Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters.

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.

Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells.

The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance. Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight. Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose. Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation. We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation. This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions. Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs. Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.

Molecular cloning and sequence analysis of bovine lactotransferrin.

The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 2115 nucleotides and a 3' non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.

Properties of the iron-binding site of the N-terminal lobe of human and bovine lactotransferrins. Importance of the glycan moiety and of the non-covalent interactions between the N- and C-terminal lobes in the stability of the iron-binding site.

The recent determination by X-ray diffraction of the tridimensional structure of human lactotransferrin has underlined the presence of two lobes, each composed of two domains, I and II, as well as the involvement of five ligands in the binding of iron. Only one of the ligands (Asp-61) is located in domain I (residues 1-90 and 252-320), while the others [two tyrosine, one histidine and one (bi)carbonate ion linked to an arginine residue] belong to domain II (residues 91-251). On the basis of these data and of our previous results concerning the isolation of the 30 kDa N-tryptic fragment (residues 4-281) and the 20 kDa N2-glycopeptide (N-terminal domain II; residues 91-253) from human and bovine lactotransferrins, we have compared the iron-binding properties of these two fragments. The results demonstrate that Asp-61, which is missing from domain II, does not take part in the stability upon protonation of the iron complex of both human and bovine lactotransferrins. Furthermore, by comparing the iron-binding properties of human and bovine lactotransferrins to those of isolated 30 kDa N-tryptic and 50 kDa C-tryptic fragments and of the reassociated N,C-tryptic complex of both proteins, it has been shown that the non-covalent interactions which occurred between the two lobes of lactotransferrins and in the reassociated N,C-tryptic complex can explain in part the high affinity of lactotransferrins for iron. Finally, deglycosylation experiments on the 30 kDa N-tryptic fragment and N-terminal domain II from human and bovine lactotransferrins demonstrate that full removal of the glycan moiety leads to the loss of iron-binding capacity and so underlines the importance of the glycan moiety in the stability upon protonation of the N-terminal iron-binding site of both lactotransferrins.

Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae.

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.