigate the dissemination of this new IS among E . faecium strains . We analyzed 131 clinical E . faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates . The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.

Appl Environ Microbiol, 1998 Apr, 64(4), 1541 - 4
An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp . lactis ML3; Mills DA et al.; Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp . lactis ML3 suggested that an origin of transfer (oriT) was present . Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans . The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE . Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.

Appl Environ Microbiol, 1998 Apr, 64(4), 1230 - 6
Specificity of milk peptide utilization by Lactococcus lactis; Juillard V et al.; To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L . lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of alpha s2-casein as the source of amino acids . Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatogrpaphy . Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L . lactis MG1363 and the inability to use large, acidic peptides . These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred . Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L . lactis MG1363 in milk was due to deprivation of leucine and methionine.

Biochim Biophys Acta, 1998 Mar 3, 1383(1), 63 - 70
Catalytic properties of the cysteine aminopeptidase PepC, a bacterial bleomycin hydrolase; Mistou MY et al.; PepC is a cytoplasmic thiol aminopeptidase widely conserved among lactic acid bacteria . PepC from Lactococcus lactis shares 35-38% identity with aminopeptidases of eukaryotic origins: the yeast and mammalian bleomycin hydrolases (BLMase) . In this work we investigated the hydrolytic activity of PepC towards various substrates: bleomycin A2, aminoacyl-p-nitroanilides (pNA) and peptides . First, we found the bleomycin hydrolase activity of lactococcal PepC and measured similar kinetics parameters to those reported for the mammalian BLMase . Second, the results obtained on aminoacyl-pNA confirmed the capacity of the enzyme to release a broad range of amino acids and the pH activity profile suggests the presence of an ionic interaction between the enzyme and the free alpha-amino group of the substrate . Third, the aminopeptidase activity measured on peptide substrates revealed that PepC possesses an extended binding site which interacts with the peptidic backbone of the substrate . The hydrolytic efficiency is highly dependent on the length of the peptide, optimal for tetrapeptides and further enhanced by the presence of hydrophobic residues in the P' positions of the substrate . These enzymatic properties are of importance for the design of specific inhibitors and the biological function of the bleomycin hydrolases.

J Bacteriol, 1998 Apr, 180(7), 1904 - 12
An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis; Poquet I et al.; The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters . In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria . Nuc devoid of its export signal (called delta(SP)Nuc) was used to create two fusions whose locations could be differentiated . Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of delta(SP)Nuc to report protein export . The shuttle vector pFUN was designed to construct delta(SP)Nuc translational fusions whose expression signals are provided by inserted DNA . The capacity of delta(SP)Nuc to reveal and identify exported proteins was tested by generating an L . lactis genomic library in pFUN and by screening for Nuc activity directly in L . lactis . All delta(SP)Nuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches . The function of some of the predicted signals was confirmed by cell fractionation studies . The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L . lactis . Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding . Our results with L . lactis show that delta(SP)Nuc is well suited to report both protein export and membrane protein topology.

J Bacteriol, 1998 Apr, 180(7), 1895 - 903
A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis; Le Loir Y et al.; Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins . We examined the secretion efficiency and capacity of L . lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein . When expressed in L . lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter . The remaining 40% was found in a cell-associated precursor form . The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT) . We identified a modification which improved secretion efficiency of both native Nuc and NucT . A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site . In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter) . The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90% . Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA . These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L . lactis.

Mol Microbiol, 1998 Mar, 27(5), 1009 - 20
Control of expression of LlaI restriction in Lactococcus lactis; O'Sullivan DJ et al.; The plasmid encoded LlaI R/M system from Lactococcus lactis ssp . lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex . Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon . In this study, the 5' end of the llal 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llalM . Deletion of this promoter region abolished LlaI restriction in L . lactis . Analysis of the intervening sequence revealed a 72-amino-acid open reading frame, designated llalC, with a conserved ribosome binding site and helix-turn-helix domain . Overexpression of llalC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa . Mutation and in trans complementation analyses indicated that C-LlaI positively enhanced LlaI restriction activity in vivo . Northern analysis and transcriptional fusions of the llal promoter to a lacZ reporter gene indicated that C x LlaI did not enhance transcription of the llal operon . Databank searches with the deduced protein sequence for llalC revealed significant homologies to the E . coli Rop regulatory and mRNA stabilizer protein . Investigation of the effect of C x LlaI on enhancement of LlaI restriction in L . lactis revealed that growth at elevated temperatures (40 degrees C) completely abolished any enhancement of restriction activity . These data provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.

J Dairy Res, 1998 Feb, 65(1), 101 - 7
Changes in the concentrations of free amino acids in milk during growth of Lactococcus lactis indicate biphasic nitrogen metabolism; Niven GW et al.; Analysis of the concentrations of free amino acids in milk during growth of Lactococcus lactis subsp, lactis revealed a biphasic pattern of change during the logarithmic phase . During the first period there was little overall change in the total concentration of amino acids in the medium . The second phase was characterized by increased net liberation of free amino acids . There were also qualitative differences in the amino acids that were taken up and utilized during each period . The concentrations of Val, Leu and Ile decreased only during the early phase, while those of Ser, Arg, Thr and Met decreased only during the second phase . Gly and Ala were utilized throughout logarithmic growth . Gly uptake appeared to be greater during the second period and accounted for the largest proportion of free amino acid utilization at this time . It is possible that the biphasic nature of amino acid nutrition was due to increased consumption in late log phase of peptides derived from milk proteins by proteolysis . Increased activity of the arginine deiminase pathway during late log phase was inferred from increased utilization of Arg and liberation of citrulline and ornithine.

FEMS Microbiol Lett, 1998 Feb 15, 159(2), 331 - 6
Nucleotide sequence and analysis of the new chromosomal abortive infection gene abiN of Lactococcus lactis subsp . cremoris S114; Prevots F et al.; A 7.275-kb DNA fragment which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis subsp . cremoris S114 . The genetic determinant for abortive infection was subcloned from this fragment . This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I homology) and for small isometric-headed bacteriophage phi 59 (group III homology) . This new gene, termed abiN, is predicted to encode a polypeptide of 178 amino acid residues with a deduced molecular mass of 20,461 Da and an isoelectric point of 4.63 . No homology with any previously described genes was found . A probe was used to determine the presence of this gene only in S114 from 31 strains tested.

Appl Environ Microbiol, 1998 Mar, 64(3), 1147 - 52
Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species; Walker SA et al.; A phage-inducible middle promoter (P15A10) from the lytic, lactococcal bacteriophage phi 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end . Sequence analysis revealed extensive homology (> 95%) to the right cohesive ends of two temperate phages of the P335 species, phi r1t and phi LC3 . Sequencing upstream and downstream of P15A10 showed that the high degree of homology between phi 31 and phi r1t continued beyond the phage promoter . With the exception of one extra open reading frame in phi 31, the sequences were highly homologous (95 to 98%) between nucleotides 13,448 and 16,320 of the published phi r1t sequence . By use of a beta-galactosidase (beta-Gal) gene under the control of a smaller, more tightly regulated region within the P15A10 promoter, P566-888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage phi r1t induced the P566-888 promoter, as determined from an increase in beta-Gal activity . Hybridization of nine other lactococcal strains with 32P-labeled P566-888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter . Mitomycin C induced the resident prophages in all these strains and concurrently induced the P566-888 promoter, as determined from an increase in beta-Gal activity . DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of phi r1t . In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P566-888 from the lytic phage phi 31 . These results point to a conserved mechanism in the regulation of gene expression between the lytic phage phi 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.

Appl Environ Microbiol, 1998 Mar, 64(3), 850 - 7
The citrate transport system of Lactococcus lactis subsp . lactis biovar diacetylactis is induced by acid stress; Garcia-Quintans N et al.; Citrate transport in Lactococcus lactis subsp . lactis biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene . We have shown previously that citP is included in the citQRP operon, which is mainly transcribed from the P1 promoter in L . lactis subsp . lactis biovar diacetylactis . furthermore, transcription of citQRP and citrate transport are not induced by the presence of citrate in the growth medium . In this work, we analyzed the influence of the extracellular pH on the expression of citP . The citrate transport system is induced by natural acidification of the medium during cell growth and by a shift to media buffered at acidic pHs . This inducible response to acid stress takes place at the transcriptional level and seems to be due to increased utilization of the P1 promoter . Increased transcription correlates with increased synthesis of CitP and results in higher citrate transport activity catalyzed by the cells . Finally, this acid stress response seems to provide L . lactis subsp . lactis biovar diacetylactis with a selective advantage resulting from cometabolism of glucose and citrate at low pHs.

Appl Environ Microbiol, 1998 Mar, 64(3), 818 - 23
Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory apparatus; Horn N et al.; The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp . lactis bv . diacetylactis WM4 have a number of features in common . They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions -1 and -2) . Translocation of both bacteriocins occurs via a dedicated secretory system . Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry . In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1 . A vector containing an inframe fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L . lactis IL1403 . This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus . The resulting L . lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P . acidilactici 347 . A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.

Yi Chuan Xue Bao, 1997 Oct, 24(5), 471 - 9
{Cloning and expression of promoter and signal peptide function fragments from Lactococcus lactis in Escherichia coli}; Huan L et al.; Promoter and signal peptide function fragments from Lactococcus lactis were cloned in E . coli using a promoter-signal sequence probe vector pGPB14 . Forty-two clones were obtained, whose level of resistance to ampicillin ranged from 100 to 8000 micrograms/ml . Eight clones were selected for beta-lactamase distribution assay . The beta-lactamase activity was found mainly in the periplasm, which indicated successful secretion of the enzyme . Southern hybridization test demonstrated that the inserted fragments were indeed from L . lactis . Restriction enzyme analysis revealed that the size of inserted fragments ranged from 80bp to 400 bp, four of which were sequenced on the vector pGEM-3Zf . It was found that the inserted fragments of pSEQ8 and pSEQ12 turned out to be part of the inserted fragments of pSEQ4 and pSEQ17 respectively . Promoter and translation initiation codon were found among all four fragments signal sequenced . One typical S . D . sequence and one atypical signal sequence were found in pHSB4 and pHSB8, while the other two contained no typical S . D . sequence and signal peptide sequence . In addition, it was found that the upstream regions of the promoter contributed to the efficiency of transcription initiation.

Eur J Biochem, 1998 Feb 1, 251(3), 565 - 72
Amphiphilic alpha-helices are important structural motifs in the alpha and beta peptides that constitute the bacteriocin lactococcin G--enhancement of helix formation upon alpha-beta interaction; Hauge HH et al.; Lactococcin G (LcnG) is an antimicrobial substance (bacteriocin) consisting of two peptides, LcnG-alpha and LcnG-beta . The structures of intact LcnG-alpha and LcnG-beta as well as various fragments of these peptides were studied by circular dichroism (CD) under several conditions . All peptides had a non-structured conformation in aqueous solutions . In the presence of trifluoroethanol, dodecylphosphocholine micelles and (negatively charged) dioleoylglycerophosphoglycerol (Ole2GroPGro) liposomes, varying amounts of alpha-helical structure were induced . Comparisons of the various fragments showed that helicity was concentrated in those parts of LcnG-alpha and LcnG-beta that would become amphiphilic if an alpha-helical structure was adopted . In the presence of zwitterionic dioleoylglycerophosphocholine (Ole2GroPCho) liposomes, the peptides were much less (if at all) structured, suggesting that the excess of positive charge on the antimicrobial peptides needs to be compensated by an excess of negative charge on the membrane . The structuring of LcnG-alpha and LcnG-beta in the presence of Ole2GroPGro liposomes was considerably enhanced when both peptides were presented simultaneously to the membranes . Consecutive addition of the two peptides to Ole2GroPGro liposomes did not give this additional structuring, indicating that the individual LcnG-alpha and LcnG-beta peptides associate with the membrane in a virtually irreversible manner that makes them inaccessible for interaction with the complementary peptide . The results suggest that upon arrival at and interaction with the target membrane, LcnG-alpha and LcnG-beta form a complex that consists of approximately 50% amphiphilic alpha-helices.

