In Bacillus subtilis stress proteins are induced in response to different environmental conditions such as heat shock, salt stress, glucose and oxygen limitation or oxidative stress. These stress proteins have been previously grouped into general stress proteins (Gsps) and heat-specif ic stress proteins Abteilung Mikrobiologi Universitilt Osnabrilck, 49076 Osnabrilck, Germany e, (Hsps). In this investigation the N-terminal sequences of 13 stress proteins of B. subtilis were determined. The quantification of the mRNA and the analysis of the protein synthesis pattern support the initial hypothesis that the chaperones DnaK and GroEL are Hsps in B. subtilis. In contrast, the recently described proteins GsiB, Ctc and RsbW belong to a class of Gsps that are induced by various stresses including heat shock. The main part of the Gsps described in this study failed to be induced in the sigB deletion mutant ML6 in response to heat shock. However, all the five Hsps were induced in this mutant in response to heat shock. These data indicate that SigB plays a crucial role in the induction of general stress genes, but is dispensable for the induction of Hsps.

The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated. Immediately after upshock the cells took up a large amount of K+ and synthesized approximately half the equivalent amount of glutamate concomitantly. After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate. Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells. The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmol x min-1 x g-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells. These results indicate that for osmoadaptation of growing cells of E. coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K+ and glutamate as osmoprotectants. We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K+ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation. Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E. coli. However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).

Bacteria respond to a decrease in temperature with the induction of proteins that are classified as coldinduced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15؇C, the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, IlvC, Gap, and triosephosphate isomerase).Bacteria must adapt to continuous changes in the environment, such as changes in the availability of nutrients or oxygen. Invariably, the response to drastic chemical and physical changes in the surroundings involves the induction of sets of specific proteins. This has been shown for various environmental stresses, including an increased salt concentration, a rise in temperature (heat shock), phage infection, or ethanol treatment (1a, 12, 17, 56). The functions and regulation of some stress-induced proteins, particularly the well-characterized heat shock proteins that act as molecular chaperones (61), have been elucidated. However, little is known about the functions of proteins induced after a decrease in temperature. A cold shock response has been found in Escherichia coli (25), and the induction of proteins in response to cold shock was monitored in Bacillus (30,58,60), Listeria (41), and Rhizobium (10) species. In E. coli, the number of proteins synthesized decreases drastically after a shift from 37 to 10ЊC, and cellular growth arrests. After 2 h, only 28 proteins are detectably produced, 14 at rates higher than preshift rates. Three hours after a cold shock, the number of proteins increases again, until after 4 h, normal protein synthesis and growth are resumed. Cold-induced proteins (CIPs) include mainly transcriptional and translational proteins, components of the pyruvate dehydrogenase complex, and CspA, the only protein that is not present at 37ЊC. Most CIPs are synthesized at a 2-to 10-fold higher level compared with preshift levels, in contrast to CspA, which is induced about 200-fold (25). Other proteins that are continually synthesized after a cold shock have been identified as ribosomal proteins L7, L12, S1, S6B, and S6A, trigger factor, elongation factor Tu (EF-Tu), EF-Ts, EF-G, and the ␤ subun...

High-affinity progesterone-binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non-denaturing zwitterionic detergent 3-[(3-~holamidopropyl)dimethylammonio]-l -propanesulfonic acid (Chaps, 20 mM, detergent/protein mass ratio 4 : l ) at a yield of 75-80%. Using [3H]progesterone as radioligand, binding studies showed high-affinity and low-affinity binding sites in microsomal preparations with an apparent Kdl of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high-affinity binding sites were present at an apparent Kd of 69 nM. In both preparations, progesterone binding was time-dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion-exchanger procedures. The purified fraction as identified by maximum specific progesterone-binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.Keywords: progesterone ; membrane-binding site ; liver; amino acid sequence.For the past decade, nonclassical actions of steroid hormones and related signal transduction pathways have gained increasing scientific interest. Evidence for nongenomic steroid effects are provided for all classes of steroid hormones including the secosteroid vitamin D3 and triiodothyronine (for review see [I, 21).As an example, the sex hormone progesterone has been found to act on oocyte maturation in a nontranscriptional manner in Xenopus laevis 131. An important instant effect of progesterone is the rapid stimulation of ion fluxes in human sperm. Blackmore et al. [4, 51 showed a rapid Ca2+ and Turner and Meizel [6] demonstrated CI-effluxes during the acrosome reaction induced by Progesterone. Moreover, in hepatocytes a rapid progesterone-induced increase of cytosolic CaZ+ was seen resulting from Caz+ influx [7].Another prominent example for a nongenomic steroid effect is the rapid stimulation of Na+/H ' -exchanger in human mononu- So far, none of these membrane steroid-binding proteins has been purified in sufficient amounts to allow molecular analysis and cloning; it appears that the lack of a satisfactory solubilization procedure for these labile proteins is one of the major obstacles in this regard. Here, progesterone-specific binding proteins are identified and characterized in porcine liver microsomes, solubilized, purified and partially sequenced from the N-terminus. Materials and Methods

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of a t least 42 general stress proteins absolutely required the alternative sigma factor oB. However, a t least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stressinducible in a sigB mutant. Such a detailed analysis also permitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response. The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon. Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specif ic proteins is shown and discussed.

The alkaline shock protein Asp23 was identified as a sigmaB-dependent protein in Staphylococcus aureus. In Bacillus subtilis, the asp23 promoter from S. aureus is regulated like other sigmaB-dependent promoters, which are strongly induced by heat and ethanol stress. However, almost no induction of asp23 expression was found after heat or ethanol stress in S. aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high. Under the same experimental conditions the sigmaB gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress. In contrast, sigmaB activity was increased by heat stress when the cells were grown in a complex medium. The constitutive expression of sigB and sigmaB-dependent stress genes in S. aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of sigmaB activity in B. subtilis, and needs further investigation. A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S. aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate sigmaB in complex medium, and thus reduce the initiation of transcription at sigmaB-dependent promoters in this strain.

Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the cholineglycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline-or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

Cells of the non-diazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 acclimate to nitrogen deprivation by differentiating into non-pigmented resting cells, which are able to survive prolonged periods of starvation. In this study, the physiological properties of the long-term nitrogen-starved cells are investigated in an attempt to elucidate the mechanisms of maintenance of viability. Preservation of energetic homeostasis is based on a low level of residual photosynthesis; activities of photosystem II and photosystem I were approximately 0.1% of activities of vegetatively growing cells. The low levels of photosystem I activity were measured by a novel colorimetric assay developed from the activity staining of ferredoxin:NADP ϩ oxidoreductase. Photosystem II reaction centers, as determined by chlorophyll fluorescence measurements, exhibited normal properties, although the efficiency of light harvesting was significantly reduced compared with that of control cells. Long-term chlorotic cells carried out protein synthesis at a very low, but detectable level, as revealed by in vivo [35 S]methionine labeling and two-dimensional gel electrophoresis. In conjunction with the very low levels of total cellular protein contents, this implies a continuous protein turnover during chlorosis. Synthesis of components of the photosynthetic apparatus could be detected, whereas factors of the translational machinery were stringently downregulated. Beyond the massive loss of protein during acclimation to nitrogen deprivation, two proteins that were identified as SomA and SomB accumulated due to an induced expression following nitrogen reduction.