• Volume 23, Issue 4

      October 1998,   pages  285-531

    • Preface

      S C Lakhotia A K Tripathi

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    • Heat induced stress proteins and the concept of molecular chaperones

      Christoph Forreiter Lutz Nover

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      Heat stress proteins can be assigned to eleven protein families conserved among bacteria, plants and animals. Most of them aid other proteins to maintain or regain their native conformation by stabilizing partially unfolded states. Hence, they are called molecular chaperones. Experimental data indicate that many of them form heterooligomeric complexes, so-called chaperone machines, interacting with each other to generate a network for maturation, assembly and intracellular targeting of proteins. In this review we summarize the essential information on the structure and function of chaperone and chaperone complexes. In addition we present a compilation ofin vivo andin vivo test systems used in the preceding ten years of chaperone research.

    • Biochemical events in the activation and attenuation of the heat shock transcriptional response

      Sanjeev H Satyal Richard I Morimoto

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      The expression of heat shock proteins in response to cellular stress is mediated by a family of heat shock transcription factors (HSFs). The transcriptional activity of these HSFs is regulated by multiple redundant regulatory mechanisms which ensure the fine tuned expression of heat shock genes. These mechanisms include cis-regulatory domains and trans-acting proteins which modulate HSF activity and control the heat shock response. Heat shock gene expression is also regulated by selective use of various HSFs under distinct developmental and growth conditions.

    • Heat stress response and heat stress transcription factors

      Klaus-Dieter Scharf Ingo Höhfeld Lutz Nover

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      Expression of heat shock protein (HSP)-coding genes is controlled by heat stress transcription factors (Hsfs). They are structurally and functionally conserved throughout the eukaryotic kingdom. In addition to the DNA-binding domain with the helix-turn-helix motif essential for DNA recognition, three functional parts in the C-terminal activator domain were characterized: (i) the HR-A/B region is responsible for oligomerization and activity control, (ii) the nuclear localizing signal (NLS) formed by a cluster of basic amino acid residues which is required and sufficient for nuclear import and (iii) short C-terminal peptide motifs with a central Trp residue (AHA elements). These three parts are indispensible for the activator function. A peculiaritiy of plants is the heat shock-inducible new synthesis of Hsfs. In tomato HsfA1 is constitutively expressed, whereas Hsfs A2 and B1 are heat shock-inducible proteins themselves. We used Hsf knock-out strains of yeast and transient reporter assays in tobacco protoplasts for functional analysis of Hsf-coding cDNA clones and mutants derived from them. HsfA2, which in tomato cell cultures is expressed only after heat shock induction, tends to form large cytoplasmic aggregates together with other HSPs (heat stress granules). In the transient expression assay its relatively low activator potential is evidently due to the inefficient nuclear import. However, the intramolecular shielding of the NLS can be released either by deletion of a short C-terminal fragment or by coexpression with HsfA1, which forms hetero-oligomers with HsfA2.

    • Modulation of polymerase II composition: A possible mode of transcriptional regulation of stress response in eukaryotes

      Parag Sadhale Nimisha Sharma P Beena Aparna Katoch Narottam Acharya Sanjay K Singh

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      Regulation of stress response in prokaryotes is mainly achieved at the transcriptional initiation level. Prokaryotes use alternative holoenzymes, consisting of the core polymerase associated with different sigma factors, which confer on it altered specificity of transcriptional initiation. Stress response being probably one of the most inevitable features of life, it would be interesting to find if eukaryotes also use a similar strategy at this level of regulation. Since the yeastSaccharomyces cerevisiae is a model system for studying many different phenomena in eukaryotes we review the transcriptional regulation of stress in this system. Based on published observations in the literature and our own studies, we have analysed the regulation of stress response, in the yeastS. cerevisiae. Two of the core subunits of the yeast RNA polymerase II, which show altered stoichiometry within the polymerase under different conditions appear to be involved specifically in regulating the stress response. In a very broad sense then, the altered subunit composition of the core polymerase or a different holoenzyme, appears to correlate with gene expression specific to stress response inS. cerevisiae and probably reflects the scenario in other eukaryotes.

