Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk.

The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Simultaneous Loss of Proteinase- and Lactose-Utilizing Enzyme Activities in Streptococcus lactis and Reversal of Loss by Transduction

During studies on spontaneous loss of lactose metabolism in Streptococcus lactis C2, it was found that the lactose-negative (lac(-)) mutants were also proteinase negative (prt(-)). This pleiotropic effect was observed in S. diacetilactis 18-16, but not in S. cremoris B1. The lac(-)prt(-) mutants from S. lactis C2 were able to grow in milk, but no pH change or measurable protein breakdown occurred. When the milk was supplemented with glucose, a slow decline in pH occurred. Addition of a protein hydrolysate to milk did not stimulate acid production. When both supplements were added to milk, normal growth and pH change were obtained. When the lac(-)prt(-) mutant of S. lactis C2 was transduced with the temperate phage from the lac(+)prt(+) parent culture, approximately equal numbers of lac(+)prt(-) and lac(+)prt(+) transductants were obtained. When the spontaneous lac(+)prt(-) strain of S. lactis C2 was converted to a lac(-)prt(-) derivative and transduced, similar results were obtained. The co-transduction of the lactose and proteinase markers suggest they are closely associated. The findings indicate that the transducing phage from S. lactis C2 can be used to examine the causes of instability in both the lactose and proteinase enzyme systems of this organism.     

Proteinase Activity in Slow Lactic Acid-Producing Variants of Streptococcus lactis

Variants of Streptococcus lactis that produce lactic acid slowly in milk were isolated by inducing plasmid loss in the wild type at 39 to 40 C. Such strains had lost most of their surface-bound proteinase activity and were designated prt(-). The specific proteinase activities of S. lactis C10 prt(+) whole cells and solubilized cell walls were 7 and 18 times, respectively, those of the prt(-) strain, but spheroplast lysates of prt(+) and prt(-) strains contained similar proteinase activity. S. lactis H1 showed a similar relative distribution of activity between prt(+) and prt(-) cellular fractions, although the overall level was lower. The limited growth in milk, characteristic of prt(-) strains, can be explained in terms of their low surface-bound proteinase activity.     

Regulation of Proteolytic Enzyme Activity in Lactococcus lactis

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.     

Evaluation of lactic acid Streptococci for the preparation of frozen concentrated starter cultures

Eight strains of Streptococcus diacetilactis and Streptococcus lactis were examined for viability, growth rate, lactic acid and diacetyl production in milk and proteolytic activity before and after freezing at --30 degrees C. Concentrations of yeast autolysate, peptone, lactose and citrate as well as the usefulness of milk and whey culture media for active biomass production were investigated. After freezing and storage at --30 degrees C, with the use of non-fat milk as a cryoprotective agent, high survival and endocellular proteolytic activity of the frozen concentrate was achieved. S. diacetilactis sp. and S. lactis 115 were shown to be more biologically active than other strains. Their physiological properties remained unaffected by freezing.     

Proteinase genes of cheese starter cultures.

The proteolytic enzymes of lactococci are of eminent importance for milk fermentations. By the combined action of proteinases and peptidases milk protein is degraded to peptides and amino acids which are required for cell growth and contribute to the organoleptic properties of the foods. The importance of the proteolytic system for dairy product quality has resulted in an increased fundamental research of the enzymes and genes involved. Proteinase plasmids have been identified and plasmid stability problems offered an explanation for the apparent instability of proteolysis in certain strains of lactococci. Chromosomal integration has recently been used to stably anchor the proteinase genes in the chromosome of Lactococcus lactis. The structural proteinase genes of a number of strains have been cloned and sequenced, and some of the properties of the enzymes they specify will be discussed. The product of a second gene is necessary for the activation of the proteinase, a proteinase maturation process that is unique in the bacterial world.     

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.     

Effect of a lactic starter culture on the growth and protease activity of Aeromonas hydrophila

Lactococcus lactis subsp. lactis strain Lac 388 (isolated from goats' cheese made without starter) had inhibitory activity against three strains of Aeromonas hydrophila either by the plating method or by the associative culture approach (broth, skim milk and ewes' milk). Low pH was only partially responsible for the antagonism. Inhibition due to hydrogen peroxide and nutrient depletion was excluded. This inhibitory compound(s) was not destroyed by proteolytic enzymes. In mixed cultures the strains of Aer. hydrophila did not produce detectable amounts of protease.     

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.     

CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.     

Effect of Culture Medium Composition and pH on the Production of M Protein and Proteinase by Group A Streptococci

The effects of pH, yeast extract, and neopeptone on the production of extracellular proteinase and M protein by group A streptococci were studied with a type 1 strain capable of producing both M protein and proteinase. The strain DS 2036-66 grew moderately well in a semisynthetic broth. M protein was produced without adding peptides to the medium. When added to a medium with 1% glucose, yeast extract (0.1%) was found to stimulate both growth and proteinase formation. Limiting the glucose to 0.25% prevented a drop in pH below 6.7 and prevented proteinase formation. Although less growth occurred with limited glucose, M protein of high specific activity was produced with an actual increase in acid-extractable M protein during the stationary phase of growth. When the medium was buffered at pH 7.85 with tris(hydroxymethyl)aminomethane buffer, 0.5% neopeptone prevented proteinase formation. This was true even in the presence of 1% glucose and 0.1% yeast extract, which resulted in a fall in pH to about 4.8 by 48 hr. Growth was greater than in Todd Hewitt broth, but the specific activity of M protein was considerably less than that found in the medium with glucose limited to 0.25%. Neopeptone was found to have little direct action on crude streptococcal proteinase. Instead, the evidence suggested that neopeptone somehow prevents proteinase elaboration. Yeast extract, on the other hand, appears to stimulate proteinase elaboration. To prevent proteinase formation, neopeptone must be added early, during the logarithmic phase of growth or at the start. In contrast, when yeast extract was added as late as 24 hr, it resulted in the elaboration of extracellular proteinase and in the decline of M protein. When 38 M nontypable strains from the diagnostic laboratory were tested for proteinase activity under conditions similar to those used in the diagnostic laboratory, only six produced much proteinase.     

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (P_I versus P_III type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing α_s1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the P_I-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a P_III-type proteinase.     

Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein.

The chlorated dipeptide L-alanyl-beta-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant of L. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di- and tripeptide transport. Uptake of di- and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.     

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.