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The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate ([V.sub.max]/[K.sub.m] ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the D-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the [NAD.sup.+] necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

A novel plasmid named pGdh442 had previously been isolated from a plant Lactococcus lactis strain. This plasmid encodes two interesting properties with applications in the dairy industry: a glutamate dehydrogenase activity that stimulates amino acid conversion to aroma compounds, and cadmium/zinc resistance that can be used as a selectable marker. Moreover, this plasmid can be transferred naturally to other strains, but appears to be incompatible with certain other lactococcal plasmids. During this study, the complete sequence of pGdh442 (68 319 bp) was determined and analysed. This plasmid contains 67 ORFs that include 20 IS elements that may have mediated transfer events between L. lactis and other genera living in the same biotope, such as Streptococcus, Pediococcus and Lactobacillus. Even though it is a low-copy-number plasmid, it is relatively stable due to a theta replication mode and the presence of two genes involved in its maintenance system. However, pGdh442 is incompatible with pSK08-derived protease/lactose plasmids because both possess the same replication and partition system, pGdh442 is not self-transmissible, but can be naturally transmitted via mobilization by conjugative elements carried by the chromosome or by other plasmids, such as the 712-type sex factor, which is widely distributed in L. lactis. In addition to several genes already found on other L. lactis plasmids, such as the oligopeptide transport and utilization genes, pGdh442 also carries several genes not yet identified in L. lactis. Finally, it does not carry genes that would trigger concern over its presence in human food.

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine [beta]-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine [gamma]-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an [DELTA]yrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a [DELTA]yrhA [DELTA]cysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.

The gene responsible for the uncommon glutamate dehydrogenase (GDH) activity of Lactococcus lactis was identified and characterized. It encodes a GDH of family I that is mainly active in glutamate biosynthesis, is carried by a large plasmid, and is included, with functional cadmium resistance genes, in a remnant Tn3-1ike transposon.

Research has been conducted on Propionibacterium freudenreichii, a component of the Swiss cheese secondary flora. The ability of this bacterium to produce flavor compounds during leucine catabolism has been investigated and the results suggest that this bacterium plays important role in the isovaleric acid formation during ripening.

Researchers created a strain of Lactococcus lactis that can convert amino acids to aroma compounds in the absence of alpha-ketoglutarate. This was done by incorporating a heterologous glutamate dehydrogenase gene into the bacterium.

Results demonstrate that branched-chain aminotransferase from Lactococcus lactis subsp. cremoris NCDO 763 is active under cheese-ripening conditions and imparts flavor to cheese. Data further show that the enzyme is a homodimer and exhibits aminotransferase activity.

Research conducted on the role of aromatic aminotransferase (AraT) in amino acid biosynthesis and amino acid conversion to aroma compounds is presented. Results indicate that AraT is responsible for the aromatic amino acid degradation.

The aminotransferase from Lactococcus lactis transaminates the aromatic amino acids, leucine and methionine, producing flavor compounds, thus giving flavor to cheese. The aminotransferase uses ketoacids and alph-ketoglutarate in this process. The enzyme does not use use oxaloacetate as second substrate and fails to act on aspartate. It is a pyridoxal 5'-phosphate-dependent enzyme, optimally active between pH 6.5 and 8 and between 35 and 45 degree celsius. It is active under cheese-ripening conditions also.

The efficiency of colostral protein digestion was studied in nine newborn lambs fed one meal of bovine colostrum 3 h after birth. The results were compared with those obtained in two unfed lambs and four lambs fed bovine milk. The protein and peptide composition (immunoglobulins G1 (IgG1), beta-lactoglobulin, alpha-lactalbumin, caseins and peptides resulting from casein hydrolysis) of digesta, gastrointestinal tissues, blood and urine were determined in samples taken 0.75 or 4 h after feeding. The amounts of ingested proteins in lambs fed colostrum were much higher than in those fed the mild diet, and their abomasal emptying was faster. alpha-Lactalbumin was highly degraded by abomasal and intestinal proteases, whereas beta-lactoglobulin and in particular the immunoglobulins were less sensitive. The gastric emptying of caseins was delayed in and the kinetics of appearance of peptides originating from casein hydrolysis was comparable to that observed in lambs fed milk and in 1-mo-old preruminant calves. Thirty-five percent of dietary amino acids ingested as colostrum were available within 4 h for amino acid metabolism; this percentage was 54% in the milk-fed lambs. In the lambs fed colostrum, these amino acids were provided by beta-lactoglobulin, casein and IgG1 (0.52, 0.43 and 0.30 g/kg body wt, respectively), whereas in milk-fed animals casein and beta-lactoglobulin were the most important sources of these amino acids (0.40 and 0.20 g/kg, respectively). J. Nutr. 123: 586-596, 1993).

A study was conducted to examine the effect of inactivation of genes encoding NADH oxidases (naxE and ahpF) and components of the electron transport chain (ETC) (menC and noxAB) on the ability of Lactococcus lactis to decrease the redox potential of ultrahigh-temperature (UHT) skim milk during growth under aerobic and anaerobic conditions. The results revealed that ETC plays a significant role in the reduction of oxidizing compounds other than oxygen and is also responsible for the decrease in the milk redox potential when the oxygen concentration reaches zero or under anaerobic conditions.

Glutamate dehydrogenase activity that catalyzes the reversible glutamate deamination to small letter alpha-ketoglutarate was detected in Lactococcus lactis strains isolated from a vegetal source was studied. The study shows that although the gdh gene of L. lactis encodes a GDD mainly active in glutamate biosynthesis, it is responsible for the capacity of L. lactis strains to catabolize amino acids to aroma compounds.

A study is conducted to identify and characterize the gene responsible for L-methionine to methanethiol (MTL) production from L-methionine in Brevibacterioum linens. For this purpose, a putative l-methionine-gamma-lyase gene is inactivated in B. linens and the consequences on VSC production is investigated.

The bacteria, which exist in the Swiss type cheeses, give out some important aroma compounds from the amino acid catabolism. In this study, the in vitro capabilities of lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus and streptococcus thermophilus to create the odorants from the amino acids are compared.

Research has been conducted on Lactococcus lactis growth in milk. The authors have demonstrated that double inactivation of aminotransferases AraT and BcaT, isolated from L. lactis, reduce L. lactis growth in milk.

Research indicates that the flavor development in the cheese is conferred by the starter Lactococcus lactis and glutamic dehydrogenase-positive lactobacilli and that carboxylic acids are important for cheese aroma. Results show that in the presence of alpha-ketoglutarate, the lactobacillus strains degrade less amount of amino acids than L. lactis subsp. cremoris NCD0763 and produce mainly nonaromatic metabolites.