Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac^- and Prt^-, the Lac⁺ Prt⁺ determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP⁺ strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.     

Rapid isolation, purification and characterisation of type II restriction enzymes from Lactococcus spp.

An inexpensive procedure that uses small volumes (5–10 ml) of cell culture for the rapid isolation of restriction enzymes, sufficiently pure to allow preliminary characterisation, is presented. The method was designed initially to screen for Type II restriction enzymes, but different assays can be devised to screen for other types of restriction enzymes. Although initially optimised in Lacotococcus lactis subsp. cremoris LC17-1, this method potentially holds wider applications in other lactococcal species as was shown by its successful application to Lactococcus lactis subp. lactis. Without the necessity for chromatographic techniques that are often expensive and time consuming, the convenience of the technique makes it suitable for rapid, routine screening of a large number of lactic acid bacterial strains, or restriction and modification systems cloned into them, for restriction enzyme activity.     

DNA fingerprinting of lactococci and streptococci used in dairy fermentations

Summary A DNA fingerprinting procedure was developed for strains of Lactococcus lactis subsps. lactis and cremoris, biovar. diacetylactis, and Streptococcus salivarius subsp. thermophilus, used in dairy fermentations. Total cellular DNA was extracted and digested with restriction endonucleases, HindIII or HaeIII, followed by separation of the fragments using agarose gel electrophoresis. L. lactis C2 was used as a representative strain for examining the effect of growth phase and cell concentration, cell washing conditions prior to lysis, type and concentration of the enzyme used to digest the cell wall, composition of the lysis buffer, and gel electrophoresis conditions. Following optimization of the fingerprinting procedure, electrophoretic migration of fragments from 23 strains produced reproducible gel patterns. L. lactis subsp. lactis strains ML3 and C2 appeared to be identical when restrricted with either Hind III or HaeIII. Similarly, S. salivarius subsp. thermophilus strains 19987 and 19258, and L. lactis subsp. cremoris strains 134 and C3, appeared to have identical DNA fingerprints following digestion with HindIII. To determine the usefulness of this technique for monitoring population changes during fermentation, various ratios of two closely related strains were inoculated into milk and allowed to grow for 16 h at 32° C. The initial inoculum ratios were determined by standard plate counts, and the final ratio was deterimined by DNA fingerprinting. DNA fingerprinting will be useful in the identification, characterization, and comparison of food fermentation microorganisms.Published as paper No. 17,803 of the contribution series of the Minnesota Agricultural Experiment Station Offprint requests to: S. K. Harlander     

Ultrasound Treatment for Harvesting an Aminopeptidase from Lactic Acid Bacteria and Quantitation of the Enzyme by Enzyme-Linked Immunosorbent Assays

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

Characterization of starter lactic acid bacteria from the Finnish fermented milk product viili

Aims: Phenotypic and molecular methods were used to identify and compare the strain composition of three industrial dairy starters used for the manufacture of viili. Methods and Results: Preliminary differentiation was made by phenotypic methods. Genotypic differentiation was carried out using polymerase chain reaction (PCR) and further characterization at strain level by pulsed‐field gel electrophoresis (PFGE). The isolates could be assigned as acid‐producing Lactococcus lactis strains of both lactis and cremoris subspecies, and aroma producers, identified as L. lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides. PCR analysis discriminated between the lactococcal subspecies, and cluster analysis of the digestion patterns of PFGE analysis revealed different genotypes in each subspecies. Each Leuconostoc‐genotype seemed to be specific to only a single starter mix. Conclusions: The work proved that in addition to L. lactis subsp. lactis biovar diacetylactis and Leuc. mesenteroides subsp. cremoris, commercial viili starters of traditional origin may contain (i) only L. lactis subsp. cremoris, (ii) both L. lactis subsp. cremoris and L. lactis subsp. lactis as a minority, and – as a new discovery – (iii) only L. lactis subsp. lactis. Significance and Impact of the Study: The results obtained give an overview of the microbial population of viili starters and can be exploited in the development of optimized starter cultures for industrial‐scale manufacture of viili.     