    • Plant Hsp100 family with special reference to rice

      Sneh Lata Singla Ashwani Pareek Anil Grover

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      Heat shock proteins (Hsps) represent a group of specific proteins which are synthesized primarily in response to heat shock in almost all biological systems. Members of Hsp100 family have been directly implicated in induction of thermotolerance in microbial and animal cells. Yeast cells harbouring defectivehsp104 gene do not show thermotolerance under conditions in which the normal cells do. Several plant species have been shown to synthesize Hsps in the range of 100 kDa. Rice Hsp104 (OsHsp104) is rapidly and predominantly accumulated in heat-shocked cells. Western blotting analysis show that anti rice Hsp104 antibodies (generated against purified Hsp104 protein from cultivated riceOryza sativa L.) cross-react with the same-sized high temperature inducible protein in 15 different wild rices. It was further found that anti rice Hsp104 antibodies also cross-react with a major high temperature regulated protein ofEscherichia coli. We have previously shown that a 110 kDa stress regulated protein in rice (OsHsp110) is immunologically related to yeast Hsp104 protein. In this paper, we present a comparative account of characteristics of the OsHsp104 and OsHsp110 proteins.

    • Characterization of the nucleotide binding properties of the 90 kDa heat shock protein (Hsp90)

      Csaba Sőti Péter Csermely

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      The recent crystallization and structural analysis of the ATP(ADP)-complex of the N-terminal domain of the 90 kDa heat shock protein (Hsp90) confirmed our earlier findings on the ATP-binding properties of Hsp90. Here we further characterize the nucleotide binding of Hsp90 by demonstrating that surface plasmon resonance measurements also indicate a low-affinity binding of ATP to Hsp90 and that [α-32P]ATP seems to have an equal preference for monomers, dimers and oligomers of Hsp90 on native polyacrylamide gels. Finally we discuss some of our results which raise the possibility that Hsp90 has two nucleotide binding sites (one in its N-terminal and another in the C-terminal domain) and that the nucleotide binding to Hsp90 dimers may display a positive cooperativity under some special conditions. The submillimolar binding affinity of ATP to Hsp90 allows the regulation of some Hsp90-related functions just in the range of ATP-level fluctuations during stress or during the cell cycle.

    • Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

      Jayanta K Pal

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      Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.

    • Plant Hsp90 family with special reference to rice

      Ashwani Pareek Sneh Lata Singla Anil Grover

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      Hsp90 family represents a group of highly conserved and strongly expressed proteins present in almost all biological species. Heat shock proteins in the range of 90 kDa have been detected in a range of plant species andhsp90 genes have been cloned and characterized in selected instances. However, the expression characteristics of plant Hsp90 are poorly understood. Work on expression characteristics of rice Hsp90 is reviewed in this paper. Experimental evidence is provided for indicating that while the rice 87 kDa protein is transiently synthesized within initial 2 h of heat shock, high steady-state levels of this protein are retained even under prolonged high temperature stress conditions or recovery following 4 h heat shock. It is further shown that fifteen different wild rices accumulate differential levels of these proteins in response to heat shock treatment.

    • Small heat shock proteins ofDrosophila: Developmental expression and functions

      D R Joanisse S Michaud Y Inaguma R M Tanguay

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      In addition to their induced expression under various stress conditions, small heat shock proteins (sHsps) are also expressed during normal development in a wide array of organisms. Members of the sHsp family ofDrosophila melanogaster are individually expressed at many stages of development, and their developmental expression contrasts sharply with their stress-induced expression. First, the developmental expression of sHsps is uncoordinated and each of the sHsps shows its own pattern of expression. Secondly this expression is highly regulated in a tissue-, cell lineage- and/or developmental stage-specific manner. An example of such regulation is during male gametogenesis when Hsp23, Hsp26 and Hsp27 are expressed in the absence of stress. However the expression of Hsp23 is restricted to cells of the somatic lineage while Hsp27 is mainly expressed in germ line cells and in some somatic cells. Heat shock does not alter the level nor the specificity of expression of these sHsps in this tissue while other HSPs, i.e., Hsp22 and Hsp70, are induced through signaling via the unique heat shock transcription factor (HSF). The HSF is shown to be present at a much lower level in testes than in other tissues and shows cell-specific distribution. The highly regulated expression of sHsps during development and differentiation is dependent on transcription factors other than the HSF. The specific expression patterns of individual sHsps suggest that these proteins may fulfill distinct functions during normal development. A search for partners of sHsps suggests that these proteins may function in cell proteolytic processes.

