Response of the estuarine cyanobacterium <Emphasis Type="Italic">Lyngbya aestuarii</Emphasis> to UV-B radiation

Growth response and changes in the spectral properties of methanolic extract of an estuarine cyanobacterium, Lyngbya aestuarii Agardh, to UV-B radiation were studied. Increase in growth accompanied by increase in chlorophyll a, protein and carbohydrate content was observed up to 12 h of UV-B irradiation followed by a decline with further increase in the duration of UV exposure. Carotenoid content progressively increased with the UV-B dose. The organism synthesized, to a significant extent, mycosporine amino acid-like substances (MAAs) upon UV-B exposure. The cells in the trichome became coiled followed by formation of small bundles as a response to UV-B radiation. SDS protein profile of the UV irradiated cells showed repression of 20 and 22 kDa proteins. However, irradiation with UV-B for 6–24 h led to overproduction of 84, 73, 60, 46, 40, 37 KDa proteins, possibly conferring protection to the organism from UV-B. UV irradiated cells cultured in florescent light for up to 7 days showed revival from UV damage of the pigments and macromolecular contents, suggesting existence of a repair mechanism in the organism.     

Sublethal stress: Impact of solar UV radiation on protein synthesis in the copepod Acartia tonsa

Aquatic organisms respond to environmental challenges such as thermal stress with the rapid induction of highly conserved polypeptides known as stress proteins or heat shock proteins (Hsps). Solar ultraviolet radiation (UVR, 280-400 nm) is an important environmental stressor in marine ecosystems. Here, we present results of experiments conducted with the marine copepod Acartia tonsa to follow the de novo protein synthesis and measure the level of constitutive and inducible isoforms of the Hsp70 gene family of stress proteins after UV exposure. Animals were collected from Tampa Bay, Florida (USA), and exposed to solar radiation (full spectrum), UV-A (320-400 nm) and PAR (400-700 nm), or PAR only, for periods of 0.5-4 h. Controls were kept in the dark. Protein synthesis was robust under all treatments when the copepods were exposed to low solar radiation intensities. Conversely, high solar radiation intensities (both UV-B and UV-A) caused an overall suppression in the protein synthesis of the copepods with no detectable induction of stress-inducible isoforms of Hsps. Immunochemical assays (western blotting) showed that UVR increased levels (3.5-4-fold increase compared to the dark control) of the constitutively expressed 70 kDa heat-shock (Hsc70) protein in A. tonsa, without indication of inducible isoform upregulation.     

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.     

The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity

The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.     

MSMEG_4626 ribonuclease from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The MSMEG_4626 gene was cloned from Mycobacterium smegmatis MC2 155. It codes for a protein of 1,037 amino acids, identified as ribonuclease E by matching to the protein family HMM TIGR00757. The protein was expressed and purified. Although its calculated molecular weight is 112.7 kDa, it has an aberrant mobility in SDS-polyacrylamide gels, like other ribonuclease E enzymes (it migrates as a 180 kDa protein). The central part of the protein displays high similarity to the catalytic domains of other RNase E enzymes. Mass spectrometric analysis revealed the presence of the chaperonin GroEL, ribosomal proteins, a negative regulator of genetic competence and GTP pyrophosphokinase in the affinity-purified preparation. It is a very unstable protein; despite the use of protease inhibitors in addition to the full-length RNase E its proteolytic fragments were detected.     

Identification and expression of a cloned yeast heat shock gene

We have isolated the yeast HSP90 gene which encodes the Mr = 90,000 heat shock-inducible protein of this organism. When this gene is introduced into yeast on a multicopy plasmid vector, a dramatic increase is observed in the level of synthesis of the Mr = 90,000 heat shock-inducible protein. This protein overproduction is due to expression of the plasmid-borne HSP90 gene, which is under the same heat shock regulation as its chromosomal counterpart. The presence of an increased dosage of the HSP90 gene has no effect on the synthesis of the other major heat shock-inducible proteins and does not alter the heat shock-associated phenotype of thermal tolerance.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Heat shock proteins of barley mitochondria and chloroplasts. Identification of organellar hsp 10 and 12: putative chaperonin 10 homologues.

Tissue slices from barley seedlings were subjected to heat shock and metabolically labelled with [35S]methionine and [35S]cysteine. Mitochondria and chloroplasts were isolated and shown to contain two novel heat shock proteins of 10 and 12 kDa, respectively. The possibility that these proteins, like a mitochondrial 10 kDa stress protein recently isolated from rat hepatoma cells [(1992) Proc. Natl. Acad. Sci. 89, in press] represent eukaryotic chaperonin 10 homologues is discussed.     