Mol Microbiol, 1998 Jan, 27(2), 299 - 310
A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation; Sanders JW et al.; Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride . Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB . GadC is homologous to putative glutamate-gamma-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains . GadB shows similarity to glutamate decarboxylases . A L . lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present . Expression of gadCB in L . lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression . Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L . lactis that is optimally active under conditions in which it is needed to maintain viability . Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii . gadR expression is chloride and glutamate independent . A gadR mutant did not produce the 3kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Appl Environ Microbiol, 1998 Feb, 64(2), 439 - 45
Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential; McAuliffe O et al.; Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp . lactis DPC3147 (M . P . Ryan, M . C . Rea, C . Hill, and R . P . Ross, Appl . Environ . Microbiol . 62:612-619, 1996) . Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity . Lacticin 3147 is bactericidal against L . lactis, Listeria monocytogenes, and Bacillus subtilis; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized . This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations . These pores were shown to be selective for K+ ions and inorganic phosphate . The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death . Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.

Gene, 1998 Jan 5, 206(1), 37 - 43
Sequence analysis of mutA and mutM genes involved in the biosynthesis of the lantibiotic mutacin II in Streptococcus mutans; Woodruff WA et al.; Streptococcus mutans, along with many other gram-positive bacteria produce small antibacterial peptides called bacteriocins . Bacteriocins elaborated by S . mutans, termed mutacins, may provide a selective force necessary for initial or sustained colonization in dental plaque by this major dental pathogen . Previously, we purified and characterized mutacin II, the first lantibiotic found in S . mutans . Specific oligonucleotides designed according to the N-terminal amino acid sequence permitted amplification of 0.7 kb upstream and 2.1 kb downstream of the N-terminus, using single-specific-primer PCR (SSP-PCR) . The gene encoding the mutacin II prepeptide, mutA, was subsequently cloned and sequenced . The complete prepeptide consists of 53 amino acids, including the 26 amino acid amphipathic leader peptide with the Gly(-2)-Gly(-1) sequence at the processing site . The prepeptide showed similarity to the lantibiotics lacticin 481, variacin, salivaricin and streptococcin A-FF22 . A 3 kb open reading frame immediately downstream of mutA, denoted mutM, showed sequence similarities to LCNDR2 from Lactococcus lactis . By analogy, mutM is probably involved in post-translational modification of the mutacin prepeptide . Gene disruption with an insertional vector pVA891 showed that intact copies of mutA and mutM are required for production of mutacin II.

Mol Microbiol, 1997 Oct, 26(1), 49 - 64
Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1; Chandry PS et al.; Bacteriophage sk1 is a small isometric-headed lytic phage belonging to the 936 species . It infects Lactococcus lactis, a commonly used dairy starter organism . Nucleotide sequence data analysis indicated that the sk1 genome is 28,451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions . The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric-headed and prolate-headed morphotype . Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified . Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo . The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L . lactis of a plasmid lacking a functional Gram-positive ori . The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47 . Although no similarity between phage sk1 and coliphage lambda at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the lambda structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product . It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric-headed phages that can be used to predict the functions of the sk1 gene products.

Nature, 1998 Jan 15, 391(6664), 291 - 5
A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene; van Veen HW et al.; Bacteria have developed many fascinating antibiotic-resistance mechanisms . A protein in Lactococcus lactis, LmrA, mediates antibiotic resistance by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane . Unlike other known bacterial multidrug-resistance proteins, LmrA is an ATP-binding cassette (ABC) transporter . The human multidrug-resistance P-glycoprotein, encoded by the MDR1 gene, is also an ABC transporter, overexpression of which is one of the principal causes of resistance of human cancers to chemotherapy . We expressed lmrA in human lung fibroblast cells . Surprisingly, LmrA was targeted to the plasma membrane and conferred typical multidrug resistance on these human cells . The pharmacological characteristics of LmrA and P-glycoprotein-expressing lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and magnesium-ATP were indistinguishable . Blockers of P-glycoprotein-mediated multidrug resistance also inhibited LmrA-dependent drug resistance . Kinetic analysis of drug dissociation from LmrA expressed in plasma membranes of insect cells revealed the presence of two allosterically linked drug-binding sites indistinguishable from those of P-glycoprotein . These findings have implications for the reversal of antibiotic resistance in pathogenic microorganisms . Taken together, they demonstrate that bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this type of multidrug-resistance efflux pump is conserved from bacteria to man.

J Bacteriol, 1998 Jan, 180(2), 407 - 11
A type IC restriction-modification system in Lactococcus lactis; Schouler C et al.; Three genes coding for the endonuclease, methylase, and specificity subunits of a type I restriction-modification (R-M) system in the Lactococcus lactis plasmid pIL2614 have been characterized . Plasmid location, sequence homologies, and inactivation studies indicated that this R-M system is most probably of type IC.

Biochemistry, 1997 Dec 23, 36(51), 16197 - 205
Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis; Bjornberg O et al.; The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis . The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site {Rowland et al . (1997) Structure 2, 239-252} . In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer . By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal . Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity . Mutants of Lys 43, Asn 132, and Lys 164 were also constructed . Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding . Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold . Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme . The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme . The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate . In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity . All of the mutant enzymes, except K164A, contained normal levels of flavin . The results are discussed in relation to the structures of DHODA and other flavoenzymes . The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.

Biochem J, 1997 Dec 1, 328 ( Pt 2), 343 - 7
Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase; Mata L et al.; PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases . The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis . The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity . The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal alpha-carboxy group plays a key role in the strict aminopeptidase activity.

Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 626 - 31
Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence; el Karoui M et al.; Studies of RecBCD-Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria . However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E . coli . We developed an in vivo model to examine components of dsDNA break repair in various microorganisms . Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E . coli, and provide evidence for exonuclease-Chi interactions . Insertional mutants of L . lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency . The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E . coli recBCD mutant . In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5'-GCGCGTG-3'), but not by the E . coli Chi (5'-GCTGGTGG-3') site . RexAB-mediated recombination was shown to be stimulated approximately 27-fold by lactococcal Chi . Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence . We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.

Appl Environ Microbiol, 1998 Jan, 64(1), 342 - 5
Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A; Steidler L et al.; In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis . A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S . aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L . lactis . The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface . L . lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.

Appl Environ Microbiol, 1998 Jan, 64(1), 82 - 7
The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters; Jensen PR et al.; We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized . The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism . The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well . DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the -35 and -10 sequences . The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained . Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.

J Bacteriol, 1998 Jan, 180(1), 96 - 9
Mechanistic properties of the two-component bacteriocin lactococcin G; Moll G et al.; Lactococcin G is a bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta . Biologically active, synthetic lactococcin G was used to study the mode of action on sensitive cells of Lactococcus lactis . The alpha and beta peptides can bind independently to the target cell surface, but activity requires the complementary peptide . Once bound to the cell surface, the peptides cannot be displaced to the surfaces of other cells . A complex of alpha and beta peptides forms a transmembrane pore that conducts monovalent cations but not protons . Efflux of potassium ions is observed only above pH 5.0, and the rate of efflux increases steeply with the pH . The consequences of cation fluxes for the viability of the target cells are discussed.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 31 - 8
Determinant role of E . coli RNase III in the decay of both specific and heterologous mRNAs; Santos JM et al.; A comparative analysis of mRNA decay was carried out in Escherichia coli using the wild-type and an isogenic RNase III deletion strain . We have studied the mRNA degradation from the Escherichia coli gene bolA, the Lactococcus lactis biovar diacetylactis citQRP operon and the Desulfovibrio vulgaris Hildenborough gene cyc . As seen by a dramatic stabilization of the specific mRNAs in the mutant strain, RNase III was crucial for the decay process of these three messages . Since RNase III, unlike RNase E, is not essential for bacterial viability we think that there is potential for using RNase III mutant strains to modulate gene expression.

Appl Environ Microbiol, 1997 Dec, 63(12), 4877 - 82
A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis; Sanders JW et al.; A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J . W . Sanders, G . Venema, J . Kok, and K . Leenhouts, Mol . Gen . Genet., in press) was exploited for the inducible expression of homologous and heterologous genes . An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR . The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L . lactis, acmA . Basal activity of Pgad resulted in a low level of expression of all three proteins . Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity . The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth . Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX . Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl . Induction of acmA expression resulted in slower release of PepX from the cells . The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.

Appl Environ Microbiol, 1997 Dec, 63(12), 4872 - 6
Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2; Simitsopoulou M et al.; A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography . The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da . Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C . The peptidase hydrolyzed all tripeptides tested . Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives . Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity . Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme . The 20 N-terminal amino acids of the tripeptidase from P . pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp . cremoris Wg2.

Appl Microbiol Biotechnol, 1997 Oct, 48(4), 449 - 53
The effect of nisin concentration and nutrient depletion on nisin production of Lactococcus lactis; Kim WS et al.; The kinetics of nisin production was studied in batch cultures using a construct of Lactococcus lactis subsp . lactis C2SmPrt-, containing a transposon (TnNip) that encodes nisin production . The introduction of TnNip into C2SmPrt- significantly lowered the specific growth rate and the maximum A620 reached was reduced from 15.2 to 11.0 . The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt-(TnNip), was examined . Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating . However, in low nutrient concentrations nisin production was limited by nutrient depletion . The specific growth rate of C2SmPrt-(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth . Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h-1 or less, the production ceased.

Appl Environ Microbiol, 1997 Nov, 63(11), 4370 - 6
Bacteriophage-triggered defense systems: phage adaptation and design improvements; Djordjevic GM et al.; A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation . The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette . Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31 . The efficiency of plaquing (EOP) on L . lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages . The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter . Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2 . The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene . Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered . Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system . When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay.

Appl Environ Microbiol, 1997 Nov, 63(11), 4252 - 60
Characterization of the lacticin 481 operon: the Lactococcus lactis genes lctF, lctE, and lctG encode a putative ABC transporter involved in bacteriocin immunity; Rince A et al.; The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains . The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid . We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A . Rince, A . Dufour, S . Le Pogam, D . Thuault, C . M . Bourgeois, and J.-P . Le Pennec, Appl . Environ . Microbiol . 60:1652-1657, 1994) . The operon contains three additional open reading frames: lctF, lctE, and lctG . The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter . When the three genes were coexpressed into a lacticin 481-sensitive L . lactis strain, the strain became resistant to the bacteriocin . This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481 . The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation . The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.

Appl Environ Microbiol, 1997 Nov, 63(11), 4210 - 5
Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis; O'Sullivan E et al.; This study demonstrates that exposure of log-phase Lactococcus lactis subsp . cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol . The intracellular pH (pHi) played a major role in the induction of this multistress resistance response . The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo . When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death . Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo . Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells . Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred . Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.