    • Interaction of the non-protein-coding developmental and stress-induciblehsrω gene withRas genes ofDrosophila melanogaster

      Pritha Ray S C Lakhotia

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      The93D orhsrω (hsr-omega) is an unusual non-protein-coding gene which shows a dynamic developmental expression in most cell types ofDrosophila melanogaster and which, besides being a member of the heat shock gene family, is uniquely induced in polytene cells by a variety of amides. We briefly review the various aspects of this gene's organization, regulation and inducible properties and present our recent data to show that, similar to our earlier report of interaction between thehsrω andhsp83 genes, mutations at theRas1 or theRas3 gene ofD. melanogaster also dominatly enhance the pre-pupal and pupal lethality of embryos that are nullosomic for this gene. In the absence of any protein product, thehsrw transcripts are suggested to interact with the Ras 1 signaling cascade. The interaction of a non-translatable, developmentally produced as well as heat and other stress inducible RNA with the Ras signaling proteins and with the Hsp83 chaperone protein opens new possibilities for understanding of functions of this intriguing gene.

    • Mechanism of heat induction of albumin in early embryonic rat liver

      Usha K Srinivas S K Swamynathan C J Revathi

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      Albumin is an adult liver specific protein whose induction in rats starts at day 19 or 20 of normal gestation. Our studies on the effect of heat stress on embryonic development showed premature induction of a 67 kDa protein at day 12 or 13 in embryonic liver cells, in addition to the induction of usual heat shock proteins. Immunoblotting with anti-albumin antibody confirmed the prematurely induced protein to be albumin. RNA dot blot showed that albumin induction upon heat shock is regulated at transcriptional level and northern blot confirmed the size of heat induced albumin transcript to be similar to the constitutively induced albumin RNA transcript.

      During heat stress, heat shock proteins are induced by the interaction of a specific heat shock transcription factor (HSF) with specific DNA sequences (heat shock elements, HSEs) present in the promoters of all heat shock genes.

      The functional significance of HSF-HSE interaction is confirmed by transient transfection assays using plasmids carrying chloramphenicol acetyl transferase reporter gene under the control of different deletion fragments of the rat albumin promoter. These assays identified the HSEs to be within −450 base pairs of the rat albumin promoter. Deletion of these HSE sequences from rat albumin promoter abolished its heat inducibility. Electrophoretic mobility shift assays with synthetic oligonucleotides, representing putative HSEs in the rat albumin promoter, and H4II-E-C3 cell extracts showed that the heat shock factor binds this region in a sequence specific and reversible manner. Gel super-shift assays with antibodies to HSF1 and HSF2 demonstrated that the HSEs present in the rat albumin promoter are bound by HSF1 but not by HSF2.

      In addition to the HSEs, we have identified a putative GAGA factor binding site in the rat albumin promoter at −228 bp to −252 bp position. These GAGA repeats are bound in a sequence-specific and reversible manner by two factors in non-stressed cells, whereas only one of these two factors continues to bind the GAGA repeats under heat shock conditions.

      We thus show that rat albumin promoter contains (i) functional HSEs to which the HSF1 binds and (ii) GAGA factor binding sites to which the GAGA factor binds and that the promoter activity can be modulated by temperature.

    • Expression and possible role of stress-responsive proteins inAnabaena

      S K Apte T Fernandes H Badran A Ballal

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      Nitrogen-fixingAnabaena strains offer appropriate model systems to study the cellular and molecular responses to agriculturally important environmental stresses, such as salinity, drought and temperature upshift. Sensitivity to stresses results primarily from reduced synthesis of vital cellular proteins such as phycocyanin and dinitrogenase reductase leading to impairment of photosynthesis and nitrogen fixation. Exposure to stresses induces the synthesis of a large number of general stress proteins and a few unique stress-specific proteins through transcriptional activation of stress-responsive genes. Using a subtractive RNA hybridization approach a large number of osmoresponsive genes have been cloned fromAnabaena torulosa. The expression of general stress proteins has been shown to form the basis of adaptation and cross-protection against various stresses inAnabaena. Prominent among such proteins are the K+-scavenging enzyme, KdpATPase, and the molecular chaperone, GroEL. Unlike heterotrophs, carbon starvation does not appear to evoke a global stress response inAnabaena. Supplementation of combined nitrogen or K+ improves inherent tolerance ofAnabaena strains to many environmental stresses.

