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PubMed1996

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.

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.

Amino Acid Sequence↗
PubMed1993

Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region.

The biochemical and genetical diversity of the subtilisin-like cell envelope proteinase (CEP) among Lactococcus lactis strains was investigated. The specificities of the proteinases of 16 strains toward the important cheese peptide alpha s1-casein fragment 1 to 23 and toward two differently charged chromophoric peptides have been determined. On the basis of the results, these strains could be classified into seven groups. The contribution to the specificity of specific residues in the large C-terminal segment, which differentiates this proteinase from most other members of the subtilisin family, was established with hybrid proteinases, even in the case of the small substrates. These remote residues and the subtilisin-like substrate-binding region are therefore assumed to be spatially close to each other and together constitute most of the binding region of CEP. DNA sequence analysis of fragments of the gene (prtP) encoding segments of the proteinase which contain the relevant residues of the substrate-binding region shows that among the strains studied, this binding region is the most negatively charged in the CEP group represented by strain HP and the positively charged in the CEP group represented by strains AM1 and SK11. Consequently, these two proteinase groups show the most divergent specificities. Each of the proteinases of the other groups shows a different intermediate specificity which in part is the reflection of an intermediate charge in the binding region. However, the results suggest that amino acid residues outside the segments known to be part of the CEP-binding region also contribute to specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Characterization of a novel plasmid-encoded HsdS subunit, S.LlaW12I, from Lactococcus lactis W12.

A novel type I restriction-modification specificity subunit, S. LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

Amino Acid Sequence↗
PubMed2006

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host.

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H(2)O(2)-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H(2)O(2) (150 mM) and a superoxide generator, menadione (30 microM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H(2)O(2)-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.

Biotechnology↗
PubMed2001

Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis.

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP(Nuc)) by that of L. lactis protein Usp45 (SP(Usp)) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP(Usp) than when directed by SP(Nuc). This SP(Usp) effect on Nuc secretion is not due to a better antifolding activity, since SP(Usp):Nuc precursor proteins display enzymatic activity in vitro, while SP(Nuc):Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP(Usp) and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

Amino Acid Sequence↗
PubMed2003

Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage.

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.

Amino Acid Sequence↗
PubMed2005

Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis.

The ~93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l- of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l-1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy.

Bacterial Proteins↗
PubMed2003

Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway.

A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

Base Sequence↗
PubMed2003

Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis.

We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an approximately 100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.

Carbon↗
PubMed1995

Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions.

The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was studied under different fermentation conditions. Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ2700. Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate. When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L. lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed. Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol. These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.

Acetolactate Synthase↗
PubMed2006

Recovery of nuclease produced by Lactococcus lactis using expanded bed ion exchange chromatography.

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml(-1). The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield.

Bioreactors↗
PubMed1999

Effects of metabolic flux on stress response pathways in Lactococcus lactis.

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.

ATP-Binding Cassette Transporters↗
PubMed1999

Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis.

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides. Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martínez, J. M. Martínez, P. E. Hernández, M. J. Gasson, J. M. Rodríguez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818-823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.

Anti-Bacterial Agents↗
PubMed2006

Plasmid content and bacteriocin production by five strains of Lactococcus lactis isolated from semi-hard homemade cheese.

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.

Bacteriocins↗
PubMed2000

Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben.

AIM: Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. METHODS AND RESULTS: A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. CONCLUSIONS: In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

Antibiosis↗
PubMed2005

Production of nisin by Lactococcus lactis in media with skimmed milk.

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30 degrees C for 36 h) in both M17 and MRS standard broth media (pH 6.0-7.0) supplemented with sucrose (1.0-12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30 degrees C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of "standard nisin" (Nisaplin, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.

Animals↗
PubMed2002

Dynamic response of catabolic pathways to autoacidification in Lactococcus lactis: transcript profiling and stability in relation to metabolic and energetic constraints.

The dynamic response of the central metabolic pathways to autoacidification (accumulation of organic acid fermentation products) in Lactococcus lactis was investigated in a global manner by integrating molecular data (cellular transcript concentrations, mRNA turnover) within physiological investigations of metabolic and energetic parameters. The decrease in pH associated with the accumulation of organic acids modified the physiological state of the cell considerably. Cytoplasmic acidification led to inhibition of enzyme activities and, consequently, to a diminished catabolic flux through glycolysis and a decreased rate of biochemical energy synthesis. This decrease in energy production together with the increased energy expenditure to counter cytoplasmic acidification led to energetic limitations for biomass synthesis. In these conditions, the specific growth rate decreased progressively, and growth ultimately stopped, although a diminished catabolic flux was maintained in the absence of growth. The cellular response to this phenomenon was to maintain significant levels of mRNA of catabolic genes, involving both continued transcription of the genes and also, in certain cases, an increase in transcript stability. Thus, translation was maintained, and intracellular concentration of certain enzymes increased, partially compensating for the inhibition of activity provoked by the diminished pH. When catabolic activity ceased after prolonged exposure to stress-induced stationary phase, endogenous RNA catabolism was observed.

Acids↗