Structure, organization, and expression of the lct gene for lacticin 481, a novel lantibiotic produced by Lactococcus lactis.

The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 51-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in beta-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

Purification and Partial Characterization of Lacticin 481, a Lanthionine-Containing Bacteriocin Produced by Lactococcus lactis subsp. lactis CNRZ 481.

Lacticin 481, a bacteriocin produced during the growth of Lactococcus lactis subsp. lactis CNRZ 481, was purified sequentially by ammonium sulfate precipitation, gel filtration, and preparative and analytical reversed-phase high-pressure liquid chromatography. Ammonium sulfate precipitations resulted in a 455-fold increase in total lacticin 481 activity. The entire purification protocol led to a 107, 506-fold increase in the specific activity of lacticin 481. On the basis of its electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gels, lacticin 481 appeared as a single peptide band of 1.7 kDa. However, dimers of 3.4 kDa also exhibiting lacticin activity were detected. Derivatives of the lacticin-producing strain which did not produce lacticin 481 (Bac) were sensitive to this bacteriocin (Bac) and failed to produce the 1.7-kDa band. Amino acid composition analysis of purified lacticin 481 revealed the presence of lanthionine residues, suggesting that lacticin 481 is a member of the lantibiotic family of antimicrobial peptides. Seven residues (K G G S G V I) were sequenced from the N-terminal portion of lacticin 481, and these did not shown any homology with nisin or other known bacteriocin sequences.

Effect of Fermentation Conditions on Growth of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in Pure and Mixed Cultures.

Two strains of mesophilic lactic acid bacteria, Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091, were grown in pure and mixed cultures in the presence or absence of citrate (15 mM) and at controlled (pH 6.5) or uncontrolled pH. Microbial cell densities at the end of growth, maximum growth rates, the pH decrease of the medium resulting from growth, and the corresponding acidification rates were determined to establish comparisons. The control of pH in pure cultures had no effect on L. lactis CNRZ 1091 populations. The final populations of S. cremoris AM2, however, were at least five times higher than when the pH was not controlled (4 x 10 vs. 2 x 10 CFU . ml). The pH had no effect on the growth rate of either strain. That of S. cremoris AM2 (0.8 h) was about twice that of L. lactis CNRZ 1091. When the pH fell below 5, the growth of both strains decreased or stopped altogether. Citrate had no effect on S. cremoris AM2, while final populations of L. lactis CNRZ 1091 were two to three times higher (3 x 10 CFU . ml); it had no effect on the maximum growth rates of the two strains. Citrate attenuated the pH decrease of the medium and reduced the maximum acidification rate of the culture by 50%, due to the growth of S. cremoris AM2. Acidification due to L. lactis CNRZ 1091, however, was very slight. Regardless of the conditions of pH and citrate, the total bacterial population in mixed culture was lower (by 39%) than that of the sum of each pure culture. Mixed culture improved the maximum growth rate of L. lactis CNRZ 1091 (0.6 h) by 50%, while that of S. cremoris AM2 was unaffected. The acidification rate of the growth medium in mixed culture, affected by the presence of citrate, resulted from the development and activity of S. cremoris AM2.

Cell-Wall-Associated Proteinases in Lactobacillus casei and Lactobacillus plantarum.

Information concerning cell-wall associated proteinases of lactobacilli is limited. In Lactobacillus casei and Lactobacillus plantarum , presence of such proteinase is clearly shown. Differences between several strains were noticed. Higher cell-wall-associated proteinase activity can be measured in extracts obtained from milk-grown cells when compared to MRS-grown cells. No aminopeptidase, dipeptidase or carboxypeptidase activities were detected in the cell-wall-associated proteinase fraction. Isoelectric focusing of αs1-casein hydrolysates obtained by the action of this fraction from L. casei grown in milk revealed the presence of a major hydrolysis product and three minor degradation products with isoelectric points more acidic than αS1. Beta-casein was also degraded by the cell-wall extract with formation of one major product and several minor products with isoelectric points more acidic than ß-casein. Two major hydrolysis products with isoelectric points higher than ß-casein were also detected. Isoelectric focusing of αs1- and ß-casein hydrolysates obtained by the action of the intracellular extracts of L. casei grown either in milk or in MRS broth shown identical patterns. As with L. casei , two strains of L. plantarum exhibited cell-wall proteinase activity. Milk-grown cells were more proteolytic than MRS-grown cells. Generally L. plantarum was significantly less proteolytic than L. casei .

Survival of Brevibacterium linens during nutrient starvation and intracellular changes.

The present work reports the survival capacity of a strain of Brevibacterium linens isolated from a French camembert cheese and the ensuing changes in cell composition. Exponentially growing cells were harvested, washed and resuspended with shaking in pH 8.0 buffer at 21 degrees C in the absence of a carbon source. The viability of this strain, assessed with slide cultures, is much less than that of coryneform bacteria isolated from soil samples, even though no cell lysis was detected. Intracellular RNA was rapidly consumed during the first few days although magnesium levels remained high. The quantity of DNA initially increased by 17% within 24 h and then remained stable during the 30 days of the experiment. During the same period, absorbance of the medium at 260 nm reached 2 absorbance units. Reserve polysaccharides in this strain are less abundant than in Arthrobacter and were rapidly consumed. Proteolysis was regular and thus maintained a pool of free amino acids which was greater than 60% of the initial value. There was a parallel accumulation of ammonia in the medium. Catalase activity decreased regularly during the first 80 h whereas the quantity of Adenosine-5'-triphosphate (ATP) dropped by 47% in 10 h, stabilizing at less than 10% of its initial value. Cell respiration declined very rapidly and was very low after 24 h.