Genes Dev, 1997 Nov 1, 11(21), 2910 - 24
A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron; Matsuura M et al.; The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion . Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli . The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein . As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA . Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand . The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E . coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA . Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component . Expression in E . coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

J Bacteriol, 1997 Nov, 179(21), 6741 - 8
A triggered-suicide system designed as a defense against bacteriophages; Djordjevic GM et al.; A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed . The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria . The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H . Restriction activity was not apparent in E . coli or L . lactis prior to phage infection . In phage challenges of L . lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size . Center-of-infection assays revealed that only 15% of infected cells released progeny phage . In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31 . The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host . This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.

Appl Microbiol Biotechnol, 1997 Sep, 48(3), 331 - 8
Heterologous gene expression of bovine plasmin in Lactococcus lactis; Arnau J et al.; Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis . Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain . When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization . The intracellular presence of plasmin led to physiological stress . Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420 . Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin . The plasmin produced was shown to be biologically active.

Lett Appl Microbiol, 1997 Sep, 25(3), 169 - 71
Nisin production by Lactococcus lactis using two-phase batch culture; Kim WS; Nisin production by Lactococcus lactis subsp . lactis C2SmPrt-(TnNip) was investigated using two-phase batch culture . A solvent (phenyl-methyl silicone oil) was introduced in addition to the aqueous phase . The partition coefficient of this solvent in aqueous nisin varied as the cultivation medium changed, with a minimum value being 1.04 . The two-phase batch culture supported a 21% increase in growth and a 24% increase in the level of nisin produced compared to the single-phase batch culture.

J Appl Microbiol, 1997 Oct, 83(4), 499 - 507
Isolation and characterization of nisin-producing Lactococcus lactis subsp . lactis from bean-sprouts; Cai Y et al.; Bacterial isolates from bean-sprouts were screened for anti-Listeria monocytogenes bacteriocins using a well diffusion method . Thirty-four of 72 isolates inhibited the growth of L . monocytogenes Scott A . One, HPB 1688, which had the biggest inhibition zone against L . monocytogenes Scott A, was selected for subsequent analysis . Both ribotyping and DNA sequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp . lactis . Polymerase chain reaction and nucleotide sequencing revealed that the genomic DNA of the bean-sprout isolates contained a nisin Z structural gene . In MRS broth, bean-sprout isolate HPB 1688 survived at 3-4.5 degrees C for at least 20 d, grew at 4 degrees C and produced anti-listerial compounds at 5 degrees C . When co-cultured with L . monocytogenes in MRS broth, the isolate inhibited the growth of L . monocytogenes at 4 degrees C after 14 d and at 10 degrees C after 2 d . When co-inoculated with 10(2) cells g-1 of L . monocytogenes on fresh-cut ready-to-eat Caesar salad, L . lactis subsp . lactis (10(8) cells g-1) was able to reduce the number of L . monocytogenes by 1-1.4 logs after storage for 10 d at 7 zero and 10 degrees C . A bacteriocin-producing Enterococcus faecium was also able to reduce the numbers of L . monocytogenes on Caesar salad, but did not act synergistically when co-inoculated with L . lactis subsp . lactis.

FEBS Lett, 1997 Sep 29, 415(2), 206 - 11
A new family of K+ transporters from Arabidopsis that are conserved across phyla; Quintero FJ et al.; Transport of K+ in higher plants, as in bacteria and fungi, is mediated by two broad classes of transport proteins that operate in the millimolar and micromolar K+ concentration ranges . A search of the Expressed Sequence Tag database using amino acid consensus sequences for the K+ transporters HAK1 from Schwanniomyces and Kup of Escherichia coli yielded two homologous sequences for Arabidopsis . Cloning and sequencing of these genes gave single open reading frames for the putative transporters, AtKT1 and AtKT2, with predicted molecular weights of 79 and 88 kDa . The predicted gene products showed a high degree of homology at the amino acid level (56% identity) and exhibited significant hydrophobic stretches in their N-terminal halves, consistent with 12 membrane-spanning, alpha-helical domains . Database searches using AtKT1 and AtKT2 identified 10 additional sequences in Arabidopsis as well as additional homologous sequences in the plant species Oryza and Allium, the bacterium Lactococcus lactis, and in Homo sapiens . Expression of AtKT2 rescued growth on low millimolar {K+} in Saccharomyces cerevisiae carrying deletions for the genes encoding the K+ transporters TRK1 and TRK2 . Rescue was associated with a 2-fold stimulation of Rb+ uptake and was sensitive to competition with external Na+ but not to extracellular pH, indicating that the gene encodes a low-affinity K+ transporter . These and additional results suggest that AtKT1 and AtKT2 belong to a superfamily of cation transporters that have been conserved through evolution.

J Clin Microbiol, 1997 Nov, 35(11), 2778 - 81
Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources; Teixeira LM et al.; This study presents phenotypic and genotypic data for seven isolates of Vagococcus fluvialis, including four strains recovered from human clinical sources, one strain isolated from an environmental source, and two strains isolated from pigs . On the basis of phenotypic characteristics, most isolates were initially classified as "unidentified enterococci," because they resembled atypical arginine-negative enterococcal species . All seven strains as well as the type strain of V . fluvialis reacted with the AccuProbe Enterococcus genetic probe . The seven isolates had virtually indistinguishable whole-cell protein profiles that were similar to that of the V . fluvialis type strain and distinct from those of Enterococcus and Lactococcus species . DNA-DNA reassociation experiments confirmed that the strains were V . fluvialis . They were 71% or more related to the V . fluvialis type strain under optimum and stringent conditions, with 2.5% or less divergence within related sequences . All strains were susceptible to ampicillin, cefotaxime, trimethoprim-sulfamethoxazole, and vancomycin and were resistant to clindamycin, lomefloxacin, and ofloxacin . Strain-to-strain variation was observed in relation to susceptibilities to 18 other antimicrobial agents . Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI . Distinctive PFGE patterns were generated, suggesting the nonclonal nature of V . fluvialis strains . Although the number of strains was small, this report provides molecular characterization of V . fluvialis and the first evidence of a possible connection of this species with human infections.

Plasmid, 1997, 38(2), 115 - 27
Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids; Gravesen A et al.; Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids . Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094 . The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced . Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences . Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA . In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA . A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication . This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

Plasmid, 1997, 38(2), 79 - 90
Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis; Liu CQ et al.; The 8.8-kb Lactococcus lactis plasmid pND302 encodes resistance to cadmium (CdR) . Regions of pND302 involved in replication and CdR were subcloned and sequenced . The replication region is localized on a 1.5-kb region and consists of an open reading frame (repB) preceded by a noncoding AT-rich sequence (ori) which is highly homologous to lactococcal theta-type replicons . The CdR determinant is localized on a 2.9-kb region and encodes putative proteins similar to the Cd(2+)-specific P-type efflux ATPase (CadA) and the transcriptional regulatory repressor (CadC) identified in Staphylococcus aureus, Bacillus firmus, and Listeria monocytogenes . Similar CdR determinants were also detected by PCR in other CdR plasmids isolated from different L . lactis strains.

J Bacteriol, 1997 Oct, 179(19), 6107 - 11
Homing of a group II intron from Lactococcus lactis subsp . lactis ML3; Mills DA et al.; Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1 . In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene . Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed . In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion . Efficient homing was observed in the absence of a functional host homologous recombination system . This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.

J Bacteriol, 1997 Oct, 179(20), 6285 - 93
Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp . lactis; Goupil-Feuillerat N et al.; The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp . lactis . It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter . The product of aldB is responsible for leucine sensibility under valine starvation . In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis . AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS . The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L . lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria . Thus, the alpha-acetolactate decarboxylase from L . lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.

Nat Biotechnol, 1997 Oct, 15(10), 980 - 3
Design of thermolabile bacteriophage repressor mutants by comparative molecular modeling; Nauta A et al.; Comparative molecular modeling was performed with repressor protein Rro of the temperate Lactococcus lactis bacteriophage r1t using the known 3D-structures of related repressors in order to obtain thermolabile derivatives of Rro . Rro residues presumed to stabilize a nonhomologous but structurally conserved hydrophobic pocket, which was shown to be important for thermostability of the Escherichia coli bacteriophage lambda repressor CI, were randomized . Of the derivatives that exhibited various temperature-sensitive phenotypes, one was shown to hold promise for both fundamental and industrial applications that require the controlled production of (heterologous) proteins in L . lactis.

Nat Biotechnol, 1997 Oct, 15(10), 976 - 9
Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening; de Ruyter PG et al.; An attractive approach to accelerate cheese ripening is to induce lysis of Lactococcus lactis starter strains for facilitated release of intracellular enzymes involvement in flavor formation . Controlled expression of the lytic genes lytA and lytH, which encode the lysin and the holin proteins of the lactococcal bacteriophage phi US3, respectively, was accomplished by application of a food-grade nisin-inducible expression system . Simultaneous production of lysin and holin is essential to obtain efficient lysis and concomitant release of intracellular enzymes as exemplified by complete release of the debittering intracellular aminopeptidase N . Production of holin alone leads to partial lysis of the host cells, whereas production of lysin alone does not cause significant lysis . Model cheese experiments in which the inducible holinlysin overproducing strain was used showed a fourfold increase in release of L-Lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the system for cheese applications.

Biochem Mol Biol Int, 1997 Sep, 43(1), 153 - 60
The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase; Giovannini PP et al.; o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit . This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis . Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+ . The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain . The isoindole derivative does not form with two mutants of the T . brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking . The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Mol Microbiol, 1997 Aug, 25(4), 717 - 25
The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin; Sheehan MM et al.; We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1 . The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages . The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity . Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate . Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate . These peculiar characteristics have been ascribed to a modified C-terminal domain . The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 236 - 41
Effect of citrate on growth of Lactococcus lactis subsp . lactis in milk; Haddad S et al.; The effect of citrate on the growth of Lactococcus lactis subsp . lactis var . diacetylactis in milk has been investigated . Five strains of Lactococcus lactis subsp . lactis var . diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease . In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly . Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains . Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate . Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp . lactis var . diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains . At 26 degrees C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant . These results show that citrate metabolism slightly stimulates the growth of lactococci in milk.

J Bacteriol, 1997 Sep, 179(18), 5884 - 91
Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase; Arnau J et al.; The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized . The deduced amino acid sequence of the L . lactis PFL . protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL . The genetic organization of the chromosomal pfl region in L . lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene . The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L . lactis . Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used . During growth with galactose, pfl showed the highest levels of expression . Constructed L . lactis pfl strains were unable to produce formate under anaerobic growth . Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp . lactis biovar diacetylactis pfl strain.

J Bacteriol, 1997 Sep, 179(18), 5795 - 801
Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli; Jeong JY et al.; A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli . This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin . SSI activity is orientation specific with respect to the direction of replication . We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E . coli . The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did . Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance . These results suggest that in E . coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21 . Primer RNA synthesis in vitro suggests that E . coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E . coli single-stranded DNA binding (SSB) protein . This region contains two stem-loop structures, stem-loop I and stem-loop II . Deletion of stem-loop I portion results in loss of priming activity by E . coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E . coli RNA polymerase on the SSB-coated single-stranded DNA template.

