Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus.

The effect of heat shock on Myxococcus xanthus was investigated during both glycerol- and starvation-induced development. Cells heat shocked at 40 degrees C for 1 h prior to a development-inducing signal displayed an accelerated rate of myxospore formation at 30 degrees C. Additionally, M. xanthus cells heat shocked prior to glycerol induction formed a greater total number of myxospores when sporulation was complete than did control cells maintained at 30 degrees C. However, in starvation-induced fruiting cells the total number of myxospores in control and heat-shocked populations was about equal when fruiting body and myxospore formation was complete. When extended heat shock (3 h) was applied to cells prior to development, no acceleration of myxospore formation was observed. Heat shock elicited the premature expression of many developmentally regulated proteins. Cell fractionation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the subcellular location and molecular weights of the 18 glycerol-induced and 9 starvation-induced developmental proteins. Comparison with previously identified M. xanthus heat shock proteins showed that nine of the developmental proteins found in glycerol-induced cells and three of the developmental proteins found in starvation-induced cells were heat shock proteins. Furthermore, heat shock increased the activity of alkaline phosphatase, a developmentally regulated enzyme, in vegetative cells, glycerol-induced cells, and starvation-induced cells.     

Effects of heat shock upon the expression of developmentally regulated genes in Myxococcus xanthus

Abstract The effects of heat shock upon the expression of several developmentally regulated genes of Myxococcus xanthus were examined. No effects were observed on levels or timing of developmentally regulated β-galactosidase expression in eight randomly selected Tn5lac insertion mutants. However, heat shock significantly affected the fruiting behavior of temperature-sensitive aggregation ( tag ) mutants of M. xanthus . The tag mutant phenotype exhibits the normal aggregation of cells to form fruiting bodies at temperatures < 34°C, but cells fail to aggregate at temperatures ⩾ 34°C. Heat shock administered to tag mutant strains prior to starvation prohibited fruiting body formation at permissive temperatures. Additionally, tag mutant strains were found to be extremely sensitive to killing at 40°C. Heat shock was also found to increase tagA and tagE expression by 22 and 47%, respectively. Mutations in tagA blocked heat shock induced expression of tagE .     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

SigB, SigC, and SigE from Myxococcus xanthus homologous to sigma32 are not required for heat shock response but for multicellular differentiation

Myxococcus xanthus has been known to have multiple sigma factors which are considered to play important roles in regulation of gene expression in development. A new gene encoding a putative sigma factor, sigE, was cloned by using a degenerate oligonucleotide corresponding to the conserved region 2.2 of M. xanthus SigA. In the 2.0-kb nucleotide sequence, an open reading frame consisting of 280 amino acid residues was identified. The amino acid sequence of SigE shows high similarity to heat shock sigma factors in bacteria. However, the sigE gene is not induced by heat shock and deletion of sigE does not affect production of heat shock proteins. SigE is expressed during both vegetative growth and fruiting body development. In the deletion mutant of the sigE gene fruiting body formation is initiated earlier and fewer spores are produced than in the parent strain. Interestingly, the deltasigE mutant shows defects in fruiting body formation at 37 degrees C. In addition to SigE, SigB and SigC show high sequence similarity to heat shock sigma factors. However, even if all three sigma factor genes are disrupted, heat shock proteins are still normally induced. A deltasigBdeltasigCdeltasigE triple deletion strain forms fruiting bodies earlier, but sporulats later than the parent strain. Spores from the triple deletion mutant are aberrant and their viability is less than 0.001% compared with that of the parent strain, suggesting that these sigma factors may have redundant functions in multicellular differentiation of M. xanthus.     