    • Conserved function and regulation ofσ32 homologues in Gram-negative bacteria

      K Nakahigashi M Kanemori M Morita H Yanagi T Yura

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      The heat shock response inEscherichia coli and related bacteria is primarily mediated byσ32 or its homologue (RpoH protein) specifically required for transcription of heat shock genes encoding molecular chaperones and proteases. Extensive work inE. coli revealed some of the mechanisms controlling the cellular level and activity ofσ32 during the heat shock response. Recent isolation of a number of RpoH homologues from γ, β and α proteobacteria provided an opportunity to examine evolutionary conservation and diversity of regulatory mechanisms in these bacteria. We here summarize the present status of this aspect of the stress response not only by comparative sequence analysis but by examining the response of representative RpoH homologues of the γ subgroup to heat shock stress. Current evidence indicates that the basic strategy of enhancing RpoH level as a primary response to heat shock stress is well conserved, but the detailed mechanisms for enhancement of the heat shock σ factor level vary among different species that may reflect diverse ecological niches.

    • The GroE chaperonin machine is the major modulator of the CIRCE heat shock regulon ofBacillus subtilis

      Wolfgang Schumann Georg Homuth Axel Mogk

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      Regulation of the heat shock response in bacteria has been studied extensively inEscherichia coli where heat shock genes are classified into three classes and where each class is regulated by a different alternate sigma factor.Bacillus subtilis serves as a second model bacterium to study regulation of the heat shock response in detail. Here, four classes of heat shock genes have been described so far where two are controlled by two different repressor proteins and the third by the alternate sigma factorσB. Class I heat shock genes consists of two operons, the heptacistronicdnaK and the bicistronicgroE operon. Transcription of thednaK operon is complex involving two promoters, premature termination of transcription, mRNA processing and different stabilities of the processed transcripts to ensure the appropriate amounts of heat shock proteins under different growth conditions. The translation product of thehrcA gene, the first gene of thednaK operon, binds to an operator designated CIRCE element, and its activity is modulated by the GroE chaperonin system. We assume that the HrcA protein, uponde novo synthesis and upon dissociation from its operator, is present in an inactive form and has to be activated by the GroE chaperonin system resulting in an HrcA-GroE reaction cycle. Induction of class I heat shock genes occurs by the appearance of denatured proteins within the cytoplasm which titrate the GroE system. This results in accumulation of inactive HrcA repressor and thereby in induction of class I heat shock genes. Upon removal of the non-native proteins from the cytoplasm, the GroE chaperonin will interact with HrcA and promote folding into its active conformation resulting in turning off of class I heat shock genes. This mechanism ensures adequate adjustment of class I heat shock proteins depending on their actual need.

    • Adaptation to low temperature and regulation of gene expression in antarctic psychrotrophic bacteria

      Malay K Ray G Seshu Kumar Kamala Janiyani K Kannan Pratik Jagtap Malay K Basu S Shivaji

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      Exposure to extremes of temperatures cause stresses which are sometimes lethal to living cells. Microorganisms in nature, however, are extremely diverse and some of them can live happily in the freezing cold of Antarctica. Among the cold adapted psychrotrophs and psychrophiles, the psychrotrophic bacteria are the predominant forms in the continental Antarctica. In spite of living in permanently cold area, the antarctic bacteria exhibit, similar to mesophiles, ‘cold-shock’ response albeit at a much lower temperatures, e.g., at 0–5°C. However, because of permanently cold condition and the long isolation of the continent, the microorganisms have acquired new adaptive features in the membranes, enzymes and macromolecular synthesis. Only recently these adaptive modifications are coming into light due to the efforts of various laboratories around the world. However, a lot more is known about adaptive response to low temperature in mesophilic bacteria than in antarctic bacteria. Combined knowledge from the two systems is providing useful clues to the understanding of basic biology of low temperature growing organisms. This article will provide an overview of this area of research with a special reference to sensing of temperature and regulation of gene expression at lower temperature.

    • Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

      Arne R Strøm

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      Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.

    • Stress responses ofBacillus subtilis to high osmolarity environments: Uptake and synthesis of osmoprotectants

      Bettina Kempf Erhard Bremer

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      A decrease in the water content of the soil imposes a considerable stress on the voil-living bacteriumBacillus subtilis: water exits from the cells, resulting in decreased turgor and cessation of growth. Under these adverse circumstances,B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated viade novo synthesis, butB. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute ofB. subtilis is the potent osmoprotectant glycine betaine. This trimethylammonium compound can be taken up by the cell through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment,B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence ofB. subtilis against high osmolarity stress.

    • Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

      M Østeras E Boncompagni A Lambert L Dupont M C Poggi D Le Rudulier

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      To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.

    • Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

      A K Tripathi B M Mishra P Tripathi

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      In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.

