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Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

Amino Acid Sequence↗

Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages.

A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined. Approximately 95-99% of all L. lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment. Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns. The complete nt sequence for a 1.6-kb region from nine independent L. lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp. This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein. Three out of the five changes occur in a 187-bp region, 5' to this large ORF. The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein. The encoded protein is extremely charged and shares some homology with yeast translation initiation factor. In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.

Amino Acid Sequence↗

Restriction-modification systems in Lactococcus lactis.

Several restriction-modification (R-M) systems have been identified in Lactococcus lactis. Most of the systems have been plasmid encoded and function as phage-resistance mechanisms. At least five different type-II R-M systems, LlaAI, LlaBI, LlaCI, LlaDI and LlaEI, were identified in isolates from a mixed Cheddar starter culture. LlaAI and LlaBI recognized the DNA sequences 5'- decreases GATC-3' and 5'-C decreases TRYAG-3', respectively. The genes coding for the LlaAI and LlaBI R-M systems have been cloned and sequenced. The LlaAI R-M system had two genes coding for methyltransferases (MTases) and one gene coding for a restriction endonuclease (ENase). The MTases showed high homology to the MTases from DpnII. The LlaBI R-M system had one gene coding for a MTase and one gene coding for an ENase.

Base Sequence↗

Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria.

Brazzein is an intensely sweet-tasting plant protein with good stability, which makes it an attractive alternative to sucrose. A brazzein gene has been designed, synthesized, and expressed in Escherichia coli at 30 degrees C to yield brazzein in a soluble form and in considerable quantity. Antibodies have been produced using brazzein fused to His-tag. Brazzein without the tag was sweet and resembled closely the taste of its native counterpart. The brazzein gene was also expressed in Lactococcus lactis, using a nisin-controlled expression system, to produce sweet-tasting lactic acid bacteria. The low level of expression was detected with anti-brazzein antibodies. Secretion of brazzein into the medium has not led to significant yield increase. Surprisingly, optimizing the codon usage for Lactococcus lactis led to a decrease in the yield of brazzein.

Amino Acid Sequence↗

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.

Amino Acid Sequence↗

Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

Adenosine Diphosphate↗

Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages.

The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them.

2-Propanol↗

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways.

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.

Amino Acids↗

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp. lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis. The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation. DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A. The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA. The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems. lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators. LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus. The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs. On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.

Adenosine Triphosphate↗

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris.

Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 with a 5.8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41,600. Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position. No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue. However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

Aminopeptidases↗

Effects of diverse environmental conditions on {phi}LC3 prophage stability in Lactococcus lactis.

The effects of various growth conditions on spontaneous phiLC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4(4) factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous phiLC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected phiLC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous phiLC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.

Attachment Sites, Microbiological↗

Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression.

The transport of citrate in Lactococcus lactis biovar diacetylactis is mediated by the citrate permease P. This polypeptide is encoded by the citP gene carried by plasmid pCIT264. In this report, we characterize the citP transcript, identify a cluster of two genes cotranscribed with citP and describe their post-transcriptional regulation. The transcriptional promoter is located 1500 nucleotides upstream of the citP gene and the transcriptional terminator is positioned next to the 3'-end of this gene. The DNA sequence was determined of the region upstream of the citP gene, including the promoter. Two partially overlapping open reading frames, citQ and citR were identified, which could encode polypeptides of 3.9 and 13 kDa respectively. These two genes, together with citP, constitute the cit cluster. Moreover, an IS-like element located between the cit promoter and the citQ open reading frame was identified. This element includes an open reading frame ORF1, which could encode a 33 kDa polypeptide. A translational fusion between the citP and a cat reporter gene showed that translation of citR and citP is coupled, and regulated by CitR. The cit mRNA was subjected to specific cleavage after addition of rifampicin to the bacterial cultures. We propose that expression of the cit cluster is controlled at the post-transcriptional level by mRNA processing at a putative complex secondary structure and by translational repression mediated by CitR.

Amino Acid Sequence↗

LlaFI, a type III restriction and modification system in Lactococcus lactis.

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactis LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

Amino Acid Sequence↗

Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis.

The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Amino Acid Sequence↗

Production of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp. lactis biovar. diacetylactis, isolated from Zimbabwean naturally fermented milk.

