Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH₂PO₄), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH₂PO₄ from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO₄, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH₂PO₄, yeast autolysate (an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Physical analysis of in vivo pCI301 fusion plasmids in Lactococcus lactis subsp. lactis

Abstract In vivo fusion plasmids identified following conjugative mobilization of pCI301, the 75-kilobase (kb) lactose-proteinase plasmid of Lactococcus lactis subsp. lactis UC317, were characterized. These plasmids (95 kb) were generated from fusion-deletion events involving pCI301 and the 38-kb UC317-derived cryptic plasmid, pCI303. Recombinant plasmids were separable into distinct classes based on their associated phenotypes and restriction maps. The formation of pCI301: : pCI303 composite plasmids within strain UC317 was also demonstrated.     

Proton-dependent kinetics of citrate uptake in growing cells of Lactococcus lactis subsp. lactis bv. diacetylactis

Abstract The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.     

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus

Abstract Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus . Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis . No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Identification and sequence analysis of the replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503

Abstract The replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503 was localised to within a 10-kb Hin dIII restriction fragment. A 6.3-kb Bgl II- Hin dIII subclone of this fragment, cloned into a replication probe vector, allowed replication in Lactococcus but not in Bacillus or Lactobacillus . Sequence analysis revealed an ORF of 1152 bp preceded by a putative ori region containing a 22-bp sequence tandemly repeated three and three-quarter times, a second smaller direct repeat and two inverted repeats. Extensive homology was observed with the well characterised replication region of the small cryptic plasmid pCI305 (Hayes, F., Vos, P., Fitzgerald, G.F., de Vos, W. and Daly, C. Plasmid 25, 16–26).     

sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc⁺Nis⁻ strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc⁻ derivatives from Suc⁺Nis⁻ strains after conjugation and curing experiments.     

Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against influenza virus infection

Aims: To evaluate the protective effects of oral administration of milk fermented with a Lactococcus strain against influenza virus (IFV) infection in a mouse model. Methods and Results: Milk fermented with exopolysaccharide‐producing Lactococcus lactis subsp. cremoris (L. cremoris) FC was orally administered to BALB/c mice for 12 days. Mice were intranasally infected with IFV A/New Caledonia/20/99 (H1N1) on day 8, and survival was determined for 14 days after IFV infection. Survival rate and body weight loss after IFV infection in the L. cremoris FC fermented milk‐administered group were significantly improved compared with those in the control group. In the unfermented milk‐administered group, survival rate was not improved, whereas body weight loss was slightly improved compared with that in the control group. The mean virus titre in the lung of the L. cremoris FC fermented milk‐administered group 3 days after infection was significantly decreased compared with that in the control group. Conclusions: These results suggest that oral administration of milk fermented with L. cremoris FC protects mice against IFV infection. Significance and Impact of the Study: These results demonstrate that oral administration of milk fermented with exopolysaccharide‐producing Lactococcus strains might protect host animals against IFV infection.     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

Aims:  Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions.
Methods and Results:  A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis .
Conclusions:  The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions.
Significance and impact of the study:  This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.     

Near infrared spectroscopy coupled with radial basis function neural network for at-line monitoring of Lactococcus lactis subsp. fermentation

In our previous work, partial least squares (PLSs) were employed to develop the near infrared spectroscopy (NIRs) models for at-line (fast off-line) monitoring key parameters of Lactococcus lactis subsp. fermentation. In this study, radial basis function neural network (RBFNN) as a non-linear modeling method was investigated to develop NIRs models instead of PLS. A method named moving window radial basis function neural network (MWRBFNN) was applied to select the characteristic wavelength variables by using the degree approximation (Da) as criterion. Next, the RBFNN models with selected wavelength variables were optimized by selecting a suitable constant spread. Finally, the effective spectra pretreatment methods were selected by comparing the robustness of the optimum RBFNN models developed with pretreated spectra. The results demonstrated that the robustness of the optimal RBFNN models were better than the PLS models for at-line monitoring of glucose and pH of L. lactis subsp. fermentation.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Dynamics of pyruvate metabolism in Lactococcus lactis.

The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.     

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

Abstract A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF -deficient strain produced a Lac⁻ phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF -deficient strain resulting in a Lac⁺ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.