J Bacteriol, 1997 Sep, 179(17), 5589 - 93
Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene; Chapot-Chartier MP et al.; Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli . This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis . One of them, cspB, was cloned and sequenced . It encodes a 66-residue protein which possesses 60% sequence identity with E . coli CspA . Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization . In addition, induction of cspB-directed beta-galactosidase activity was observed . These results indicate that the L . lactis cspB gene is cold shock inducible.

J Bacteriol, 1997 Sep, 179(17), 5282 - 7
Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio; Garrigues C et al.; During batch growth of Lactococcus lactis subsp . lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate . This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio . The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity . Under such conditions, metabolism was homolactic . Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio . Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase . Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased . However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion . Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.

J Appl Microbiol, 1997 Aug, 83(2), 139 - 46
Proteolytic enzyme activity in lactococci grown in different pretreated milk media; Meijer WC et al.; Lactococcus lactis ssp . lactis MG1363, harbouring plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, was used to study the effect of two different treatments of the growth medium milk on the activity levels of PrtP and the intracellular localized aminopeptidase PepN and X-prolyl-dipeptidyl aminopeptidase PepXP . All three proteolytic enzymes showed lower activity levels in cells grown in high heat-treated milk as compared to cells grown in non-heat-treated milk . Highest activity levels of the three studied enzymes were found in cells grown in milk heat treated for 30 min at 63 degrees C . Using cells of strain L . lactis ssp . lactis MG1363, harbouring plasmid pNZ544, which encodes reporter gene gusA under control of the prtP promoter, it was demonstrated that the regulation of PrtP takes place at the transcription initiation level . After separation of the pH 4.6 soluble fraction of high heat-treated milk with reverse phase HPLC, it was found that the hydrophilic small peptide fraction of the milk was responsible for this regulation . Amino acid analysis of this fraction confirmed that this fraction consisted of peptides only . Ultrafiltration of milk, which increases the dry matter of the milk specifically through increase of its protein content, only significantly affected the levels of PrtP and PepN in cells of strain MG1363 . Highest activity was found during growth in unconcentrated milk, and the lowest level was found during growth in four times concentrated retentate . Using cells of strain MG1363(pNZ544), it was demonstrated that also in this case regulation of PrtP takes place at the level of transcription initiation . Approximately 40% of the decrease in activity of PrtP and PepN could be explained by the presence of higher amounts of purified whey proteins and higher amounts of dry mass . This suggests the presence of another factor, concentrated by ultrafiltration, which controls the production different proteolytic enzymes in concentrated retentate.

J Appl Microbiol, 1997 Aug, 83(2), 133 - 8
Note: genetic and biochemical characterization on nisin Z produced by Lactococcus lactis ssp . lactis biovar . diacetylactis UL 719; Meghrous J et al.; The bacteriocin produced by Lactococcus lactis ssp . lactis biovar . diacetylactis UL 719 was purified and characterized . Two peaks exhibiting antimicrobial activity were obtained after purification . Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis . The molecular mass as determined by mass spectrometry was 3346.39 +/- 0.40 Da for peak 1 and 3330.39 +/- 0.27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z . The two purified peaks exhibiting antimicrobial activity appear to correspond with oxidized and native forms of nisin Z.

J Dairy Sci, 1997 Aug, 80(8), 1528 - 36
Expression of ropy and mucoid phenotypes in Lactococcus lactis; Dierksen KP et al.; Strains of Lactococcus lactis ssp . lactis and Lactococcus lactis ssp . cremoris were cultured under aerobic and anaerobic conditions on plates of whey agar, Elliker agar, and M17L agar at 15, 20, and 30 degrees C to determine the environmental conditions required for the expression of the ropy phenotype . Two strains, L . lactis ssp . cremoris Ropy 352 and L . lactis ssp . cremoris Hollandicus, exhibited two distinct polysaccharide phenotypes, ropy and mucoid . Expression of these phenotypes could be induced individually or simultaneously . The inducible nature of this response suggests that genetic regulators were present . Western blots were used to determine whether or not Lon protease and RcsA, two regulators of polysaccharide expression in Escherichia coli, were present in lactococci . Lon, a negative regulator, and RcsA, an unstable positive regulator, have been shown at the structural level to be conserved in a number of Gram-negative and Gram-positive microorganisms . The present study found evidence for structural conservation of Lon protease in lactococci . Less of the Lon-like protein was observed in the ropy strains than in the nonropy strains.

J Dairy Res, 1997 Aug, 64(3), 433 - 43
Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese; Singh TK et al.; Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry . Most of the peptides were from the N-terminal half of the beta-casein, but peptides from alpha s1- and alpha s2-caseins were also identified; the extract also contained alpha-lactalbumin . Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from alpha s1- and beta-caseins, produced by chymosin and plasmin respectively . Plasmin seemed to be involved in the hydrolysation of alpha s2-casein . Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.

J Dairy Res, 1997 Aug, 64(3), 399 - 407
Purification and characterization of aminopeptidase P from Lactococcus lactis subsp . cremoris; Mc Donnell M et al.; Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococcus lactis subsp . cremoris AM2 with a 5.8% yield . The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41,600 . Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively . The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position . No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides . On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue . However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues . As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

Microbiology, 1997 Aug, 143 ( Pt 8), 2575 - 82
Spatial interactions between subsurface bacterial colonies in a model system: a territory model describing the inhibition of Listeria monocytogenes by a nisin-producing lactic acid bacterium; Thomas LV et al.; The effect of spatial separation on interactions between subsurface bacterial colonies was tested using a model system: the inhibition of Listeria monocytogenes by nisin-producing and nisin-non-producing Lactococcus lactis subsp . lactis . Separation distance was controlled by altering the number of inoculum organisms within the agar . Mean separation distance was calculated by determining the mean volume available to each cell at the start of the experiment . Inhibition was assessed by comparing the growth of L . monocytogenes in pure culture with its growth in the presence of Lac . lactis subsp . lactis . Increasing the distance between colonies resulted in an exponential decrease in inhibition . When L . monocytogenes and Lac . lactis subsp . lactis colonies were within 100 microns of each other, the increase in cell numbers per L . monocytogenes colony was only 0.6 c.f.u . (which indicated some cells had become non-viable) . This was a log reduction of 3.5 compared to the pure culture control . A separation distance of 1000 microns resulted in a L . monocytogenes colony growth increment of 2.5 x 10(2) c.f.u . per colony, a log reduction of 3.0 compared to the control . Increasing the separation distance to 3000 microns resulted in a L . monocytogenes colony growth increment of 1.3 x 10(6) c.f.u . per colony, a log reduction of 0.9 compared to the control . The effects of nisin and acidity were investigated by using a nisin-non-producing strain of Lac . lactis subsp . lactis and by buffering the medium . Data were obtained for the effect of separation on inhibition, as well as competition between colonies of the same species . The inhibition was mathematically described in terms of a simplified 'territory' model of immobilized bacterial growth . There was a strong qualitative agreement between the mathematical model and the experimental data . It was concluded that the phenomenon of propinquity is of important consideration when modeling and predicting microbial growth within solid food systems.

J Biol Chem, 1997 Jul 18, 272(29), 18140 - 6
Membrane potential-generating malate (MleP) and citrate (CitP) transporters of lactic acid bacteria are homologous proteins . Substrate specificity of the 2-hydroxycarboxylate transporter family; Bandell M et al.; Membrane potential generation via malate/lactate exchange catalyzed by the malate carrier (MleP) of Lactococcus lactis, together with the generation of a pH gradient via decarboxylation of malate to lactate in the cytoplasm, is a typical example of a secondary proton motive force-generating system . The mleP gene was cloned, sequenced, and expressed in a malolactic fermentation-deficient L . lactis strain . Functional analysis revealed the same properties as observed in membrane vesicles of a malolactic fermentation-positive strain . MleP belongs to a family of secondary transporters in which the citrate carriers from Leuconostoc mesenteroides (CitP) and Klebsiella pneumoniae (CitS) are found also . CitP, but not CitS, is also involved in membrane potential generation via electrogenic citrate/lactate exchange . MleP, CitP, and CitS were analyzed for their substrate specificity . The 2-hydroxycarboxylate motif R1R2COHCOOH, common to the physiological substrates, was found to be essential for transport although some 2-oxocarboxylates could be transported to a lesser extent . Clear differences in substrate specificity among the transporters were observed because of different tolerances toward the R substituents at the C2 atom . Both MleP and CitP transport a broad range of 2-hydroxycarboxylates with R substituents ranging in size from two hydrogen atoms (glycolate) to acetyl and methyl groups (citromalate) for MleP and two acetyl groups (citrate) for CitP . CitS was much less tolerant and transported only citrate and at a low rate citromalate . The substrate specificities are discussed in the context of the physiological function of the transporters.

J Biol Chem, 1997 Jul 18, 272(29), 18044 - 50
Characterization of Escherichia coli NrdH . A glutaredoxin-like protein with a thioredoxin-like activity profile; Jordan A et al.; Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases . Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes . Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase . Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis . The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH . Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function . Overexpression of E . coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase . Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione . We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site . We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.

Eur J Biochem, 1997 Jul 15, 247(2), 581 - 7
Amplified expression, purification and functional reconstitution of the dipeptide and tripeptide transport protein of Lactococcus lactis; Hagting A et al.; Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a proton-motive-force-driven peptide-transport protein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g . from yeasts, plants, and the kidney and small intestine of rabbit, man and rat . The expression level of DtpT protein in L . lactis was increased (20-40-fold) to approximately 10% of total integral membrane protein by means of a low-copy-number vector and selecting the appropriate growth conditions . Membrane vesicles bearing the DtpT-His6 protein (containing a C-terminal factor-Xa cleavage site and a six-histidine-tag) showed a Pro-Ala uptake activity that was half that of membranes containing the wild-type protein . The activity in the DtpT-His6 membrane vesicles increased at least 50% upon removal of the His6 tag from the protein . More than 95% DtpT was solubilized from L . lactis membranes in the presence of 1% (mass/vol.) n-dodecyl-beta-D-maltoside, and approximately 2 mg DtpT-His6 was purified by Ni2+-chelate affinity chromatography from 100 mg membrane protein . Purified DtpT-His6 was reconstituted unidirectionally into detergent-saturated formed liposomes, which were prepared from Escherichia coli phospholipid and egg phosphatidylcholine; the detergent was removed by adsorption to polystyrene beads . The highest uptake activities were obtained when DtpT was incorporated into liposomes that were treated with a low amount of n-dodecyl-beta-D-maltoside (onset of liposome solubilization) . The uptake activity could be improved by addition of NaCl (200 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps.

J Mol Biol, 1997 Jul 11, 270(2), 179 - 87
Splicing and intron-internal RNA editing of trnK-matK transcripts in barley plastids: support for MatK as an essential splice factor; Vogel J et al.; Group II introns frequently require assistance by specific factors, maturases, for folding and effective splicing in vivo . The only putative maturase of higher plant chloroplasts is encoded by matK, located in the intron of trnK . We show that in barley matK transcripts are modified at a first codon base by C-to-U RNA editing . The resulting H --> Y substitution restores a sequence motif that is present in maturases of yeast and plant mitochondria and of Lactococcus ltrA and that is positioned within the X domain . Processing of trnK-matK transcripts was further investigated in plastids lacking functional ribosomes due to a mutation . Absence of the intron-encoded matK gene product in these plastids is correlated with the accumulation of precursor transcripts for tRNALys(UUU)-matK, processed to different degrees, and by the lack of mature and spliced tRNA molecules . These results suggest an essential role of MatK for splicing of its own transcript in vivo . Processing of the 5' end of trnK exon 1 was found to proceed efficiently also in the mutant plastids although the two tRNA exons were separated by the 2481 nt intron . Consequently, presence of the intron does not interfere with the formation of mature 5' termini.

