When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.
The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.
The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-Methionine-␥-lyase (MGL) can convert Lmethionine to methanethiol (MTL), ␣-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiolproducing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via ␥-elimination is a key step in the formation of VSCs in B. linens.Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to the typical aromas of different cheeses (12,14,33). VSCs arise primarily from the degradation of L-methionine to methanethiol (MTL) by the cheese microflora. This thiol is a common precursor for a variety of other sulfur-bearing compounds including the auto-oxidation products (11), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters, primarily arising from chemical reaction of MTL with acyl coenzyme A (acyl-CoA) (22). Numerous studies have therefore been done to control and/or diversify VSC synthesis during the ripening process by the use of properly selected microorganisms (4, 6, 15, 43). Many cheese microorganisms are capable of producing VSCs from L-methionine. Some of them, such as brevibacteria, especially Brevibacterium linens (17), are known to be very good VSC producers while others, such as lactic acid bacteria (LAB), can produce only limited amounts of VSCs (14).The most direct route for MTL biosynthesis, is the L-methionine ␥-elimination that directly produces MTL, ␣-ketobutyrate, and ammonia from L-methionine. This L-methionine ␥-elimination activity is quite high in B. linens and corynebacteria (17) and is also suspected in several other cheese surface bacteria, such as Micrococcus luteus, Arthrobacter sp., and Staphylococcus equorum (8). In contrast, such activity is quite low in LAB (14). In B. linens, the methionine ␥-elimination is catalyzed by a L-methionine-␥-lyase (MGL), a pyridoxal phosphate (PLP)-dependent enzyme for which L-methionine is the best substrate (16). In contrast, in LAB the reaction is catalyzed by a cystathionine -lyase (CBL) and a cystathionine ␥-lyase (CGL) which are only slightly active towards L-methionine (1, 10, 18). In LAB, another pathway for L-methionine conversion to VSCs also exists but produces limited amounts of MTL (7,35).Coryneform bacteria are generally found on the surface of smear cheeses and give the typical sulfur notes to cheeses such as Limburger, Tilsiter, Livarot, Epoisses, and Munster. To date, B. linens is the only food-grade bacterium from which MGL has been purified and characterized (16,26,31,38,39), but neither its protein sequence nor its gene...
In milk, Streptococcus thermophilus displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated. During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity.The concentrations in milk of the essential amino acids glutamic acid and methionine (45 and Ͻ1 mg per liter, respectively) (7) are far below the requirements of Streptococcus thermophilus (200 and 60 mg per liter, respectively) (9). Consequently, S. thermophilus has to find complementary sources of amino acid in order to grow in milk to high cell densities. The presence of a cell wall proteinase, PrtS (1), an oligopeptide transport system, Ami (2), and a large set of intracellular peptidases (13) enables S. thermophilus to use milk proteins in a pathway similar to that described for lactococci (6,8). Nevertheless, the relationship between proteolysis and growth of S. thermophilus in milk has not yet been characterized.The possible limitation of the growth rate of S. thermophilus related to proteolysis was studied. The growths in milk of eight industrial strains and one laboratory strain were evaluated by spiral plating (Fig.
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