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PubMed2003

Identification of the host determinant of two prolate-headed phages infecting Lactococcus lactis.

A gene responsible for host determination was identified in two prolate-headed bacteriophages of the c2 species infecting strains of Lactococcus lactis. The identification of the host determinant gene was based on low DNA sequence homology in a specific open reading frame (ORF) between prolate-headed phages with different host ranges. When a host carrying this ORF from one phage on a plasmid was infected with another phage, we obtained phages with an altered host range at a frequency of 10(-6) to 10(-7). Sequencing of phage DNA originating from 10 independent single plaques confirmed that a genetic recombination had taken place at different positions between the ORF on the plasmid and the infecting phage. The adsorption of the recombinant phages to their bacterial hosts had also changed to match the phage origin of the ORF. Consequently, it is concluded that this ORF codes for the host range determinant.

Amino Acid Sequence↗
PubMed2004

The effect of Lactococcus lactis starter cultures on the oxidative stability of liquid whey.

The oxidative stability of liquid Cheddar cheese whey was evaluated using 2 Lactococcus lactis starter cultures in combination and alone along with a control, utilizing glucono-delta-lactone for acid development. Fresh and stored whey were evaluated for volatile composition, free fatty acids, and flavor by descriptive sensory analysis. A significant increase in volatile lipid oxidation products, most notably, hexanal, occurred during storage, and a corresponding decline in the free fatty acid linoleic acid was found. The flavor and aroma characteristic, cardboardy, was correlated to the increase in volatile lipid oxidation products and the decline in linoleic acid. Evidence strongly suggested that lipid oxidation was initiated during whey production and escalated during storage and that the starter cultures significantly influenced the level of volatile lipid oxidation products. Further understanding of the impact of starter cultures on whey may allow for the production of higher quality whey ingredients with wider food application.

Cheese↗
PubMed2004

New expression system tightly controlled by zinc availability in Lactococcus lactis.

Here we developed the new expression system P(Zn) zitR, based on the regulatory signals (P(Zn) promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn(2+) high-affinity uptake and regulation. A P(Zn) zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic beta-galactosidase. Nuclease and beta-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of P(Zn) zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn(2+), availability in the environment. Our results demonstrate that P(Zn) zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn(2+). The efficiency of the P(Zn) zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either P(Zn) zitR or P(nisA) nisRK control. lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the P(Zn) zitR system. An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection. P(Zn) zitR proved to be a powerful expression system for L. lactis, as it is tightly controlled by the zinc concentration in the medium.

Cations, Divalent↗
PubMed2001

Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate.

Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h(-1). More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, alpha-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.

Alcohol Dehydrogenase↗
PubMed2003

Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity.

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.

ATP-Binding Cassette Transporters↗
PubMed2004

Respiration metabolism reduces oxidative and acid stress to improve long-term survival of Lactococcus lactis.

The impact of oxygen on a cell is strongly dependent on its metabolic state: survival in oxygen of free-living Lactococcus lactis, best known as a fermenting, acidifying bacterium, is generally poor. In contrast, if haem is present, L. lactis uses oxygen to switch from fermentation to respiration metabolism late in growth, resulting in spectacularly improved long-term survival. Oxygen is thus beneficial rather than detrimental for survival if haem is provided. We examined the effects of respiration on oxygen toxicity by comparing integrity of stationary phase cells after aerated growth without and with added haem. Aeration (no haem) growth caused considerable cellular protein and chromosomal DNA damage, increased spontaneous mutation frequencies and poor survival of recA mutants. These phenotypes were greatly diminished when haem was present, indicating that respiration constitutes an efficient barrier against oxidative stress. Using the green fluorescent protein as an indicator of intracellular oxidation state, we showed that aeration growth provokes significantly greater oxidation than respiration growth. Iron was identified as a main contributor to mortality and DNA degradation in aeration growth. Our results point to two features of respiration growth in lactococci that are responsible for maintaining low oxidative damage: One is a more reduced intracellular state, which is because of efficient oxygen elimination by respiration. The other is a higher pH resulting from the shift from acid-forming fermentation to respiration metabolism. These results have relevance to other bacteria whose respiration capacity depends on addition of exogenous haem.

Bacterial Proteins↗
PubMed2006

Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis.

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.

Catalysis↗
PubMed1999

Lactococcus lactis contains only one glutamate decarboxylase gene.

Glutamate decarboxylase, which is associated with a glutamate-dependent acid-resistance mechanism, was purified from Lactococcus lactis subsp. lactis by a three-step procedure. The specific activity was increased about 114-fold with a yield of 16%. The N-terminal amino acid sequence of the enzyme was determined. The gene encoding this enzyme was cloned in Escherichia coli, and its nucleotide sequence was determined. The deduced amino acid sequence suggests that the enzyme is produced as a mature form (466 amino acid residues), not as a precursor protein. The subunit molecular mass of L. lactis glutamate decarboxylase was calculated to be 53 926 Da. The enzyme was maximally active at pH 4.7 and reacted only with L-glutamate among 20 alpha-amino acids. The apparent Km value was calculated to be 0.51 mM. The activity was stable at acidic pH values; there was no activity in the neutral pH range. At pH 4.1 the enzyme activity was retained at temperatures up to 70 degrees C in 10 min incubations. L. lactis glutamate decarboxylase behaved as a single protein when the enzyme was purified. A single band corresponding to the glutamate decarboxylase gene was detected on Southern blot analysis. These data suggest that there is one glutamate decarboxylase gene in L. lactis.