J Appl Microbiol, 1997 Jul, 83(1), 85 - 90
An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages; Lillehaug D; This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis . Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested . Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed . By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates . Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.

Microbiology, 1997 Jul, 143 ( Pt 7), 2277 - 86
Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp . cremoris UC503; Twomey DP et al.; The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp . cremoris UC503 was completed . The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5' CCNGG 3' sites, cleaving after the second cytosine and the degenerate central base . The ENase gene (scrFIR; 362 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encodes proteins that independently confer protection against ScrFI digestion . scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzymes . The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric Ssoll ENase from Shigella sonnei . The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs . The ability of ScrFI to cleave non-canonically modified 5' CCNGG 3' sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.

J Bacteriol, 1997 Jul, 179(14), 4473 - 9
Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition; Duwat P et al.; Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair . However, other species, particularly those which exist under different environmental conditions than does E . coli, may have rather different responses . Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis . Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C . DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames . Seven mutants were chosen for further characterization . They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L . lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination . Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.

Appl Environ Microbiol, 1997 Jul, 63(7), 2722 - 8
Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA; Buist G et al.; The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase . Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA . An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did . Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant . PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant . A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t . After mitomycin induction of an exponential-phase culture of L . lactis LL302 carrying this plasmid, the cells became subject to autolysis, resulting in the release of intracellular proteins.

Appl Environ Microbiol, 1997 Jul, 63(7), 2702 - 7
Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes; Erlandson K et al.; A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations . The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend . The blend consisted of two closely related Lactococcus lactis subsp . cremoris strains, 160 and 331, and one L . lactis subsp . lactis strain, 210 . Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers . Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend . These strain-specific probes were used in a HGMF colony hybridization assay . Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population . When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains . The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.

J Bacteriol, 1997 Jul, 179(13), 4164 - 71
Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis; Nardi M et al.; The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF {V . Monnet, M . Nardi, A . Chopin, M.-C . Chopin, and J.-C . Gripon, J . Biol . Chem . 269:32070-32076, 1994}) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp . cremoris NCDO763 . Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp . lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763 . From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters . The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases . Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403 . Traces of several recombination events are visible on the lactose-proteinase plasmid . This suggests that the duplication of pepF occurred by recombination from the chromosome of an L . lactis subsp . lactis strain followed by gene transfer . We discuss the possible functions of PepF and the role of its amplification.

FEBS Lett, 1997 Jun 30, 410(2-3), 452 - 6
Metabolic analysis of S . cerevisiae strains engineered for malolactic fermentation; Bony M et al.; A complete malolactic fermentation was achieved using Saccharomyces cerevisiae strains coexpressing the genes mleS and mae1 coding for the Lactococcus lactis malolactic enzyme and the Schizosaccharomyces pombe malate permease under the control of yeast promoters . The expression level of mae1 greatly influences the kinetics of the reaction by controlling the rate of malate uptake meanwhile a high expression level of mleS induces a partial consumption of malate derived from glucose by the malolactic enzyme . A strain expressing several copies of mae1 and one copy of mleS degrades 3 g/l of malate almost exclusively through the malolactic pathway in 4 days under enological conditions, without metabolic side effects.

J Mol Biol, 1997 Jun 27, 269(5), 851 - 60
Crystal structures and mechanism of 6-phospho-beta-galactosidase from Lactococcus lactis; Wiesmann C et al.; The initial structural model of 6-phospho-beta-galactosidase from Lactococcus lactis was refined to an R-factor of 16.4% (R{free} = 23.6%) to 2.3 A resolution (1 A = 0.1 nm), and the structures of three other crystal forms were solved by molecular replacement . The four structural models are essentially identical . The catalytic center of the enzyme is approximately at the mass center of the molecule and can only be reached through a 20 A long channel, which is observed with an "open" or "closed" entrance . The closed entrance is probably too small for the educt lactose-6-phosphate to enter, but large enough for the first product glucose to leave . Among the presented structures is a complex between an almost inactive mutant and the second product galactose-6-phosphate, which is exclusively bound at side-chains . A superposition (onto the native enzyme) of galactose-6-phosphate as bound to the mutant suggests the geometry of a postulated covalent intermediate . The binding mode of the educt was modeled, starting from the bound galactose-6-phosphate . A tightly fixed tryptophan is used as a chopping-board for splitting the disaccharide, and several other aromatic residues in the active center cavity are likely to participate in substrate transport/binding.

Structure, 1997 Jun 15, 5(6), 775 - 88
The structure of enzyme IIAlactose from Lactococcus lactis reveals a new fold and points to possible interactions of a multicomponent system; Sliz P et al.; BACKGROUND: The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the binding, transmembrane transport and phosphorylation of numerous sugar substrates . The system is also involved in the regulation of a variety of metabolic and transcriptional processes . The PTS consists of two non-specific energy coupling components, enzyme I and a heat stable phosphocarrier protein (HPr), as well as several sugar-specific multiprotein permeases known as enzymes II . In most cases, enzymes IIA and IIB are located in the cytoplasm, while enzyme IIC acts as a membrane channel . Enzyme IIAlactose belongs to the lactose/cellobiose-specific family of enzymes II, one of four functionally and structurally distinct groups . The protein, which normally functions as a trimer, is believed to separate into its subunits after phosphorylation . RESULTS: The crystal structure of the trimeric enzyme IIAlactose from Lactococcus lactis has been determined at 2.3 A resolution . The subunits of the enzyme, related to each other by the inherent threefold rotational symmetry, possess interesting structural features such as coiled-coil-like packing and a methionine cluster . The subunits each comprise three helices (I, II and III) and pack against each other forming a nine-helix bundle . This helical bundle is stabilized by a centrally located metal ion and also encloses a hydrophobic cavity . The three phosphorylation sites (His78 on each monomer) are located in helices III and their sidechains protrude into a large groove between helices I and II of the neighbouring subunits . A model of the complex between phosphorylated HPr and enzyme IIAlactose has been constructed . CONCLUSIONS: Enzyme IIAlactose is the first representative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined . Some of its structural features, like the presence of two histidine residues at the active site, seem to be common to all enzymes no overall structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank . Enzyme IIAlactose shows surface complementarity to the phosphorylated form of HPr and several energetically favourable interactions between the two molecules can be predicted.

FEMS Microbiol Lett, 1997 Jun 15, 151(2), 237 - 44
Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136; Kantor A et al.; The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis . Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194 . The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment . Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids . The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame . Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids . Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136 . The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.

Biochemistry, 1997 Jun 3, 36(22), 6777 - 85
Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis; Hagting A et al.; Transport of hydrophilic di- and tripeptides into Lactococcus lactis is mediated by a proton motive force-driven peptide transport protein (DtpT) that shares similarity with eukaryotic peptide transporters, e.g., from kidney and small intestine of rabbit, man, and rat . Hydropathy profiling in combination with the "positive inside rule" predicts for most of the homologous proteins an alpha-helical bundle of 12 transmembrane segments, but the positions of these transmembrane segments and the location of the amino and carboxyl termini are by no means conclusive . The secondary structure of DtpT was investigated by analyzing 42 DtpT-alkaline phosphatase fusion proteins, generated by random or directed fusions of the corresponding genes . These studies confirm the presence of 12 transmembrane segments but refute several other predictions made of the secondary structure . Data obtained from the fusion proteins were substantiated by studying the accessibility of single cysteine mutants in putative cytoplasmic or extracellular loops by membrane (im)permeant sulfhydryl reagents . The deduced topology model of DtpT consists of a bundle of 12 alpha-helixes with a short amino and a large carboxyl terminus, both located at the cytoplasmic site of the membrane . On the basis of sequence comparisons with DtpT, it seems likely that the structure model of the amino-terminal half of DtpT also holds for the eukaryotic peptide transporters, whereas the carboxyl-terminal half is largely different.

Appl Environ Microbiol, 1997 Jun, 63(6), 2454 - 9
Cloning and sequence analysis of putative histidine protein kinases isolated from Lactococcus lactis MG1363; O'Connell-Motherway M et al.; Eight recombinant plasmids harboring chromosomal fragments of Lactococcus lactis MG1363 were shown to phenotypically suppress a histidine protein kinase (HPK) deficiency in either of two different E . coli strains . Sequence analysis of the plasmid inserts revealed five different complete or partial open reading frames (ORFs) specifying proteins with high similarity to HPKs . One of the plasmids also harbored an additional ORF, unrelated to HPKs, with suppressing activity.

Appl Environ Microbiol, 1997 Jun, 63(6), 2439 - 41
Construction of specific erythromycin resistance mutations in the temperate lactococcal bacteriophage TP901-1 and their use in studies of phage biology; Koch B et al.; A method for the construction and isolation of specifically designed mutations of the temperate lactococcal phage TP901-1 has been developed . Two different erm-labeled mutants were isolated . One was shown to be defective in lysogenization and excision . The other, showing normal lysogenization, was used for host range studies.

Appl Environ Microbiol, 1997 Jun, 63(6), 2293 - 9
Isolation and properties of Lactococcus lactis subsp . lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose; Boumerdassi H et al.; Following treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, three mutants of Lactococcus lactis subsp . lactis biovar diacetylactis CNRZ 483 that produced diacetyl and acetoin from glucose were isolated . The lactate dehydrogenase activity of these mutants was strongly attenuated, and the mutants produced less lactate than the parental strain . The kinetic properties of lactate dehydrogenase of strain CNRZ 483 and the mutants revealed differences in the affinity of the enzyme for pyruvate, NADH, and fructose-1,6-diphosphate . When cultured aerobically, strain CNRZ 483 transformed 2.3% of glucose to acetoin and produced no diacetyl or 2,3-butanediol . Under the same conditions, mutants 483L1, 483L2, and 483L3 transformed 42.0, 78.9, and 75.8%, respectively, of glucose to C4 compounds (diacetyl, acetoin, and 2,3-butanediol) . Anaerobically, strain CNRZ 483 produced no C4 compounds, while mutants 483L1, 483L2, and 483L3 transformed 2.0, 37.0, and 25.8% of glucose to acetoin and 2,3-butanediol . In contrast to the parental strain, the NADH balance showed that the mutants regenerated most of the NAD via NADH oxidase under aerobic conditions and by ethanol production under anaerobic conditions.

Appl Environ Microbiol, 1997 Jun, 63(6), 2131 - 5
Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk; Flambard B et al.; The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied . Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study . An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected . The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction . Extension of the study to wild-type proteinase-positive L . lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.

