Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk.

AIM: The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant. METHODS AND RESULTS: The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617(T) and its derivative, carbamoylphosphate synthetase-negative mutant A17(DeltacarB), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO(2) depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l-arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine. CONCLUSIONS: Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.

Characterization of the primary structure of H-protein from Pisum sativum and location of a lipoic acid residue by combined liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

A purified extract of H-protein, a subunit of the glycine cleavage complex of the pea leaf mitochondria, was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardment (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Determination of the molecular weight of the entire protein, a 14 kDa subunit of the glycine decarboxylase complex, was achieved by ESI mass spectrometry and revealed covalent binding of the protein to the stabilizing agent beta-mercapto-ethanol. On-line LC/MS analysis of peptides arising from the endoproteinase Glu-C digestion of the H-protein was achieved using capillary columns (0.25 mm i.d.), and permitted confirmation of the previously reported sequence deduced from cDNA cloning experiments. The detailed interpretation of data extracted from these LC/MS experiments facilitated identification of peptides containing modified amino acid residues. In particular the identification of a lipoic acid cofactor, a rather unusual modified lysine residue which interacts with different active sites in the enzyme complex, was achieved using both LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified lysine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.