Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and succinic acid: A model

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and succinic acid as the carbon source. Growth on succinic acid was possible after long adaptation periods. The flows of glucose, succinic acid, oxygen, carbon dioxide, and biomass to and from the system were measured. It proved necessary to expand our previous model to accommodate the active transport of succinic acid by the cell. The values found for the efficiency of the oxidative phosphorylation (P/O) and the amount of ATP needed for production of biomass from monomers gave the same values as found for substrate mixtures taken up passively.     

Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain.

Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein.     

Determination of plasma bile acids by capillary gas-liquid chromatography-electron capture negative chemical ionization mass fragmentography

Combined capillary gas-liquid chromatography-electron capture negative chemical ionization mass spectrometry of pentafluorobenzyl ester-TMSi ether derivatives of bile acids and isotope dilution using deuterated internal standards are introduced as a sensitive and selective analysis technique for plasma bile acids. As a result of the high ionization efficiency of pentafluorobenzyl derivatives under electron capturing conditions and minimal fragmentation, the detection limit of this technique is low: 1 pg for each bile acid. The high sensitivity enabled the detection and quantitation of atypical bile acids in 200-microliters aliquots of plasma from fasting healthy adults as exemplified by trihydroxycoprostanic acid (0.002 +/- 0.001 mumol/l) and dihydroxycoprostanic acid (0.013 +/- 0.002 mumol/l).     

Identification of a gene required for maturation of an extracellular lactococcal serine proteinase.

Directly upstream of the Lactococcus lactis subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.     

Bile acid synthesis in cell culture

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.     

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp. lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis. The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation. DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A. The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA. The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems. lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators. LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus. The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs. On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.     

Net Uptake of Sulfate and its Transport to the Shoot in Spinach Plants Fumigated with H2S or SO2: Does Atmospheric Sulfur Affect the “Inter-Organ” Regulation of Sulfur Nutrition?*

Spinach plants (Spinacea oleracea L. cv. Estivato) were grown on nutrient solutions under deficient, normal and excess sulfate supply. In both young and mature plants net uptake of sulfate and its transport to the shoot increased with increasing sulfate supply, but both processes proceeded at a higher rate in young as compared to mature plants. The relative sulfate transport, i.e. the relative amount of the sulfate taken up that is transported to the shoot, decreased with increasing sulfate supply. Apparently, net uptake of sulfate is not strictly controlled by the sulfur demand of the shoot, but xylem loading appears to counteract excess transport of sulfate to the shoot. Fumigation with H₂S or SO₂ reduced net uptake of sulfate by the roots in sulfur-deficient plants and absolute as well as relative sulfate transport to the shoot independent of the three sulfate levels supplied to the plant. At the same time thiol contents of the shoot and the root were enhanced by fumigation with H₂S and SO₂. These findings are consistent with the idea that thiols produced in the leaves can mediate demand-driven control of sulfate uptake by the roots and its transport to the shoot.