Appl Environ Microbiol, 1997 Jun, 63(6), 2124 - 30
The effects of adding lactococcal proteinase on the growth rate of Lactococcus lactis in milk depend on the type of enzyme; Helinck S et al.; Increasing the proteolytic activity of Lactococcus lactis cultures in milk by adding the corresponding proteinase resulted in a stimulation of the growth rate regardless of the strain and the type of proteinase, demonstrating that the rate of casein degradation was responsible for the growth rate limitation of L . lactis in milk . However, the stimulation was only transient, and the reduction in growth rate in the poststimulation phase depended on the type of cell envelope proteinase . When a PI-type proteinase was added, three causes were involved in the subsequent reduction in growth rate: degradation of the added proteinase, repression of the proteolytic activity expressed by the cells, and competition for peptide uptake . When a PIII-type proteinase was added, the cessation of stimulation was due to the autoproteolysis of the added enzyme only.

J Bacteriol, 1997 Jun, 179(11), 3410 - 5
Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363; Hellendoorn MA et al.; The gene pepV, encoding a dipeptidase from Lactococcus lactis subsp . cremoris MG1363, was identified in a genomic library in pUC19 in a peptidase-deficient Escherichia coli strain and subsequently sequenced . PepV of L . lactis is enzymatically active in E . coli and hydrolyzes a broad range of dipeptides but no tri-, tetra-, or larger oligopeptides . Northern (RNA) and primer extension analyses indicate that pepV is a monocistronic transcriptional unit starting 24 bases upstream of the AUG translational start codon . The dipeptidase of L . lactis was shown to be similar to the dipeptidase encoded by pepV of L . delbrueckii subsp . lactis, with 46% identity in the deduced amino acid sequences . A PepV-negative mutant of L . lactis was constructed by single-crossover recombination . Growth of the mutant strain in milk was significantly slower than that of the wild type, but the strains ultimately reached the same final cell densities.

Arch Biochem Biophys, 1997 May 15, 341(2), 315 - 20
The N-terminal sequence of Lactococcus lactis phosphoglucose isomerase purified by affinity chromatography differs from the other species; Nomura M et al.; A specific monoclonal antibody, M3A, was produced to rapidly purify Lactococcus lactis phosphoglucose isomerase (PGI) for amino acid sequence analysis . M3A recognized the Lac . lactis PGI specifically and sensitively with both enzyme-linked immunosorbent assay and Western blot analysis . The enzyme was rapidly purified to a specific activity of 21.8 U/mg with a yield of 20% by a three-step procedure, including M3A-bound Sepharose chromatography . The specific activity of PGI was increased about 64.1-fold from the cell lysate . The molecular mass of Lac . lactis PGI was estimated to be about 50 kDa by SDS-PAGE . The N-terminal amino acid sequence of Lac . lactis PGI exhibited no significant similarity to other PGIs, except for a 52.6% identity to Bacillus stearothermophilus PGI A and PGI B . These results suggest that there might be some molecular types of PGI.

Appl Environ Microbiol, 1997 May, 63(5), 1826 - 37
Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis; Kilstrup M et al.; The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism . It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains . In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L . lactis subsp . cremoris laboratory strain MG1363, which was originally derived from a dairy strain . After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels . Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress . Two classes of heat stress proteins were identified from their temporal induction pattern . The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min . GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min . The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress . Unlike other organisms, all salt stress-induced proteins in L . lactis were also subjected to heat stress induction . DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level . These data indicate an overlap between the heat shock and salt stress responses in L . lactis.

J Bacteriol, 1997 May, 179(9), 2949 - 57
Molecular characterization of a genomic region in a Lactococcus bacteriophage that is involved in its sensitivity to the phage defense mechanism AbiA; Dinsmore PK et al.; A spontaneous mutant of the lactococcal phage phi31 that is insensitive to the phage defense mechanism AbiA was characterized in an effort to identify the phage factor(s) involved in sensitivity of phi31 to AbiA . A point mutation was localized in the genome of the AbiA-insensitive phage (phi31A) by heteroduplex analysis of a 9-kb region . The mutation (G to T) was within a 738-bp open reading frame (ORF245) and resulted in an arginine-to-leucine change in the predicted amino acid sequence of the protein . The mutant phi31A-ORF245 reduced the sensitivity of phi31 to AbiA when present in trans, indicating that the mutation in ORF245 is responsible for the AbiA insensitivity of phi31A . Transcription of ORF245 occurs early in the phage infection cycles of phi31 and phi31A and is unaffected by AbiA . Expansion of the phi31 sequence revealed ORF169 (immediately upstream of ORF245) and ORF71 (which ends 84 bp upstream of ORF169) . Two inverted repeats lie within the 84-bp region between ORF71 and ORF169 . Sequence analysis of an independently isolated AbiA-insensitive phage, phi31B, identified a mutation (G to A) in one of the inverted repeats . A 118-bp fragment from phi31, encompassing the 84-bp region between ORF71 and ORF169, eliminates AbiA activity against phi31 when present in trans, establishing a relationship between AbiA and this fragment . The study of this region of phage phi31 has identified an open reading frame (ORF245) and a 118-bp DNA fragment that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA.

J Biotechnol, 1997 Apr 25, 54(2), 95 - 104
Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp . lactis M138 encoding abortive phage infection; Su P et al.; Plasmid pND852 (56 kb) encodes nisin resistance and was isolated from Lactococcus lactis ssp lactis (L . lactis) M138 by conjugation to L . lactis LM0230 . It conferred strong resistance to the isometric-headed phage phi 712 and partial resistance to the prolate-headed phage phi c2 . A 2.6 kb HpaII fragment encoding phage resistance was cloned into the streptococcal/Bacillus hybrid vector pGB301 to generate pND817 . The mechanism of phage resistance encoded by pND817 involved abortive infection and this was illustrated by a reduction in burst size from 166 to 6 at 30 degrees C and from 160 to 90 at 37 degrees C . Partial resistance was therefore retained at 37 degrees C . DNA sequencing revealed that the abortive infection was encoded by a single open reading frame (ORF), designated abiI, encoding a 332 amino acid protein . Neither abiI nor the predicted product showed significant homology to any existing sequence in the GenBank database . Frame shift mutation at the unique EcoRI site within the ORF resulted in loss of the Abi+ phenotype, confirming that the ORF is responsible for the encoded phage resistance.

Vaccine, 1997 Apr-May, 15(6-7), 616 - 9
Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C; Norton PM et al.; Mice inoculated intranasally (i.n.) with a recombinant strain of live Lactococcus lactis expressing tetanus toxin fragment C (TTFC), produced both serum and secretory antibodies to TTFC . Killed bacteria which had accumulated TTFC intracellularly in vitro also elicited protective serum antibody responses . There was no requirement for either colonization or invasion of the mucosa . In addition secretory antibody responses in the lung and nasal tissues were elicited after i.n . inoculation in the presence of an adjuvant.

Mol Microbiol, 1997 Apr, 24(2), 387 - 97
Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis; van Kranenburg R et al.; Lactococcus lactis strain NIZO B40 produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose . A 40 kb plasmid encoding exopolysaccharide production was isolated through conjugal transfer of total plasmid DNA from strain NIZO B40 to the plasmid-free L . lactis model strain MG1614 and subsequent plasmid curing . A 12 kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified downstream of an iso-IS982 element . The predicted gene products of epsABCDEFGHIJK show sequence homologies with gene products involved in exopolysaccharide, capsular polysaccharide, lipopolysaccharide, or teichoic acid biosynthesis of other bacteria . Transcriptional analysis of the eps gene cluster revealed that the gene cluster is transcribed as a single 12 kb mRNA . The transcription start site of the promoter was mapped upstream of the first gene epsR . The involvement of epsD in exopolysaccharide (EPS) biosynthesis was demonstrated through a single gene disruption rendering an exopolysaccharide-deficient phenotype . Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.

FASEB J, 1997 Apr, 11(5), 355 - 64
Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX; Turner AJ et al.; Mammalian cell-surface peptidases participate in the postsecretory processing and metabolism of neuropeptides and peptide hormones . Neutral endopeptidase-24.11 (NEP) is the prototype of a family of zinc metallopeptidases that also includes the endothelin-converting enzymes (ECE) and which are structurally related to the bacterial enzymes thermolysin and lactococcal endopeptidase . Two other mammalian gene products exhibit strong homology with NEP: the erythrocyte cell-surface antigen, KELL; and the putative product of the PEX gene, which has been associated with X-linked hypophosphatemic rickets . No enzymic activity has yet been attributed to KELL and PEX proteins, and they remain peptidases in search of a substrate . A wide range of biologically active peptide substrates has been described for NEP, of which the enkephalins and the atrial natriuretic peptide family have assumed greatest significance . Endothelin-converting enzyme catalyses the final step in the biosynthesis of the vasoconstrictor peptide, endothelin (ET) . Like NEP, it is a type II integral membrane protein, but is expressed predominantly in endothelial cells . Isoforms of ECE (ECE-1alpha, ECE-1beta, and ECE-2) exist that differ in a number of characteristics . In particular, ECE-1, through the paracrine effects of ET-1, may contribute to the proliferation of smooth muscle after angioplasty and to the development of human atherosclerosis . Inhibitors of ECE and NEP may have important therapeutic applications in cardiovascular and renal medicine.

Gene, 1997 Apr 1, 188(2), 229 - 34
The isolation of novel heat shock genes in Lactococcus lactis using RNA subtractive hybridization; Arnau J et al.; Lactococcus lactis is subjected to heat shock (hs) during cheese manufacturing . A number of conserved hs genes have been cloned and studied in this organism, although no regulatory gene, e.g . alternative sigma factor, has been identified . RNA subtractive hybridization was used to identify genes expressed very early when L . lactis MG1363 was shifted from 30 to 43 degrees C . 32P-labeled cDNA synthesized from RNA isolated from hs cells at 43 degrees C was mixed with an excess vegetative RNA and the mixture was directly used as a probe after a short hybridization step . Northern analysis revealed a moderate induction for the probes used, and low expression was also detected in non-hs cells, demonstrating the applicability of this technique for the detection of differentially expressed genes . The probes were used to identify genomic library clones containing the corresponding genes . Among the five clones studied, a cell division operon including a putative ftsZ homolog (pJAK2) was identified . Additionally, a putative hsp86 homolog (pJAK3), three different transposase encoding genes (pJAK1 and pJAK3), a gene coding for a deoR-like transcriptional repressor (pJAK4) and a putative regulatory gene that showed homology to an alkaline shock protein (pJAK5) were characterized.

Appl Environ Microbiol, 1997 Apr, 63(4), 1434 - 40
Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures; Coakley M et al.; The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P . Ryan, M.C . Rea, C . Hill, and R.P . Ross, Appl . Environ . Microbiol . 62:612-619, 1996) . The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication . By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties . Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin . Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures . Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters . In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated . This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry . The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.

Appl Environ Microbiol, 1997 Apr, 63(4), 1274 - 83
Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis; Emond E et al.; The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp . lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L . lactis strains . It had very limited effect on prolate phages of the c2 species . The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi) . Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment . Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames . Deletion mutants showed that only orf1 was needed to produce the Abi phenotype . orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98 . DNA and protein sequence alignment programs found no significant homology with databases . However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA . No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L . lactis cells . This system is believed to act at or prior to phage DNA replication . WHen cloned into a high-copy vector, AbiK efficiency increased 100-fold . AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations.

