Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains.

A total of 34 lactic acid bacteria isolates from 4 different Brazilian kefir grains were identified and characterized among a group of 150 isolates, using the ability to tolerate acidic pH and resistance to bile salts as restrictive criteria for probiotic potential. All isolates were identified by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of representative amplicons. Eighteen isolates belonged to the species Leuconostoc mesenteroides, 11 to Lactococcus lactis (of which 8 belonged to subspecies cremoris and 3 to subspecies lactis), and 5 to Lactobacillus paracasei. To exclude replicates, a molecular typing analysis was performed by combining repetitive extragenic palindromic-PCR and random amplification of polymorphic DNA techniques. Considering a threshold of 90% similarity, 32 different strains were considered. All strains showed some antagonistic activity against 4 model food pathogens. In addition, 3 Lc. lactis strains and 1 Lb. paracasei produced bacteriocin-like inhibitory substances against at least 2 indicator organisms. Moreover, 1 Lc. lactis and 2 Lb. paracasei presented good total antioxidative activity. None of these strains showed undesirable enzymatic or hemolytic activities, while proving susceptible or intrinsically resistant to a series of clinically relevant antibiotics. The Lb. paracasei strain MRS59 showed a level of adhesion to human Caco-2 epithelial cells comparable with that observed for Lactobacillus rhamnosus GG. Taken together, these properties allow the MRS59 strain to be considered a promising probiotic candidate.

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese.

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.

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

The 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.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem.

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.

Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein.

The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase. In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein. This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae. However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment. The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions.

Trehalose accumulation in mutants of Saccharomyces cerevisiae deleted in the UDPG-dependent trehalose synthase-phosphatase complex.

In Saccharomyces cerevisiae, trehalose-6-phosphate synthase converts uridine-5'-diphosphoglucose and glucose 6-phosphate to trehalose 6-phosphate which is dephosphorylated by trehalose 6-phosphatase to trehalose. These two steps take place within a complex consisting of three proteins: trehalose-6-phosphate synthase encoded by the GGS1/TPS1 (= FDP1, = BYP1, = CIF1) gene, trehalose 6-phosphatase encoded by the TPS2 gene and by a third protein encoded by both the TSL1 and TPS3 genes. Using three different methods for trehalose determination, we observed trehalose accumulation in ggs1/tps1delta, tps2delta and tsl1delta mutants, and in the double mutants ggs1/tps1delta/tps2delta and also in ggs1/tps1delta deleted mutants suppressed for growth on glucose. All these mutants harbor MAL genes. Trehalose synthesis in these mutants is probably performed by the adenosine-5'-diphosphoglucose-dependent trehalose synthase, (ADPG-dependent trehalose synthase) which was detected in all strains tested. It is noteworthy that trehalose accumulation in these mutants was detected only in cells grown on weakly repressive carbon sources such as maltose and galactose or during the transition phase from fermentable to non-fermentable growth on glucose. alpha-Glucosidase activity was always present in high amounts. We also describe an adenosine-diphosphoglucosepyrophosphorylase (ADPG-pyrophosphorylase) activity in Saccharomyces cerevisiae which increased concomitantly with the accumulation of trehalose during the transition phase from fermentable to non-fermentable growth in MAL-constitutive (MAL2-8c) strains. The same was observed when MAL-induced (MAL1) strains were compared during growth on glucose and maltose. These results led us to conclude that maltose-induced trehalose accumulation is independent of the UDPG-dependent trehalose-6-phosphate synthase/phosphatase complex; that the ADPG-dependent trehalose synthase is responsible for maltose-induced trehalose accumulation probably by forming a complex with a specific trehalose-6-phosphatase activity and that ADPG synthesis is activated during trehalose accumulation under these conditions.

Kefir Grains Change Fatty Acid Profile of Milk during Fermentation and Storage.

Several studies have reported that lactic acid bacteria may increase the production of free fatty acids by lipolysis of milk fat, though no studies have been found in the literature showing the effect of kefir grains on the composition of fatty acids in milk. In this study the influence of kefir grains from different origins [Rio de Janeiro (AR), Viçosa (AV) e Lavras (AD)], different time of storage, and different fat content on the fatty acid content of cow milk after fermentation was investigated. Fatty acid composition was determined by gas chromatography. Values were considered significantly different when p<0.05. The highest palmitic acid content, which is antimutagenic compost, was seen in AV grain (36.6g/100g fatty acids), which may have contributed to increasing the antimutagenic potential in fermented milk. Higher monounsaturated fatty acid (25.8 g/100g fatty acids) and lower saturated fatty acid (72.7 g/100g fatty acids) contents were observed in AV, when compared to other grains, due to higher Δ9-desaturase activity (0.31) that improves the nutritional quality of lipids. Higher oleic acid (25.0 g/100g fatty acids) and monounsaturated fatty acid (28.2g/100g fatty acids) and lower saturated fatty acid (67.2g/100g fatty acids) contents were found in stored kefir relatively to fermented kefir leading to possible increase of antimutagenic and anticarcinogenic potential and improvement of nutritional quality of lipids in storage milk. Only high-lipidic matrix displayed increase polyunsaturated fatty acids after fermentation. These findings open up new areas of study related to optimizing desaturase activity during fermentation in order to obtaining a fermented product with higher nutritional lipid quality.

Catabolite inactivation of trehalose synthesis during growth of yeast on maltose.

1. The effects of catabolite inactivation upon the trehalose pathway linked to maltose utilization were investigated in Saccharomyces cerevisiae. Mutant strains devoid of UDPG-trehalose synthase activity were used in this study. 2. Trehalose accumulation was also susceptible to catabolite inactivation as has been reported for the carrier protein, one of the components of the maltose system. Reversibility was only achieved when incubation with glucose did not exceed 5 min and was dependent upon protein synthesis.