Electrochemical proton gradient of Brevibacterium linens and its relationship to phenylalanine transport.

The proton motive force generated by Brevibacterium linens was determined by the accumulation of radiolabelled tetraphenylphosphonium for transmembrane potential (delta psi) and by the accumulation of benzoate ions for the H+ chemical gradient (delta pH). In resting cells at pH 8.0, the delta psi was 172 +/- 10 mV while delta pH was 9 mV. The additions of valinomycin to B. linens in the presence of 100 mM exogenous potassium reduced the delta psi and led to a drastic inhibition of phenylalanine transport. These findings are consistent with the hypothesis that a membrane potential is essential for active phenylalanine transport in B. linens cells at pH 8.0. Phenylalanine transport in these cells (halotolerant strain) did not depend on the presence of Na+.

[Transport of phenylalanine and tyrosine in Brevibacterium linens: specificity and incorporation into proteins]. / Le transport de la phénylalanine et de la tyrosine chez Brevibacterium linens: spécificité et incorporation dans les protéines.

The specificity of phenylalanine and tyrosine carriers was investigated using actively metabolizing cells of Brevibacterium linens. The cellular protein synthesis of resting cells was very weakly inhibited, even with high concentrations of chloramphenicol or tetracycline. The nonaromatic amino acids were weak inhibitors for these carriers, while fluorinate analogues of phenylalanine and tyrosine were very potent competitive inhibitors. In practice these analogues cannot be used to replace amino acids to evaluate transport without incorporation because they are incorporated in cellular proteins.

Transport of aromatic amino acids by Brevibacterium linens.

Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of 24, 3.5, and 1.8 microM. Transport of these amino acids was optimum at pH 7.5 and 25 degrees C for phenylalanine and pH 8.0 and 35 degrees C for tyrosine and tryptophan. Crossed inhibitions were all noncompetitive. The only marked stereospecificity was for the L form of phenylalanine. Transport was almost totally inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were much more inhibitory for tryptophan transport than for transport of the other two aromatic amino acids.

[Peptide hydrolases of lactobacilli of the Thermobacterium group. I. Demonstration of these activities in Lactobacillus helveticus, L. acidophilus, L. lactis and L. bulgaricus]. / Les peptide-hydrolases des lactobacilles du groupe Thermobacterium. I. Mise en évidence de ces activités chez Lactobacillus helveticus, L. acidophilus, L. lactis et L. bulgaricus.

The intracellular peptide hydrolase activities of Lactobacillus helveticus, L. acidophilus, L. lactis, and L. bulgaricus were determined using various aminopeptidase, dipeptidase, and carboxypeptidase substrates in addition to casein and whey protein fractions. The different activities were then separated using disc gel electrophoresis. Each bacterium had aminopeptidase activity towards various amino acid beta-naphthylamides and dipeptides. The four species also showed bands of true dipeptidase activities on a large number of dipeptides. Intracellular enzymes from thermophilic lactobacilli also hydrolysed the whey proteins (alpha-lactalbumin and beta-lactoglobulin). From the results of electrophoresis on beta-casein and alpha s1-casein it was shown that beta-casein was totally hydrolysed by L. lactis while it was only partially hydrolysed by the intracellular enzymes of L. acidophilus and L. bulgaricus. On the other hand, alpha s1-casein was only partially hydrolysed by L. helveticus, L. lactis, and L. bulgaricus.

Hydrolysis of beta-casein and peptides by intracellular neutral protease of Streptococcus diacetylactis.

The endopeptidase activity of mesophilic streptococci was characterized further by investigating the specificity of an intracellular endopeptidase from Streptococcus diacetylactis for beta-casein, derived peptides, and bradykinin. The inhibitory action of phosphoramidon as well as direct determinations of metal content showed this enzyme was a metalloprotein. Hydrolysis of native beta-casein was relatively low. Peptides obtained from the fraction soluble at pH 4.6 led to the demonstration that Pro186-Ile187 and Ala189-Phe190 were hydrolyzed by the enzyme. Two peptides derived from beta-casein by the action of chymosin were hydrolyzed efficiently: we observed hydrolysis of Lys176-Ala177, Lys169-Val170, and Pro206-Ile207. The Pro7-Phe8 bond of bradykinin was hydrolyzed rapidly, showing that this enzyme was efficient for the hydrolysis of prolyl peptide bonds. The protease was slightly less sensitive to phosphoramidon than was thermolysin. Metal analyses showed the enzyme contained 580 microgram of zinc and 4,760 microgram of calcium per gram protein. This protease is thus a true metalloenzyme (E.C.3.4.24.4), and its action may complete the hydrolysis initiated by chymosin remaining active in cheese curd by hydrolyzing peptides released by chymosin.

Peptide hydrolases of Lactobacillus casei: isolation and general properties of various peptidase activities.

Discovery of an endopeptidase by gel chromatography and separation of 3 exopeptidases (a dipeptidase, an aminopeptidase and a specific carboxypeptidase) from Lactobacillus casei NCDO 151 by affinity chromatography is described. The 3 exopeptidases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline but were reactivated with Co2+ and Mn2+. The pH optima for aminopeptidase, dipeptidase and carboxypeptidase activities were 6.5, 7.6 and 7.2, respectively. Maximum activity was obtained at 45 degrees C for the aminopeptidase, at 30 degrees C for the dipeptidase and at 40 degrees C for the carboxypeptidase. The substrate specificities of the 3 enzymes were also studied. The properties of these 3 enzymes are compared with those of other bacteria.