Malty compound-producing Lactococcus lactis subsp. lactis biovar. diacetylactis strain INF-DM1, originally isolated from naturally fermented milk in Zimbabwe was used to prepare fermented milk from ordinary milk, milk enriched with 2.5% (w/v) skimmed milk powder and by 2.5% (w/v) increase in dry matter by ultrafiltration. Inoculated milk was incubated at 22, 30 and 37 degrees C. Analyses were made after 0, 9, 18 and 24 h incubation and also after 24 h incubation followed by storage for one week at 4 degrees C. Samples were analysed for volatile compounds including malty compounds and for organic acids, pH and log cfu/g. All samples were also judged for sensory attributes. Products made from enriched milks showed increased viscosity which was most marked in ultrafiltrated milk incubated at 30 and 37 degrees C. The levels of certain compounds (lactic acid, citrate and diacetyl) were significantly affected by milk type. Incubation temperature had a significant effect on starter growth rate and the rate of production and amount of the malty compounds, lactate, diacetyl, ethanol, acetoin and acetaldehyde. 3-Methyl butanal concentrations were above the taste threshold level of 0.06 ppm in almost all products, including stored products. Although initial growth rate was fastest at 37 degrees C, an uncoupling of acid production and growth was observed after 9 h incubation, suggesting that this is above the optimum temperature for this strain. In addition, products incubated at 37 degrees C showed a tendency to whey separation, indicating that this temperature is also too high to give optimum product quality. All products attained good scores in sensory analysis provided that fermentation was complete. Variation in the levels of malty compounds during the fermentation had no significant effect on the sensory score for total flavour.

Acetaldehyde↗

Anaerobic sugar catabolism in Lactococcus lactis: genetic regulation and enzyme control over pathway flux.

Lactic acid bacteria and particularly Lactococcus lactis are widely used for the production of lactic acid in fermented foods. Control of the catabolic rate in L. lactis, i.e., the rate of lactic acid production, appears to be determinant for dairy product quality. While the mechanisms involved in control have not been totally elucidated, they seem to depend upon the strain and the growth conditions. Furthermore, it remains unclear whether the catabolic rate is controlled at the level of transcription, translation or enzyme activity. The recent sequencing of the L. lactis genome has brought novel insights to physiologic studies of the bacteria. This review discusses both genetic information and metabolic studies concerning anaerobic sugar catabolism in L. lactis.

Anaerobiosis↗

[Glutathione plays an anti-oxidant role in Lactococcus lactis].

To assess the physiological function of GSH in resistance to oxidative stress in Lactococcus lactis ssp. cremoris NZ9000, the recombinant strain NZ9000 (pNZ3203) capable of producing GSH was used as the experiment materials. The anti-oxidant role of glutathione was observed under higher H2O2 dosage, i.e., 150 mmol/L H2O2 treatment for 15 min. The resistance of NZ9000 (pNZ3203) cells grown for 3 h, 5 h, and 7 h (nisin-induced for 1 h, 3 h and 5 h) were 1.8-fold, 2.6-fold, and 2.9-fold that of NZ9000 (pNZ8148) cells, respectively. In addition, the survival of NZ9000 (pNZ3203) cells grown for 5 h (nisin-induced for 3 h), upon treatment of 20 mmol/L menadione for 60 min, was 6.2-fold that of NZ9000 (pN8148) cells. Therefore, introduction a new biosynthetic pathway of glutathione could confer higher resistance to oxidative stress on L. lactis NZ9000.

Antioxidants↗

Heterologous expression of the plant coumarate: CoA ligase in Lactococcus lactis.

AIMS: To demonstrate the expression of coumarate : CoA ligase of Arabidopsis thaliana in Lactococcus lactis as a first step of cloning the vanillin pathway. METHODS AND RESULTS: The 4CL gene was amplified from a cDNA library of A. thaliana by PCR and subcloned into a multicopy lactococcal vector where the expression is under the nisA promoter. The maximum yield of the protein in the recombinant strain of L. lactis was obtained 3 h after induction with 10 ng ml(-1) of nisin. However, these levels were only fraction of those detected in cell extracts of Pseudomonas fluorescens AN103 strain which naturally expresses its own enzyme when grown in the presence of ferulic acid as a carbon source. Among different substrates examined, the enzyme was most active against coumaric acid. CONCLUSIONS: The gene encoding coumarate : CoA ligase in A. thaliana was isolated, sequenced, cloned and expressed in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first of the two steps for genetic engineering of the vanillin pathway in the GRAS (generally recognized as safe) organism L. lactis.

Arabidopsis↗