Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 µmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast

Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes

AbstractThe microbial community composition and chemical characteristics of a Brazilian milk kefir sample produced during its manufacturing and refrigerated storage were investigated by culture-dependent and -independent methods and HPLC. Lactococcus lactis ssp. cremoris and ssp. lactis, Leuconostoc mesenteroides, Acetobacter lovaniensis, and Saccharomyces cerevisiae were isolated, whereas the detected bands on denaturing gel gradient electrophoresis corresponded to Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus parakefiri, and S. cerevisiae. After fermentation, lactic acid bacteria were present at levels of 10 log units, whereas acetic acid bacteria and yeast were present at levels of 7.8 and 6 log units, respectively. The lactic acid bacteria and yeast counts remained constant, whereas acetic acid bacteria counts decreased to 7.2 log units during storage. From fermentation to final storage, the pH, lactose content and citric acid of the kefir beverage decreased, followed by an increase in the concentrations of glucose, galactose, ethanol, and lactic, acetic, butyric, and propionic acids. These microbiological and chemical characteristics contribute to the unique taste and aroma of kefir. This research may serve as a basis for the future industrial production of this beverage in Brazil

Prevalência de tuberculose bovina em animais e rebanhos abatidos em 2009 no estado de Mato Grosso, Brasil

Estimou-se a prevalência de tuberculose em bovinos, e em seus respectivos rebanhos, abatidos em 2009 no estado de Mato Grosso, utilizando como diagnóstico confirmatório o exame bacteriológico e o molecular a partir de fragmentos de tecidos lesionados. Nos sete abatedouros selecionados, detentores de serviço de inspeção federal (SIF), foram inspecionados 41.193 bovinos, sadios ao exame ante mortem, procedentes de 492 rebanhos originários de 85 (60%) municípios mato-grossenses. Um total de 198 carcaças apresentaram lesões suspeitas. Apenas três carcaças (3/198) apresentaram lesões confirmadas como tuberculosas pelos diagnósticos laboratoriais. A prevalência aparente de tuberculose bovina em animais e rebanhos abatidos no estado de Mato Grosso foi de 0,007% [IC 95% = -0,001%; 0,016%] e 0,61% [IC 95% = -0,08%; 1,30%], respectivamente. O estado do Mato Grosso possui, naturalmente, um status sanitário considerado de baixa prevalência