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PubMed1998

A new theta-type thermosensitive replicon from Lactococcus lactis as an integration vector for Enterococcus faecalis.

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42 degrees C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30 degrees C to 42 degrees C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis.

Amino Acid Sequence↗
PubMed1997

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

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.

Alleles↗
PubMed1996

Mutational analysis and chemical modification of Cys24 of lactococcin B, a bacteriocin produced by Lactococcus lactis.

Using site-directed mutagenesis the single cysteine residue at position 24 of lactococcin B was replaced by all other possible amino acids. Most of these mutant molecules retained bacteriocin activity, with the exception of those in which cysteine was replaced by a positively charged amino acid. This would seem to be in agreement with the authors' earlier observation that treatment of the wild-type molecule with HgCl2 resulted in its inactivation. The factor that causes inactivation of lactococcin B seems to be the introduction of a positive charge at position 24 by HgCl2 rather than oxidation of this residue, as treatment of the bacteriocin with other oxidative chemicals did not interfere with the ability of lactococcin B to dissipate the membrane potential of sensitive cells. Results are also reported which imply that inactive lactococcin B can still bind to its receptor. It can be replaced by an active bacteriocin molecule, resulting in dissipation of the membrane potential.

Bacteriocins↗
PubMedDate not supplied

Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp. cremoris W15.

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.

Amino Acid Sequence↗
PubMed2000

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

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

ATP-Binding Cassette Transporters↗
PubMed2005

The specificity of oligopeptide transport by Streptococcus thermophilus resembles that of Lactococcus lactis and not that of pathogenic streptococci.

Peptide transport is a crucial step in the growth of Streptococcus thermophilus in protein- or peptide-containing media. The objective of the present work was to determine the specificity of peptide utilization by this widely used lactic acid bacterium. To reach that goal, complementary approaches were employed. The capability of a proteinase-negative S. thermophilus strain to grow in a chemically defined medium containing a mixture of peptides isolated from milk as the source of amino acids was analysed. Peptides were separated into three size classes by ultrafiltration. The strain was able to use peptides up to 3.5 kDa during growth, as revealed by liquid chromatography and mass spectrometry analyses. The same strain was grown in chemically defined medium containing a tryptic digest of casein, and the respective time-course consumption of the peptides during growth was estimated. The ability to consume large peptides (up to 23 residues) was confirmed, as long as they are cationic and hydrophobic. These results were confirmed by peptide transport studies. Extension of the study to 11 other strains revealed that they all shared these preferences.

Caseins↗
PubMed2000

Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000.

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

Amino Acid Motifs↗
PubMed1996

Genetic determinants for the biosynthesis of nisin, a bacteriocin produced by Lactococcus lactis.

In the past, the genetic determinants for nisin biosynthesis were thought to be plasmid-located. However, it has been shown that production of nisin, immunity to nisin, and other properties such as the fermentation of sucrose, are encoded on 70 kb conjugative transposons that are chromosomally located. The extrachromosomal location of the nisin genes has not been substantiated by experiments that unequivocally show plasmid transfer. Two natural variants of nisin have been identified, nisin A and nisin Z, encoded by the genes nisA and nisZ, respectively. Both genes have been cloned and sequenced and differ only in a single base pair. Approximately 12 kb downstream from the structural gene has been cloned and sequenced, and a further 10 genes involved in the biosynthesis of nisin have been identified. The nisB and nisC gene products are involved in nisin maduration, the nisT in its secretion and the nisP in its processing. The nisR and nisK gene products have a regulatory role and the nisI, nisF, nisE and nisG are involved in immunity to nisin. All these genes display significant homology to the corresponding genes of the related lantibiotics subtilin and epidermin.

Amino Acid Sequence↗
PubMed2000

Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4.

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.

Angiotensin I↗
PubMed2006

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture.

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10(4) cfu/mL, and 10(5) cfu/mL) were ripened at 5 or 13 degrees C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13 degrees C developed aged flavors (brothy, sulfur, and nutty flavors) more rapidly than cheeses held at 5 degrees C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.

Aldehydes↗
PubMed2005

Substrate specificity and ionic regulation of GlnPQ from Lactococcus lactis. An ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains.

We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmic substrate-binding domains. Deletion analysis showed that the substrate-binding domain, in the primary sequence of GlnP nearest to the translocator domain, is used as the receptor that delivers the substrate to the translocator. Membrane reconstitution of the detergent-solubilized and purified GlnPQ complex yielded proteoliposomes that transported glutamine and glutamic acid at the expense of ATP. The transport activity of GlnPQ increased with lumenal salt concentration and internal pH, but the mechanism of ionic activation of the transporter is distinct from that of other osmoregulatory ATP-binding cassette transporters and does not depend on the presence of anionic lipids. The regulation of GlnPQ conforms to an electrostatic switch in which protein domain(s) and low molecular weight electrolytes participate.

ATP-Binding Cassette Transporters↗
PubMed1991

Identification of a new genetic determinant for cell aggregation associated with lactose plasmid transfer in Lactococcus lactis.