    • Genetic engineering of polyamine and carbohydrate metabolism for osmotic stress tolerance in higher plants

      M V Rajam S Dagar B Waie J S Yadav P A Kumar F Shoeb R Kumria

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      Plant growth and productivity are greatly affected by various stress factors. The molecular mechanisms of stress tolerance in plant species have been well established. Metabolic pathways involving the synthesis of metabolites such as polyamines, carbohydrates, proline and glycine betaine have been shown to be associated with stress tolerance. Introduction of the stress-induced genes involved in these pathways from tolerant species to sensitive plants seems to be a promising approach to confer stress tolerance in plants. In cases where single gene is not enough to confer tolerance, metabolic engineering necessitates the introduction of multiple transgenes in plants.

    • Regulation of iron uptake minimizes iron-mediated oxidative stress

      V Braun

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      Sequential accommodation of single electrons by the unpaired orbitals of dioxygen yields oxygen radicals, hydrogen peroxide (H2O2), hydroxide radicals (OH·), and finally water (H2O). Fe2+ catalyses the formation of the most reactive hydroxide radical from hydrogen peroxide and thus contributes substantially to the toxicity of oxygen. Insolubility of Fe3+ demands the incorporation of iron into transferrin, lactoferrin, ferritin, iron-sulphur clusters, and heme. Bacteria and fungi synthesize low molecular weight compounds, termed siderophores, which are secreted and used to transport Fe3+ into the microbial cells. Iron is economically used and iron toxicity is minimized by the synthesis of siderophores and ferric siderophore transport systems, and by induction of transport gene transcription by certain Fe3+-loaded siderophores. When cells contain sufficient iron, Fe2+-loaded Fur protein and Fe2+-loaded DtxR protein repress gene transcription in Gram-negative bacteria and in most Gram-positive bacteria, respectively. In a recently discovered novel transcription control mechanism, ferric citrate and ferric pseudobactins induce transcription of the iron transport systems by binding to cell surface receptor proteins without entering the cells. Cytoplasmic sigma factors are activated by a signaling device that involves a protein in the outer membrane and a protein in the cytoplasmic membrane. Both proteins extend into the periplasm to transduce the signal through the space between the two membranes. Intracellular iron homeostasis secured by regulation of iron uptake prevents excessive oxidative stress, which could otherwise overcome the cellular defence and repair systems and kill the cells.

    • Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

      H Ohtake J Kato A Kuroda H Wu T Ikeda

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      Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (Pi), as the preferred P source, have evolved complex systems to survive during Pi starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to Pi. The evidence that the chemotactic response to Pi (Pi taxis) was observed only with cells grown in Pi-limiting medium suggests that Pi taxis plays an important role in scavenging Pi residues under conditions of Pi starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when Pi is added to cells previously subjected to Pi starvation stress. Since polyP can serve as a P source during Pi starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during Pi starvation. In the present review, we summarize our current knowledge on regulation of bacterial Pi taxis and polyP accumulation in response to Pi starvation stress.

    • Cycles of famine and feast: the starvation and outgrowth strategies of a marineVibrio

      Sujatha Srinivasan Staffan Kjelleberg

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      Studies of starvation survival in non-differentiating bacteria have largely focused on physiological changes and regulatory aspects of a few master regulators such as the signal molecule ppGpp and the stationary phase alternative sigma factor, sigma S. Recent findings have implicated a series of novel key events for the entry as well as exit from starvation. The importance of alternative sigma factors other than sigma S is emerging. In addition, low molecular weight extracellular signals have been demonstrated to be essential for the induction and mediation of several adaptive responses. The importance of mRNA modification and stability for starvation survival as well as outgrowth is receiving renewedinterest. In this paper, we present the results obtained from studies of starvation survival and recovery ofVibrio sp. strain S14.

    • Stress-inducible transgenic nematodes as biomonitors of soil and water pollution

      R S Power H E David M H A Z Mutwakil K Fletcher C Daniells M A Nowell J L Dennis A Martinelli R Wiseman E Wharf D I de Pomerai

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      This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations. Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes is discussed.

    • Heat shock protein-peptide interaction as the basis for a new generation of vaccines against cancers and infectious diseases

      Pramod K Srivastava

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      Heat shock proteins (HSPs) are associatedin vivo with the entire repertoire of peptides (antigenic and otherwise) generated within that cell. Immunization with such HSP-peptide complexes is unusually efficient in eliciting cellular immune responses against the antigenic peptides associated with the HSPs. This broad and general principle is the basis for a new generation of vaccines against cancers and infectious diseases and circumvents the need for identification of the T cell epitopes for any given cancer or infectious agent.

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