Appl Environ Microbiol, 1997 Apr, 63(4), 1244 - 51
Identification of a recA homolog (recALP) on the conjugative lactococcal phage resistance plasmid pNP40: evidence of a role for chromosomally encoded recAL in abortive infection; Garvey P et al.; The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P . Garvey, G.F . Fitzgerald, and C . Hill, Appl . Environ . Microbiol . 61:4321-4328, 1995) . DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants . One of these ORFs encodes a recA homolog (designated recALP) . This is the first report of a recA-like determinant located to a plasmid . The second ORF (orfU) shares homology with the umuC gene of the SOS response . Analysis of a number of lactococcal strains confirmed the presence of recALP-like sequences in at least two other lactococcal strains . The proximity of the recA and umuC homologs suggested a possible role in the phase resistance encoded by the Abi determinants . However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF . Nor could the recALP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122 . Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system.

Biochemistry, 1997 Apr 1, 36(13), 3802 - 10
Structure-function relations of variant and fragment nisins studied with model membrane systems; Giffard CJ et al.; Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp . lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane . The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants . N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes . By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes . In these respects I30W nisin A is similar to native nisin . Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility . While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects . These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes . Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association . The results are discussed in the context of other recently proposed models for nisin action.

Enzyme Microb Technol, 1997 Apr, 20(5), 381 - 8
Microbial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous prefermentation of milk; Sodini I et al.; Four strains of mesophilic lactic acid bacteria were separately or coentrapped in kappa-carrageenan/locust bean gum gel beads and used for continuous prefermentation of UHT skim milk in a stirred-tank bioreactor . Lactic acid and cell productivities of the immobilized cell bioreactor were particularly high and remarkably stable during eight weeks of continuous milk prefermentation (about 18 g h-1 l-1 of lactic acid and 4.9 x 10(12) CFU h-1 l-1, respectively, but important variations of the bacterial populations is prefermented milk and gel beads occurred in any case (co-or separate entrapment) . The strain Lactococcus lactis subsp . lactis biovar diacetylactis CDII became dominant, accounting for approx . 90% (released cells) and 78% (immobilized cells) of the total population . Microscopic observations of sections of gels beads showed a progressive destructing of the bead surface with rupture and release of entrapped viable cells from peripheral cavities of the gel . It is believed that these cavities close again after releasing all or part of their cell content, entrapping the different strains of the mixed culture and initiating a new colonization step and a cross-contamination of the beads . On the other hand, experimentations over seven-week periods with pasteurized milk showed the high resistance of the immobilized cell bioreactor to psychrotrophic contamination.

Gene, 1997 Mar 25, 188(1), 129 - 36
A highly efficient and stable system for site-specific integration of genes and plasmids into the phage phiLC3 attachment site (attB) of the Lactococcus lactis chromosome; Lillehaug D et al.; An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed . A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp . lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation . Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication . Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system . This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L . lactis . The above results, the observation that the phiLC3 attB site appear to be conserved in L . lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L . lactis.

Mol Microbiol, 1997 Mar, 23(6), 1293 - 301
Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis; van Belkum MJ et al.; Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N-terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions -1 and -2 . A dedicated ATP-binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane . To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterium divergens that is secreted via the cell's general secretion pathway . Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively . The different leader peptides directed the production of divergicin in the homologous hosts . In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts . For ABC-transporter-dependent secretion in E . coli the outer membrane protein TolC was required . Using this strategy, colicin V was produced in L . lactis by fusing this bacteriocin behind the leader peptide of leucocin A.

Microbiology, 1997 Mar, 143 ( Pt 3), 855 - 65
Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene; Qian N et al.; Maltose metabolism in Lactococcus lactis involves the conversion of beta-glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM) . The gene encoding beta-PGM (pgmB) was cloned from a genomic library of L . lactis using antibodies . The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found . The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified beta-PGM (25 kDa) . A short sequence at the N-terminus was found to be similar to known metal-binding domains . The expression of beta-PGM in L lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium . This indicates that beta-PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates . The specific activity of beta-PGM during fermentation was dependent on the maltose concentration in the medium . The maximum specific activity of beta-PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g l-1 . Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased . The specific activity of alpha-PGM responded similarly to beta-PGM, but the magnitude of the response was lower . Preferential sugar utilization and alpha- and beta-PGM suppression was observed when L . lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar . In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.

Nat Biotechnol, 1997 Mar, 15(3), 253 - 7
Engineering pathways for malate degradation in Saccharomyces cerevisiae; Volschenk H et al.; Deacidification of grape musts is crucial for the production of well-balanced wines, especially in colder regions of the world . The major acids in wine are tartaric and malic acid . Saccharomyces cerevisiae cannot degrade malic acid efficiently due to the lack of a malate transporter and the low substrate affinity of its malic enzyme . We have introduced efficient pathways for malate degradation in S . cerevisiae by cloning and expressing the Schizosaccharomyces pombe malate permease (mae1) gene with either the S . pombe malic enzyme (mae2) or Lactococcus lactis malolactic (mleS) gene in this yeast . Under aerobic conditions, the recombinant strain expressing the mae1 and mae2 genes efficiently degraded 8 g/L of malate in a glycerol-ethanol medium within 7 days . The recombinant malolactic strain of S . cerevisiae (mae1 and mleS genes) fermented 4.5 g/L of malate in a synthetic grape must within 4 days.

J Bacteriol, 1997 Mar, 179(5), 1748 - 54
A C-methyltransferase involved in both ubiquinone and menaquinone biosynthesis: isolation and identification of the Escherichia coli ubiE gene; Lee PT et al.; Strains of Escherichia coli with mutations in the ubiE gene are not able to catalyze the carbon methylation reaction in the biosynthesis of ubiquinone (coenzyme Q) and menaquinone (vitamin K2), essential isoprenoid quinone components of the respiratory electron transport chain . This gene has been mapped to 86 min on the chromosome, a region where the nucleic acid sequence has recently been determined . To identify the ubiE gene, we evaluated the amino acid sequences encoded by open reading frames located in this region for the presence of sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases . One open reading frame in this region (o251) was found to encode these motifs, and several lines of evidence that confirm the identity of the o251 product as UbiE are presented . The transformation of a strain harboring the ubiE401 mutation with o251 on an expression plasmid restored both the growth of this strain on succinate and its ability to synthesize both ubiquinone and menaquinone . Disruption of o251 in a wild-type parental strain produced a mutant with defects in growth on succinate and in both ubiquinone and menaquinone synthesis . DNA sequence analysis of the ubiE401 allele identified a missense mutation resulting in the amino acid substitution of Asp for Gly142 . E . coli strains containing either the disruption or the point mutation in ubiE accumulated 2-octaprenyl-6-methoxy-1,4-benzoquinone and demethylmenaquinone as predominant intermediates . A search of the gene databases identified ubiE homologs in Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovani, Lactococcus lactis, and Bacillus subtilis . In B . subtilis the ubiE homolog is likely to be required for menaquinone biosynthesis and is located within the gerC gene cluster, known to be involved in spore germination and normal vegetative growth . The data presented identify the E . coli UbiE polypeptide and provide evidence that it is required for the C methylation reactions in both ubiquinone and menaquinone biosynthesis.

Structure, 1997 Feb 15, 5(2), 239 - 52
The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis; Rowland P et al.; BACKGROUND: . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides . Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical . One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer . Sequence alignments have identified three different families among the DHODs: the two L . lactis enzymes belong to two of the families, whereas the enzyme from E . coli is a representative of the third . As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L . lactis . The differences between the two L . lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families . RESULTS: . The crystal structure of DHODA has been determined to 2.0 resolution . The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis . The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts . Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues . This cavity is proposed as the substrate-binding site . CONCLUSIONS: . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group . Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences . The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity . The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.

Antonie Van Leeuwenhoek, 1997 Feb, 71(1-2), 117 - 28
The role of transport processes in survival of lactic acid bacteria . Energy transduction and multidrug resistance; Konings WN et al.; Lactic acid bacteria play an essential role in many food fermentation processes . They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation . In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos . In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline . Lactic acid bacteria have also developed multidrug resistance systems . In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter . This cationic ABC-transporter has surprisingly high structural and functional analogy with the human MDR1-(P-glycoprotein) . For anions an ATP-driven ABC-like excretion systems exist.

Appl Environ Microbiol, 1997 Feb, 63(2), 694 - 702
The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes; Urbach E et al.; Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L . lactis used for industrial fermentations, L . lactis subsp . cremoris and subsp . lactis . Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1% . Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L . lactis subsp . cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese . Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes . However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements . Fourteen new L . lactis subsp . cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.

Appl Environ Microbiol, 1997 Feb, 63(2), 414 - 9
An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds; Yvon M et al.; The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development . In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds . Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp . cremoris strain was purified and characterized . The enzyme transaminates the aromatic amino acids, leucine, and methionine . It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors . In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate . It is essential for the transformation of aromatic amino acids to flavor compounds . It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa . The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.

Dev Biol Stand, 1997, 90, 153 - 60
Immunization with bacterial antigens: infections with streptococci and related organisms; Bercovier H et al.; Streptococcal infections of fish have been reported from various parts of the world, including the Far East, the United States, South Africa, Australia, Israel and Europe . Classification of Gram-positive cocci (DNA-DNA hybridization studies coupled with 165 sequencing) has shown that at least five different defined species are pathogenic to fish, e.g . Streptococcus iniae (syn . S . shilot), Streptococcus difficile, Lactococcus garvieae (syn . Enterococcus seriolicida), Lactococcus piscium and Vagococcus salmoninarum . "Streptococcosis" of fish should therefore be regarded as a complex of similar diseases caused by different genera and species of Gram-positive cocci, each capable of inducing CNS damage, as well as various degrees of multisystem organ involvement . Panophthalmitis ("pop-eye") and meningitis/meningoencephalitis are the sole findings in trout infected by S . iniae and in tilapines infected by S . difficile . In contrast, L . garvieae-infected trout bear a systemic hyperacute infection with diffuse haemorrhages . Therapeutic measures are generally ineffective . Development of vaccines is therefore essential to control these diseases . In our studies, trout were vaccinated intraperitoneally with whole-cell formalin-inactivated S . iniae and L . garvieae and tilapines with whole-cell formalin-inactivated and acellular S . difficile extract . Under laboratory conditions, S . difficile-vaccinated tilapines were protected against a challenge of 100 LD50s . Protection was correlated with the development of specific agglutinins . Western blot analysis supported the hypothesis that only a few proteins act as protective antigens . S . iniae autovaccines were effective in preventing the disease in rainbow trout in Israel . Under field conditions, fish vaccinated at 50 g were protected for over four months . The qualitative analysis of the humoral response indicated that specific antibodies are directed against a few protein moieties . The fact that passive transfer of antibodies protected fish from experimental infection suggests that the basic mechanism of protection is antibody mediated . L . garvieae autovaccines developed for Italian trout farming were found to elicit a response similar to that of S . iniae . Despite the high virulence of L . garvieae (LD50 of 6 x 10(1) CFU/fish, compared with 3 x 10(4) CFU/fish of S . iniae), the protection against the experimentally induced disease lasted for five months under laboratory conditions, with survival rates of 80-90% . A single injection of the vaccine (0.1 ml/fish) resulted in specific antibody production detectable for six months . In the field, protection rates of 70-80% were obtained for a period of three months, in fish of 200-300 g reared at water temperatures of 18-21 degrees C.

J Basic Microbiol, 1997, 37(3), 187 - 96
Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables; Franz CM et al.; Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE . The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes . These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (<5.0) . They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme . These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin . Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin . The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is discussed.