Amino Acid Sequence↗
PubMed2005

High-frequency conjugation system facilitates biofilm formation and pAMbeta1 transmission by Lactococcus lactis.

The importance of conjugation as a mechanism to spread biofilm determinants among microbial populations was illustrated with the gram-positive bacterium Lactococcus lactis. Conjugation triggered the enhanced expression of the clumping protein CluA, which is a main biofilm attribute in lactococci. Clumping transconjugants further transmitted the biofilm-forming elements among the lactococcal population at a much higher frequency than the parental non-clumping donor. This cell-clumping-associated high-frequency conjugation system also appeared to serve as an internal enhancer facilitating the dissemination of the broad-host-range drug resistance gene-encoding plasmid pAMbeta1 within L. lactis, at frequencies more than 10,000 times higher than those for the non-clumping parental donor strain. The implications of this finding for antibiotic resistance gene dissemination are discussed.

Bacterial Proteins↗
PubMed1993

Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge.

To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form.

Animals↗
PubMed2006

Altering renneting pH changes microstructure, cell distribution, and lysis of Lactococcus lactis AM2 in cheese made from ultrafiltered milk.

The objective of this study was to investigate the lysis of a highly autolytic strain of Lactococcus lactis ssp. cremoris AM2 in a model cheese made from concentrated ultrafiltered milk. From the same initial ultrafiltered retentate inoculated with L. lactis AM2, 5 cheeses were made by the addition of rennet at different pH values (6.6, 6.2, 5.8, 5.4, and 5.2). Lysis was monitored by measurement of the release of lactate dehydrogenase, an intracellular marker enzyme, and by immunodetection of intracellular proteins with species-specific antibodies. Confocal scanning laser microscopy (CSLM) was used to investigate the cheese microstructure by staining for protein and fat. Dual staining with a bacterial viability kit with CSLM was performed to reveal the integrity and localization of the bacterial cells. Levels of soluble calcium significantly increased when the pH at which the rennet was added decreased. In cheese renneted at pH 6.6, CSLM revealed an open porous structure containing a dense protein network with fat globules of different sizes distributed in the aqueous phase. In cheese renneted at pH 5.2, the protein network was homogeneous, with a less dense protein network, and an even distribution of fat globules. On d 1, bacterial cells were organized into colonies in cheese renneted at pH 6.6, whereas in cheeses renneted at pH 5.2, bacteria were evenly dispersed as single cells throughout the protein network. Lysis was detected on d 1 in cheeses renneted at high pH values and continued to increase throughout ripening, whereas induction of lysis was delayed in cheeses renneted at lower pH values until the end of ripening. This study demonstrates that alterations in the microstructure of the cheese and the distribution of cells play a role in lysis induction of L. lactis AM2.

Amino Acids↗
PubMed2005

Role of mRNA stability during genome-wide adaptation of Lactococcus lactis to carbon starvation.

The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5'-stabilizer and inverted repeated sequences as a 3'-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5'-end, and exonuclease attack at the 3'-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.

Carbon↗
PubMed1992

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.

Cloning, Molecular↗
PubMed1995

A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk.

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

Amino Acid Sequence↗
PubMed2004

Protective immunity of SpaA-antigen producing Lactococcus lactis against Erysipelothrix rhusiopathiae infection.

AIMS: To develop an economical, safe and simple vaccination system against swine erysipelas using SpaA-antigen producing Lactococcus lactis. METHODS AND RESULTS: The spaA gene of Erysipelothrix rhusiopathiae was inserted into a shuttle plasmid pSECE1 to construct pSECE1.3. The SpaA produced in L. lactis maintained a stable antigenicity without degrading in growth. After mice were inoculated intranasally and orally with pSECE1.3-carrying L. lactis cells, IgG and IgA specific to SpaA were detected, and all the mice survived a challenge with 100 LD(50) of E. rhusiopathiae Tama-96 in the inner thigh. CONCLUSIONS: SpaA-producing L. lactis appears useful as an effective subunit vaccine against swine erysipelas. SIGNIFICANCE AND IMPACT OF THE STUDY: In this vaccination system, purification of the antigen and injection are unnecessary, leading to a reduced production cost, reduced labour and less stress to the animals. This vaccination system of the lactic acid bacteria should be a safe and suitable vehicle for a polyvalent vaccine.

Administration, Intranasal↗
PubMed1991

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis.

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.

Amino Acid Sequence↗
PubMed1993

LlaAI and LlaBI, two type-II restriction endonucleases from Lactococcus lactis subsp. cremoris W9 and W56 recognizing, respectively, 5'-/GATC-3' and 5'-C/TRYAG-3'.

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

Deoxyribonucleases, Type II Site-Specific↗
PubMed1996

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis.

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

Animals↗