Hypoxia Induces Synthesis of a Novel 22-kDa Protein in Neonatal Rat Oligodendrocytes

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecophysiology of the estuarine cyanobacterium Lyngbya aestuarii to varying salinity in vitro

Lyngbya aestuarii, one of the dominant cyanobacterium grows in different salinity gradients of Chilika lagoon. It was isolated in axenic culture and its ecophysiology with response to different concentrations of salinity was studied in vitro to understand its adaptation strategies in the changing salinities of the lagoon. Changes in morphological features of the organism were observed with salinity gradients higher than 28 g/L and lower than 14 g/L salinity. Increase in growth was accompanied by increase of chlorophyll-a, carotenoid and cell protein contents of the organism from 3.5 to 28 g/L. Cellular carbohydrate content was higher with increasing salinity of the medium up to 90 g/L. No detrimental effect on pigment synthesis and macromolecular content of the organism was observed at the salinity level ranging from 7 to 56 g/L salinity. Methanolic extract of L. aestuarii showed prominent absorption at 334 nm in the UV-B region of the spectrum due to mycosporine-like amino acid (MAA) and the quantity of MAA increased with increasing salinity. At 7 g/L salinity 150, 93, 58, 34 and 18 kDa proteins were up-regulated; however, at 14 g/L 37, 26 and 28 kDa proteins and in 0, 3.5 and 90 g/L 122, 32 and 20 kDa proteins were repressed; this shows similarities of salinity-induced protein modifications as observed in higher plants. Super oxide dismutase activity also increased in the cells grown at 56 g/L salinity. We conclude because of having these effective adaptation strategies, L. aestuarii cope very well with the changing salinity in different seasons and grows well in the different sectors of the lagoon.     

An archaeal chaperonin-based reactor for renaturation of denatured proteins

We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock. Received: January 24, 1999 / Accepted: August 7, 1999     

Localization of a multifunctional chaperonin (GroEL protein) in nitrogen-fixing Anabaena PCC 7120

The occurrence and distribution of a multifunctional chaperonin-60 (cpn60), the GroEL protein, was demonstrated in the cyanobacterium Anabaena PCC 7120 by using a rabbit anti-GroEL (Escherichia coli) antibody. Western-blot analysis showed a distinct cross-reaction with a protein of approx. 65 kilodaltons, analogous to the Mr of the E. coli homologue. Immunocyto-chemical studies of vegetative cells showed that a chaperonin was localized in both vegetative cells and heterocysts. In vegetative cells cpn60 was primarily detected both in the carboxysomes, and in the cytoplasm, though mainly in the thylakoid region of the latter. In heterocysts, specialized cells for nitrogen fixation, the cpn60 label was prominent and was evenly distributed throughout the cell. These results support recent findings that chaperonins are multifunctional proteins, and extend those findings by demonstrating the occurrence of cpn60 in a prokaryotic cyanobacterium and by raising the possibility of the involvement of this chaperonin in the assembly of heterocystous proteins.Abbreviations cpn60 chaperonin-60 - Mr relative molecular mass - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Heat protectors and heat-induced preferential redistribution of 26 and 70 kDa proteins in Chinese hamster ovary cells

An overall increase of 40% in nuclear-associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear-associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43 degrees C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1-0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6-1.8. The normal levels of these two proteins were restored when cells were incubated at 37 degrees C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector-treated cells, suggesting that "repair" of heat-induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new protein synthesis.     

Effect of High Temperature on Protein Expression in Strawberry Plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.     

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.     

Proteomic analysis of heat-stable proteins in Escherichia coli

Some proteins of E. coli are stable at temperatures significantly higher than 49 degrees C, the maximum temperature at which the organism can grow. The heat stability of such proteins would be a property which is inherent to their structures, or it might be acquired by evolution for their specialized functions. In this study, we describe the identification of 17 heat-stable proteins from E. coli. Approximately one-third of these proteins were recognized as having functions in the protection of other proteins against denaturation. These included chaperonin (GroEL and GroES), molecular chaperones (DnaK and FkpA) and peptidyl prolyl isomerases (trigger factor and FkpA). Another common feature was that five of these proteins (GroEL, GroES, Ahpc, RibH and ferritin) have been shown to form a macromolecular structure. These results indicated that the heat stability of certain proteins may have evolved for their specialized functions, allowing them to cope with harsh environments, including high temperatures.     

Detection of Hsp60 (GroEL)-like proteins in Vibrio parahaemolyticus and vibrio species by Western immunoblotting analysis

Detection of heat shock proteins in Vibrio parahaemolyticus was investigated by SDS-PAGE and Western immunoblotting procedure using an anti-Hsp 60 antibody. Results indicate that V. parahaemolyticus elicited at least one Hsp 60 (GroEL)-like protein with apparent molecular weight of about 58 000 (58 kDa) when submitted to a heat shift from 30 to 42C. Kanagawa phenomenon-positive and -negative strains of V. parahaemolyticus responded the same way. Six other Vibrio species also showed an increased synthesis of GroEL-like (58 kDa) protein after heat shock, while synthesis of 58 kDa protein of V. alginolyticus was at a similar level before and after heat shock. Vibrio nereis showed an increased synthesis of a 60 kDa GroEL-like protein.