Derivatives of the lactose miniplasmid pMG820 were constructed in which a staphylococcal erm gene was inserted and in which this was accompanied by subsequent deletion of the lactose genes. The resulting plasmids were thus marked with both erythromycin resistance and lactose utilization genes in pF1132 or solely erythromycin resistance in pF1133. These plasmids retained the normal conjugation properties characteristic of lactose plasmid pLP712, including the generation by intermolecular rearrangement of high-frequency-transfer Clu+ derivatives which exhibited cell aggregation. The use of such Clu+ plasmids in a variety of mating experiments between different lactococcal strains and the observation of cell aggregation when particular mating mixtures were made led to the discovery of a new component of this conjugation system named Agg. A chromosomal gene agg was postulated to be present in some but not all strains of lactococci. High-frequency conjugation and cell aggregation thus depend on the presence of both Agg and Clu, although in a mating pair these components can be in the same or in separate strains. The Agg and Clu components may be analogous to the binding substance and aggregation substance that are involved in the hemolysin plasmid transfer system of Enterococcus faecalis, although control of their expression is different.

Conjugation, Genetic↗
PubMed1999

Molecular characterization of a phage-encoded resistance system in Lactococcus lactis.

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.

Bacteriophages↗
PubMed2001

Hydrolysis of caseins and formation of hydrophilic and hydrophobic peptides by wild Lactococcus lactis strains isolated from raw ewes' milk cheese.

AIMS: To investigate the hydrolysis of alphaS1-, alphaS0-, betaB-, betaA1- and betaA2-caseins by 32 wild lactococci of different randomly amplified polymorphic DNA (RAPD) patterns, isolated from raw ewes' milk cheese, and the production of hydrophilic and hydrophobic peptides from whole casein by those strains. METHODS AND RESULTS: Most strains hydrolysed all caseins, and degraded beta-caseins to a larger extent than alphaS-caseins, when the proteolytic activity of whole cells was determined by capillary electrophoresis. Higher levels of hydrophilic than of hydrophobic peptides were produced from whole casein by all strains, according to reverse-phase high performance liquid chromatography analyses. CONCLUSIONS: Cell envelope proteinases of most lactococci isolated from raw ewes' milk cheese were CEPII, CEPII/III or CEPIII (classification of Exterkate et al. 1993). A negative correlation was found between degraded alphaS- and beta-caseins and a highly positive correlation between hydrophilic and hydrophobic peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Fast acid-producing lactococci from raw ewes' milk cheese have considerable and diverse caseinolytic activities. Their peptide production patterns do not reveal serious risks of bitter-flavour defect in cheeses if used as components of dairy starters.

Animals↗
PubMed2001

[Cloning and structure analysis of a restriction and modification system, LlaBIII from Lactococcus lactis subsp. cremoris W56].

A 22.4 kb naturally occurring plasmid pJW566, isolated from L. lactis W56, was found to encode an R/M system named LlaBIII. The LlaBIII R/M system was isolated on a chloramphenicol resistant derivative of plasmid pJW566, resulting in a plasmid pJK1. Subcloning analysis showed that the LlaBIII determinant was located on a 5 kb HindIII-Sph I fragment. The fragment was sequenced. It contained a single open reading frame (ORF), corresponding to a protein of 1584 or 1576 aa. In the deduced amino acid sequence seven helicase motifs characteristic of endonuclease type I and type III and a conserved catalysis motif X in the R subunits of type I R/M systems were located in the N-terminus, followed by four conserved motifs found in DNA N6-adenine methyltransferases. The C-terminus of the deduced amino acid sequence showed no homology to known R/M systems. Therefore, this polypeptide encoded by LlaBIII is a multifunctional protein possessing putative DNA recognition, methylation and restriction activities.

Cloning, Molecular↗
PubMed2002

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

Animals↗
PubMed2001

Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis.

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

Bacterial Proteins↗
PubMed2006

Identification of the leucine-to-2-methylbutyric acid catabolic pathway of Lactococcus lactis.

Nutrient starvation and nonculturability in bacteria lead to changes in metabolism not found during the logarithmic phase. Substrates alternate to those used during growth are metabolized in these physiological states, yielding secondary metabolites. In firmicutes and actinobacteria, amino acid catabolic pathways are induced during starvation and nonculturability. Examination of lactococci showed that the population entered a nonculturable state after carbohydrate depletion and was incapable of growth on solid media; however, the cells gained the ability to produce branched-chain fatty acids from amino acids. Gene expression profiling and in silico pathway analysis coupled with nuclear magnetic resonance spectroscopy were used to delineate the leucine catabolic pathway. Lactococci produced acetic and propionic acid during logarithmic growth and starvation. At the onset of nonculturability, 2-methylbutyric acid was produced via hydroxymethyl-glutaryl-coenzyme A (CoA) and acetyl-CoA, along with ATP and oxidation/reduction precursors. Gene expression profiling and genome sequence analysis showed that lactococci contained redundant genes for branched-chain fatty acid production that were regulated by an unknown mechanism linked to carbon metabolism. This work demonstrated the ability of a firmicute to induce new metabolic capabilities in the nonculturable state for producing energy and intermediates needed for transcription and translation. Phylogenetic analyses showed that homologues of these enzymes and their functional motifs were widespread across the domains of life.

Butyrates↗