Periplasmic trehalase from Escherichia coli--characterization and immobilization on spherisorb.

1. Trehalase was partially purified from Escherichia coli and characterized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C. 2. Trehalase represented approximately 50% of the total protein released by osmotic shock. The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others. 3. The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol. 4. Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel). Retention of total catalytic activity averaged 32%. 5. The reactor, stored for one month at -5 degrees C, retained 98% of its initial immobilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose.

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 mol 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.

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 beta-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.

Prevalência de tuberculose bovina em animais e rebanhos abatidos em 2009 no estado de Mato Grosso, Brasil / Prevalence of bovine tuberculosis in herds and animals slaughtered in 2009 in the State of Mato Grosso, Brazil

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.

The prevalence of bovine tuberculosis in cattle, and its herds, slaughtered in 2009 in the state of Mato Grosso, Brazil, was estimated using bacteriological analysis and molecular test, from fragments of injured tissues as well as direct DNA templates. 41,193 cattle, which appeared healthy in the ante mortem examination, from seven selected slaughterhouses, under Brazilian federal inspection services (SIF), were inspected. The animals were from 492 herds located in 85 (60%) different cities of Mato Grosso. A total of the 198 carcasses had suspicious lesions. Three carcasses (3/198) had lesions that were found to be tuberculous in laboratory diagnosis. The apparent prevalence of bovine tuberculosis in the total number of animals and in herds slaughtered in Mato Grosso was 0.007% [IC 95% = -0.001%; 0.016%] and 0.61% [IC 95% = -0.08%; 1.30%], respectively. The sanitation status demonstrated in Mato Grosso indicates the progress in this state toward the eradication of bovine tuberculosis.

Identification of an ADPG-dependent trehalose synthase in Saccharomyces.

Uridine diphosphoglucose is not the sole donor for trehalose synthesis in yeast cells: an ADPG-dependent trehalose synthase, has been identified in mutant strains with undetectable UDPG-dependent trehalose-6-P synthase activity. Genetic and chromatographic studies indicate that the two activities correspond to different proteins. The apparent Km for the nucleotide is similar for both enzymes, and Mg2+ is also required for both activities; however, a striking difference was observed with respect to ATP.Mg activation. This newly determined enzymatic activity in Saccharomyces clarifies previous contradictory results with mutant strains that are able to accumulate trehalose during growth yet whose UDPG-dependent trehalose synthase activity is undetectable in vitro.

Trehalase immobilization on aminopropyl glass for analytical use.

Trehalase is the enzyme which hydrolyzes the disaccharide trehalose into two alpha-D-glucose molecules. In this article, we present the immobilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 plasmid, which contains the trehalase gene. The partially purified enzyme had a specific activity of 356 U/mg and could be used for quantifying trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent coupling with retention of its catalytic activity. The support chosen for the majority of the experiments reported was aminopropyl glass, although spherisorb-5NH(2) and chitin were also tested. The immobilized enzyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, and no release of enzyme molecules was detected during this procedure. The best condition found for storing the enzyme-support complex was at 4 degrees C in the presence of 25 mM sodium maleate, containing 7 mM beta-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50% glycerol. The enzyme under these conditions was stable, retaining approximately 100% of its initial activity for at least 28 days. The immobilized enzyme can be employed to detect trehalose molecules in micromolar concentration. The optimum pH value found was 4.5 and the K(m) app. 4.9 x 10(-3) M trehalose at pH 4.6 and 30 degrees C, with V(max) of 5.88 micromol glucose x min x(-1), as calculated by a Lineweaver-Burk plot.

Ca2+/calmodulin-binding proteins in yeast. Catabolite repression and induction by carbon sources.

Soluble calmodulin-binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non-fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose-grown cells. Among the calmodulin -binding polypeptides, eight were phosphorylated in a Ca2+/calmodulin -dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [gamma-32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.

Further evidence for the alternative pathway of trehalose synthesis linked to maltose utilization in Saccharomyces.

Yeast strains bearing a deficiency in trehalose-6-phosphate synthase activity are unable to accumulate trehalose on any carbon source unless they contain one of the MAL genes. If the gene is inducible then synthesis of trehalose occurs specifically during growth on maltose: when the MAL gene is constitutive then trehalose accumulation can also be seen when cells are grown on glucose. Different systems for trehalose synthesis were suggested: one of them would require the UDPG-linked trehalose synthase whereas the second would utilize an alternative pathway. We proposed a mechanism by which the gene-product of a MAL gene would serve as a common positive regulator for the expression of the genes coding for maltose permease, alpha-glucosidase and some component of the trehalose accumulation system. In order to elucidate this novel pathway a strain lacking UDPG-linked trehalose synthase activity and harboring a defect in maltose uptake was constructed. Excessive maltose uptake resulted in accumulation of intracellular maltose, and twice as much trehalose as in a control strain. Partial inhibition of hexokinase by xylose affected the ratio between internal maltose and trehalose and significantly reduced glycogen synthesis. Sodium fluoride also blocked glycogen synthesis but allowed for trehalose accumulation. Moreover, a mutant which lacks hexokinase I and II was unable to accumulate trehalose when grown on glucose in spite of the presence of a constitutive MAL2 gene. These results suggest that trehalose synthesis would require G-6-P formation derived from maltose. Such a deviation would allow for slowing down the glycolytic flux which, in turn, would favour efficient maltose utilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.

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 percent 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 percent 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.