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PubMed2003

Overproduction and purification of the CcpA protein from Lactococcus lactis.

In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.

Bacterial Proteins↗
PubMed1993

Insertion sequence analysis of protoplast fused strains of Lactococcus lactis ssp. cremoris.

The use of insertion sequence probes for the analysis of fusants obtained following protoplast fusion is described. Hybridization of both total and plasmid DNA from parent and fusant strains with probes to IS904 and ISS1 showed that of the four protoplast fusions examined, three appeared to involve a rearrangement of genetic material while in the fourth the fusant appeared similar to one of the parental strains. This method of analysis provides more information about the changes induced by protoplast fusion than that obtained by monitoring the acquisition or loss of individual characteristics.

Base Sequence↗
PubMed2002

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments.

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.

Alanine↗
PubMed2002

Metabolic engineering of Lactococcus lactis: the impact of genomics and metabolic modelling.

Lactic acid bacteria display a relatively simple and well described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies that led to the efficient re-routing of the lactococcal pyruvate metabolism to end-products other than lactic acid, including diacetyl and alanine. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to exopolysaccharides and folic acid. Finally, the (future) impact of the developments in the area of genomics and corresponding high-throughput technologies will be discussed.

Fermentation↗
PubMed2005

Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008.

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 degrees C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.

Animals↗
PubMed1996

Tween 80 effect on bacteriocin synthesis by Lactococcus lactis subsp. cremoris J46.

Sorbitan polyoxyethylene monooleate (Tween 80) suppressed bacteriocin cell adhesion. Within the range 0-1% (v/v), there was an increase in bacteriocin production in regulated (pH 5.5 or 6.0) batch cultures with increasing Tween 80 concentration. For example, at pH 5.5 and in the presence of 1% Tween 80, bacteriocin production was about fourfold higher than in its absence. However, further increase in Tween 80 concentration did not result in a significant modification of the bacteriocin titre. It was shown that the increase was not linked to an activating effect of the surfactant on preformed enzyme, to an increase of bacteriocin availability or to a sensitization of the target cell, demonstrating that Tween 80 promoted bacteriocin production.

Bacteriocins↗
PubMed1999

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains.

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.

DNA Probes↗
PubMed1996

Presence of non-functional nisin genes in Lactococcus lactis subsp. lactis isolated from natural starters.

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.

Animals↗
PubMed1995

NisP is related to nisin precursor processing and possibly to immunity in Lactococcus lactis.

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

Base Sequence↗
PubMed1999

A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis.

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.

Base Sequence↗
PubMed1992

Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712.

High-frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.

Base Sequence↗
PubMed1999

Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon.

Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

Amino Acid Sequence↗
PubMed2003

Influence of pH drop on both nisin and pediocin production by Lactococcus lactis and Pediococcus acidilactici.

AIMS: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin production in whey. METHODS AND RESULTS: The effect of pH drop on both bacteriocin productions was tested in non-buffered whey and whey buffered at initial pH 6.3 with 0.03, 0.10 and 0.25 mol l-1 of potassium hydrogen phthalate-NaOH. An accurate description of the experimental data of nisin and pediocin obtained at different pH drops is obtained with the proposed model. CONCLUSIONS: The proposed model was able to typify both bacteriocins as pH-dependent primary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: The decisive role of pH drop for bacteriocin production on whey was demonstrated and modelled. This study contributes to a better understanding of underlying metabolic regulatory mechanisms, which could facilitate the optimization of bacteriocin production for upscaling.

Bacteriocins↗
PubMed2002

Crystallization and preliminary X-ray crystallographic studies of a complex between the Lactococcus lactis Fpg DNA-repair enzyme and an abasic site containing DNA.

For protein-DNA complex crystallization, the choice of the DNA fragment is crucial. With the aim of crystallizing the 31 kDa Fpg DNA-repair enzyme bound to DNA, oligonucleotide duplexes varying in length, sequence, end type and nature of the specific DNA target site were used. Crystals of several protein-DNA combinations grew from solutions containing both polyethylene glycol and salt. This systematic crystallization screening followed by optimization of the crystallization conditions by microseeding led to crystals of Fpg bound to a 13 base-pair duplex DNA carrying the 1,3-propanediol abasic site analogue which are suitable for crystallographic analysis. Complete native data sets have been collected to 2.1 A resolution.

Carbon-Oxygen Lyases↗
PubMed2000

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2.

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Bacteriophages↗
PubMed2002

Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA.

The formamidopyrimidine-DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.

Amino Acid Sequence↗
PubMed2003

[Food-grade expression of human Cu/Zn-superoxide dismutase gene in Lactococcus lactis].

A food-grade gene expression system in L. lactis using the lacF gene as selection marker was constructed and further used for food-grade expression of human Cu/Zn superoxide dismutase (Cu/Zn SOD). Firstly, an integrative plasmid pUCEmDE containing homologous fragments with 0.5 kb flank sequences of the lacF gene was constructed. The lacF gene was in-frame deleted by double cross-over between the plasmid pUCEmDE and the chromosomal DNA in L. lactis MG5267 and resulted in a food-grade host WZ103 that was confirmed by PCR and Lac phenotype examination. After that, a complementary plasmid pMG36eF in which the lacF gene was controlled by the strong constitutive promoter P32 was electroporated into WZ103 and resulted in the restoration of Lac+ phenotype, indicating that the lacF function in WZ103 could be complemented by the lacF gene in pMG36eF. Finally, a food-grade plasmid pWZ104 used for the expression of Cu/Zn SOD was constructed, in which the lacF gene was used as a selective marker instead of any antibiotic resistance genes. Expressed Cu/Zn SOD in WZ103 (pWZ104) was demonstrated and showed biological activity through non-denatured PAGE and SOD activity gel-staining.

Food Microbiology↗
PubMed2001

A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis.

AIMS: A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. METHODS AND RESULTS: Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure. CONCLUSION: This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.

Anti-Bacterial Agents↗