Three‐phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of ‘soidon’ (indigenous fermented food) in North‐east India were studied using cultivation‐dependent and cultivation‐independent molecular approaches. Cultivation‐dependent analyses (PCR‐amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation‐independent analyses (PCR‐DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three‐phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1–2 days) which was joined by Leuconostoc citreum during the mid‐phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5–7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation‐dependent studies complemented with cultivation‐independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 g/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 g/ml and 4.5 ± 1.5 g/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 10⁹ cfu/ml and decreased to 1.5 (± 0.2) × 10⁹ cfu/ml and 0.5 (± 0.3) × 10⁹ cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Antisense RNA directed against the major capsid protein of Lactococcus lactis subsp. cremoris bacteriophage 4-1 confers partial resistance to the host

Summary Antisense RNA targeted against the major capsid protein (MCP) of Lactococcus lactis subsp. cremoris bacteriophage F4-1 reduced bacteriophage replication by up to 50%. The region containing the mcp gene was oriented to transcribe the antisense strand using a L. lactis subsp. cremoris Wg2 promoter. The size of the mcp insert transcribed affected the level of bacteriophage inhibition and the greatest level of inhibition was achieved using a 301-bp fragment from the 5 end of the mcp. Antisense mcp RNA constructs were stable and did not alter the endogenous plasmid profile in the host, L. lactis subsp. cremoris F4-1. There were, however, some adverse effects on the host during the stationary phase as exhibited by a decline in cell density. Offprint requests to: C. A. Batt     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Inactivation of the Glutamate Decarboxylase Gene in Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.     

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

     

Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis

Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity. Received: 26 January 1999 / Accepted: 20 February 1999     

Downregulation of Salmonella Virulence Gene Expression During Invasion of Epithelial Cells Treated with Lactococcus lactis subsp. cremoris JFR1 Requires OppA

Invasion of Salmonella into host intestinal epithelial cells requires the expression of virulence genes. In this study, cell culture models of human intestinal cells (mucus-producing HT29-MTX cells, absorptive Caco-2 cells, and combined cocultures of the two) were used to determine the effects of Lactococcus lactis subsp. cremoris treatments (exopolysaccharide producing and nonproducing strains) on the virulence gene expression of Salmonella Typhimurium and its mutant lacking the oligopeptide permease subunit A (ΔoppA). During the course of epithelial cell (HT29-MTX, Caco-2, and combined) infection by Salmonella Typhimurium DT104, improved barrier function was reflected by increased transepithelial electrical resistance in cells treated with both strains of L. lactis subsp. cremoris. In addition, virulence gene expression was downregulated, accompanied with lower numbers of invasive bacteria into epithelial cells in the presence of L. lactis subsp. cremoris treatments. Similarly, virulence gene expression of Salmonella was also suppressed when coincubated with overnight cultures of both L. lactis subsp. cremoris strains in the absence of epithelial cells. However, in medium or in the presence of cell cultures, Salmonella lacking the OppA permease function remained virulent. HT29-MTX cells and combined cultures stimulated by Salmonella Typhimurium DT104 showed significantly lower secretion levels of pro-inflammatory cytokine IL-8 after treatment with L. lactis subsp. cremoris cell suspensions. Contrarily, these responses were not observed during infection with S. Typhimurium ΔoppA. Both the exopolysaccharide producing and nonproducing strains of L. lactis subsp. cremoris JFR1 exhibited an antivirulence effect against S. Typhimurium DT104 although no significant difference between the two strains was observed. Our results show that an intact peptide transporter is essential for the suppression of Salmonella virulence genes which leads to the protection of the barrier function in the cell culture models studied.

     

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.     

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     

Cloning, Sequencing, and Expression of the Pyruvate Carboxylase Gene in Lactococcus lactis subsp. lactis C2

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.