Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.     

The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards β-casein was investigated. β-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from β-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.     

Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis.

Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined. The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E. J. Smid, A. J. M. Driessen, and W. N. Konings, J. Bacteriol. 171:292-298, 1989). The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues. The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L. lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides. This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine. The oligopeptide transport system has a lower activity than the di-tripeptide transport system. Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate. The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate.     

Utilization of Oligopeptides by Listeria monocytogenes Scott A

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226–233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Construction of Lactococcus lactis thyA-null using the Red recombination system

Lactococcus lactis—a food-grade nonpathogenic lactic acid bacterium—is used widely in the food industry. In this report, we describe an approach to construct deficient strains in L. lactis utilizing the λ-Red recombination system. Three kinds of recombinant proteins, λ exonuclease, β protein and γ protein, were induced by l-arabinose in L. lactis MG1363 harboring the plasmid pKD46. A chloramphenicol-resistant cassette was amplified from pGj103 containing homology arms of 50 nt to the thyA gene. The PCR-generated DNA fragment was then electroporated into L. lactis MG1363, which expressed the recombination proteins. ThyA-null strains resistant to chloramphenicol were obtained and their growth characteristics were analyzed in relation to thymidine requirement. The results revealed that the thyA gene in L. lactis MG1363 was successfully knocked out. This is the first time that the Red system has been used in a Gram-positive bacterium, and use of the techniques presented here should prompt rapid and efficient mutagenesis or modification of L. Lactis chromosomal genes.     

Use of Native Lactococci as Vehicles for Delivery of DNA into Mammalian Epithelial Cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine β-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co²⁺ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.     

A multifunction ABC transporter (Opt) contributes to diversity of peptide uptake specificity within the genus Lactococcus

Growth of Lactococcus lactis in milk depends on the utilization of extracellular peptides. Up to now, oligopeptide uptake was thought to be due only to the ABC transporter Opp. Nevertheless, analysis of several Opp-deficient L. lactis strains revealed the implication of a second oligopeptide ABC transporter, the so-called Opt system. Both transporters are expressed in wild-type strains such as L. lactis SK11 and Wg2, whereas the plasmid-free strains MG1363 and IL-1403 synthesize only Opp and Opt, respectively. The Opt system displays significant differences from the lactococcal Opp system, which made Opt much more closely related to the oligopeptide transporters of streptococci than to the lactococcal Opp system: (i) genetic organization, (ii) peptide uptake specificity, and (iii) presence of two oligopeptide-binding proteins, OptS and OptA. The fact that only OptA is required for nutrition calls into question the function of the second oligopeptide binding protein (Opts). Sequence analysis of oligopeptide-binding proteins from different bacteria prompted us to propose a classification of these proteins in three distinct groups, differentiated by the presence (or not) of precisely located extensions.     

A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation

A mutant of fast milk-coagulating (Fmc⁺) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc⁻) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc⁺ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

GC-MS based metabolomics analysis reveals the effects of different agitation speeds on the level of proteinogenic amino acids in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> MG1363

Lactococcus lactis subsp. cremoris MG1363 is an opportunistic lactic acid bacterium (LAB) that has emerged as one of the most promising candidate cell factories. The availability of genome-level information and U.S. Federal Drug administration’s designation of ‘generally recognized as safe’ (GRAS) are two of the more important key factors for its wide-ranging applications in numerous biotechnological processes. Several studies have shown that various physiological conditions, such as temperature, salinity and pH, can influence the physiological growth of L. lactis; agitation, in particular, can increase the production of amino acids and fermentation by-products. However, the effect of different agitation speeds on the growth of L. lactis’ has rarely been examined. In the study reported here, we used a gas chromatography–mass spectrometry-based metabolomics approach to investigate the effects of different agitation speeds on the production of proteinogenic amino acids (PAAs) by L. lactis MG1363. Lactococcus lactis MG1363 was grown under four different agitation speeds (50, 100, 150 and 200 rpm) at a constant temperature of 30 °C, and the differences in the specific growth rate and levels of PAAs were determined. Approximately 15 PAAs with concentrations ranging from 0 to 50 mmol/L were detected under all conditions. Partial least squares discriminant analysis (PLS-DA) revealed a distinct difference when L. lactis was incubated at 100 and 150 rpm. Heatmap analysis showed that the levels of pyruvate-, glutamate- and aspartate-based amino acids were varied under the different agitation conditions. The time-series analysis showed an increment of lysine when L. lactis’ cells were cultured with shaking at 50, 100 and 200 rpm. Taken together, these results highlight the changes in the levels of PAAs in L. lactis cells in response to agitation. In addition, the collected dataset will be useful for optimization of ¹³C-labeling based experiments in L. lactis.     

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.

     

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 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.     

Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis

Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP⁻ PrtM⁻) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.     

Specificity and regulation of peptide transport on Neurospora crassa.

The tripeptide, glycyl-d,l-leucyl-l-tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora. Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.     

Autolysis of Lactococcus lactis Is Influenced by Proteolysis

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.