Plasmid, 1997, 37(1), 42 - 50
Site-specific nicking in vitro at ori T by the DNA relaxase of Tn5252; Srinivas P et al.; Tn5252 is a promiscuous streptococcal element capable of madiating horizontal spread of multiple antibiotic resistance . To begin understanding the functional role of a transfer-related region in Tn5252, its nucleotide sequence was determined . Sequence of this 3 . 3-kb DNA segment revealed the presence of six open reading frames . The predicted amino acid sequence of one of the open reading frames, ORF9, showed similarity to a predicted protein product of the lactococcal conjugative plasmid, pC1528 . The deduced primary protein sequence of another, ORF4, showed strong structural similarity to conserved regions of various prokaryotic DNA relaxases that initiate conjugal transfer by strand- and site-specific cleavage at the transfer origin . A hybrid protein containing the ORF4 protein fused to the carboxyl terminal end of maltose binding protein was purified from Escherichia coli and found to specifically nick plasmids carrying a 2-kb DNA segment derived from the transposon . The nicking reaction is protein concentration-dependent . These results imply that the conjugative transposition of Tn5252 may involve rolling circle replication and transfer of a unique DNA strand.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 149 - 54
A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar . diacetylactis; Deng YM et al.; A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar . diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L . lactis LM0230 . The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region . A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA.

J Bacteriol, 1997 Jan, 179(1), 135 - 40
Role of transmembrane pH gradient and membrane binding in nisin pore formation; Moll GN et al.; Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics . It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure . Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes . The rate of dissipation increases with the magnitude of the delta pH . Nisin forms pores only when the delta pH is inside alkaline . The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH . Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L . lactis cells . Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding . Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation . These findings are consistent with the wedge model of nisin-induced pore formation.

Curr Microbiol, 1997 Jan, 34(1), 23 - 6
Differential induction of the chaperonin GroEL and the Co-chaperonin GroES by heat, acid, and UV-irradiation in Lactococcus lactis subsp . lactis; Hartke A et al.; Microsequencing of a polypeptide with MW of 14.5 and pI of 5.0 induced by heat treatment at 42 degrees C and 50 degrees C in Lactococcus lactis subsp . lactis revealed that it corresponds to the co-chaperonin GroES . Quantitative analysis of analytical 2-D gels showed a relative induction of 12- and 11-fold after 30 min of heat adaptation at 42 degrees C and 50 degrees C, respectively . GroES is also induced by an acid shift from pH 7 to pH 5.5 and by UV254 nm-irradiation, with relative induction factors of 3.8 and 2.3, respectively . To our knowledge this is the first report showing induction of GroES by mild acid treatment . Contrasting to the relative induction of the groEL gene product, the second protein encoded by the groESL operon, GroES shows significantly higher induction under all stress situations.

Eur Respir J, 1996 Dec, 9(12), 2508 - 12
Proinflammatory effect of Pediococcus pentosaceus, a bacterium used as hay preservative; Duchaine C et al.; Bacterial cultures, such as Pediococcus pentosaceus, are used to treat hay with the objective of preventing hay heating and moulding, and thus, the development of the microbial growth which causes farmer's lung . The aim of this study was to investigate whether such bacterial cultures have the potential to induce a pulmonary inflammatory response . Mice were instilled 3 days week-1 for 3 weeks with either saline or nonviable preparations of P . pentosaceus, Saccharopolyspora rectivirgula, Lactococcus lactis (control bacteria) or with the combinations of S . rectivirgula and P . pentosaceus . P . pentosaceus induced a significant inflammatory response in the lung which was similar to that produced by S . rectivirgula . L . lactis produced a response of a lower intensity . The total number of cells in bronchoalveolar lavage were: S . rectivirgula: 6.4 x 10(5) cells.mL-1; P . pentosaceus: 4.3 x 10(5) cells.mL-1; S . rectivirgula + P . pentosaceus: 5.4 x 10(5) cells.mL-1, L . lactis: 6.8 x 10(5) cells.mL-1 and saline group 3.7 x 10(4) cells.mL-1 . The lung index was higher in S . rectivirgula+P . pentosaceus and P . pentosaceus groups than in S . rectivirgula, L . lactis and saline groups . The quantity of specific immunoglobulin G and A (IgG and IgA) to P . pentosaceus and L . lactis levels (in the blood and/or lavage fluid) were similar to those against S . rectivirgula . In mice, P . pentosaceus has the potential to induce a similar inflammatory response in the lung as S . rectivirgula, which is the most common antigen responsible for farmer's lung disease in Quebec . Further studies are needed to verify whether farmers can develop farmer's lung or other lung responses to this new potential antigen.

Biotechnol Appl Biochem, 1996 Dec, 24 ( Pt 3), 251 - 6
Concentration and recovery of the bacteriocin nisin from Lactococcus lactis subsp . lactis; Van't Hul JS et al.; This investigation compared various techniques to concentrate and recover nisin from Lactococcus lactis cells . Centrifugation, combined with pH manipulation, was initially investigated as it is known that nisin will adsorb to producer cells at pH 6.5 and desorb at pH < or = 3.0 . Unfortunately, centrifugation stripped producer cells of nisin even at pH 6.5; therefore a milder separation process (microfiltration) was evaluated . Results using a medium (LTB) containing peptone, tryptone, yeast extract, NaCl, Na2HPO4 and glucose demonstrated that nisin could be at least partially concentrated with cells via microfiltration . However, when a filtered stillage-based medium was used, nisin production was boosted to levels which exceeded the holding capacity of producer cells, resulting in release of nisin from cells at pH 6.5 . Since it appears unfeasible to use producer cells for nisin recovery, an alternative may be to separately immobilize cells/ fragments in a re-usable column to act as a "resin' to adsorb nisin . Microfiltration could then be used to release nisin from cells, with nisin recovered by passing the permeated material through the immobilized-cell columns at pH 6.5.

Appl Environ Microbiol, 1996 Dec, 62(12), 4536 - 41
Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881; Simmonds RS et al.; Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall . Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449 . Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall . The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S . pyogenes, Streptococcus gordonii, Streptococcus oralis, S . mutans, and Streptococcus rattus has been evaluated.

Appl Environ Microbiol, 1996 Dec, 62(12), 4493 - 8
Genetic organization of the mle locus and identification of a mleR-like gene from Leuconostoc oenos; Labarre C et al.; Characterization of the mle locus harboring the malolactic enzyme gene mleA and malate permease gene mleP from Leuconostoc oenos was completed in this study by mRNA analysis . Northern (RNA) blot experiments revealed a 2.6-kb transcript, suggesting an operon structure harboring mleA and mleP genes . Primer extension analysis showed that the mle operon has a single transcription start site located 17 nucleotides upstream of the ATG translation start site for the mleA gene . We found sequences, TTGACT and TATGAT (which are separated by 18 bp), that are closely related to the gram-positive and Escherichia coli consensus promoter sequences . Upstream of the mleA gene, an 894-bp open reading frame that transcribed divergently from the mle operon was found . Sequence analysis and expression in E . coli minicells suggest that this open reading frame encodes a polypeptide with an apparent molecular mass of 34 kDa belonging to the LysR-type regulatory protein family . Protein comparisons showed the highest level of identity with the MleR regulatory protein from Lactococcus lactis, which is involved in the expression of the malolactic genes in the presence of L-malate . However, the MleR-like protein of L . oenos seems different from the protein of Lactococcus lactis, since no regulation of the malolactic enzyme by L-malate was effective under our experimental conditions.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 217 - 24
A general system for generating unlabelled gene replacements in bacterial chromosomes; Leenhouts K et al.; A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes . The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein . Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans . Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance . Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector . The second recombination event results either in gene replacement or in restoration of the original copy of the gene . As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain . A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms . The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 27 - 32
Presence of non-functional nisin genes in Lactococcus lactis subsp . lactis isolated from natural starters; Moschetti G et al.; Sixty-four lactococcal strains isolated from natural whey starters were screened for the presence of the nisin structural gene by polymerase chain reaction . Seven of them showed a specific PCR product of 320 bp; only two produced antagonistic activity and were resistant to nisin . Southern blots of SmaI-digested DNA from PCR-positive strains hybridized with a nisA probe displayed a location of the gene on different SmaI fragments . Among PCR-positive strains, nisin producers showed specific transcript after reverse transcriptase-PCR, as well as some non-nisin-producing strains . The RT-PCR product could not be shown in one non-nisin-producing PCR-positive strain.

J Biol Chem, 1996 Nov 15, 271(46), 29359 - 65
The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and {FeS} redox centers; Nielsen FS et al.; The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene . However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme . We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L . lactis on multicopy plasmids . The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit . The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+ . The DHOdehase B purified from the E . coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the deltakappa-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e . two PyrDB and two PyrK subunits . The deltakappa-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of {2Fe-2S} redox clusters per mol of native protein as tightly bound prosthetic groups . The deltakappa-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme . Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.

J Appl Bacteriol, 1996 Nov, 81(5), 493 - 500
Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant; Duan K et al.; A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp . lactis (L . lactis) M189 . pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization . The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb . Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production . pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI . The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation . The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.

J Bacteriol, 1996 Nov, 178(21), 6158 - 65
Gene organization and transcription of a late-expressed region of a Lactococcus lactis phage; Parreira R et al.; The lactococcal phage bIL41 belongs to the small isometric-headed phages of the 936 quasi-species and is resistant to the abortive infection determined by abiB . A 10.2-kb segment from this phage, in which late transcription is initiated, has been sequenced . Thirteen open reading frames (ORFs) organized in one transcriptional unit have been identified . The location of two of them and the structural features of the proteins they code for are evocative of terminase subunits . Five other ORFs specify proteins which are highly homologous to structural proteins from the closely related phage F4-1 . By comparing the phage bIL41 sequence with partial sequences available for four related phages, we were able to deduce a chimerical phage map covering the middle- and a large part of the late-expressed regions . Phages from this quasi-species differ by the insertion or deletion of either 1 to about 400 bp in noncoding regions or an entire ORF . Transcription was initiated 9 min after infection at a promoter with a -10 but no -35 consensus sequence . Synthesis of a phage activator protein was needed for initiation of transcription . A large 16-kb transcript covering all of the late-expressed region of the genome was synthesized . This transcript gave rise to smaller units . One of these units most probably resulted from a RNase E processing.

J Bacteriol, 1996 Nov, 178(21), 6151 - 7
Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family; Ge Z et al.; In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis . H . pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities) . The H . pylori ftsH product was overexpressed in E . coli and reacted immunologically with an anti-E . coli FtsH serum (T . Tomoyasu, K . Yamanaka, K . Murata, T . Suzaki, P . Bouloc, A . Kato, H . Niki, S . Hiraga, and T . Ogura, J . Bacteriol . 175:1352-1357, 1993) . FtsH was also shown to be present in the membrane fraction of H . pylori, suggesting that it is membrane bound . Disruption of the ftsH gene led to the loss of viability of H . pylori, demonstrating that this gene is essential for cell growth . Overproduction of both H . pylori FtsH and E . coli FtsH together tremendously reduced the growth rate of the E . coli host cells, whereas the growth of the E . coli cells carrying the wild-type E . coli ftsH operon on the chromosome was not significantly affected by overproduction of H . pylori FtsH itself . This result suggests that the abnormal growth of cells results from interaction between H . pylori FtsH and E . coli FtsH.

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