Catalatic capacities in heat-shocked Euglena cells

1. Various heat treatments were applied to the wild strain Z. Klebs. of Euglena gracilis. 2. Samples of cells were taken at day 1 of the culture at 26 degrees C in a 33 mM lactate medium, when the catalatic capacities of the catalase were highest. 3. They were either submitted to heat treatments (36 and 38 degrees C), or heat-shocks (40, 42 degrees C) or non-permissive heat stress (45 degrees C) for 15 min, 1 and 2 hr. 4. After a 2-hr 45 degrees C treatment the cells were unable to recover normal physiological functions. 5. Heat treatments between 36 and 38 degrees C decreased the catalatic capacities of cells, while heat-shocks at 40 and 42 degrees C strongly reinforced these capacities of hydrogen peroxide dismutation. 6. Having been heat-shocked at 42 degrees C for 2 hr, the cells became different from control cells: (a) after several months of culture, they displayed catalatic capacities increased by 65%; (b) they were able from now on to survive a 2 hr heat shock at 45 degrees C.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Characterization of a small heat shock protein, Mx Hsp16.6, of Myxococcus xanthus

A number of heat shock proteins in Myxococcus xanthus were previously identified by two-dimensional (2D) gel electrophoresis. One of these protein was termed Mx Hsp16.6, and the gene encoding Mx Hsp16.6 was isolated. Mx Hsp16.6 consists of 147 amino acid residues and has an estimated molecular weight of 16,642, in accordance with the apparent molecular mass in the 2D gel. An alpha-crystallin domain, typically conserved in small heat shock proteins, was found in Mx Hsp16.6. Mx Hsp16.6 was not detected during normal vegetative growth but was immediately induced after heat shock. Expression of the hsp16.6 gene was not induced by other stresses, such as starvation, oxidation, and high osmolarity. Mx Hsp16.6 was mostly localized in particles formed after heat shock and precipitated by low-speed centrifugation. Furthermore, Mx Hsp16.6 was detected in highly electron-dense particles in heat-shocked cells by immunoelectron microscopy, suggesting that it forms large complexes with heat-denatured proteins. An insertion mutation in the hsp16.6 gene resulted in lower viability during heat shock and lower acquired thermotolerance. Therefore, it is likely that Mx Hsp16.6 plays critical roles in the heat shock response in M. xanthus.     

Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.

Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C. When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C. In pulse-labeling experiments with [14C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.     

Heat-shock-induced proteins from Myxococcus xanthus

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shocked M. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357-5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218-224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit are heat shock inducible.     

Heat shock response of Pseudomonas aeruginosa.

The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins.     

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.     

A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.     

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

Factors influencing the heat shock response of Xenopus laevis embryos

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.     

Cell-specific association of heat shock-induced proton flux with actin ring formation in Chenopodium cells: comparison of auto- and heterotroph cultures

A comparison of the responses of extracellular pH, buffering capacity and actin cytoskeleton in autotroph and heterotroph Chenopodium rubrum cells to heat shock revealed cell-specific reactions: alkalinization caused by the heat shock at 25-35 degrees C was higher in heterotroph cells and characterized by heat shock-induced changes in the actin cytoskeleton and ring formation at 35-37 degrees C. Rings (diameter up to 3 mum) disappeared and extracellular pH recovered after the heat-shocked cells were transferred into control medium. At 41 degrees C, no rings but a network of coarse actin filaments were induced; at higher temperatures, fragmentation of the actin cytoskeleton and release of buffering compounds occurred, indicating sudden membrane leakage at 45-47 degrees C. The calcium chelator EGTA [ethylene-glycol-bis(beta-aminoethyl-ether)-N,N,N',N'-tetraacetic-acid] increased the frequency of heat shock-induced rings. Ionophore (10 microM nigericin) and the sodium/proton antiport blocker [100 microM 5-(N-ethyl-N-isopropyl)-amiloride] mimicked the effect of the 37 degrees C heat shock. The cytoskeleton inhibitors latrunculin B, cytochalasin D and 2,3-butanedione monoxime inhibited ring formation but not alkalinization. In autotroph cells, the treatment with nigericin (10 microM) produced rings, although the actin cytoskeleton was not affected by temperatures up to 45 degrees C. We conclude that Chenopodium cells express a specific temperature sensor that has ascendancy over the organization of the actin cytoskeleton; this is probably a temperature- and potential-sensitive proton-transporting mechanism that is dependent on the culture conditions of the heterotroph cells.     

Morphogenesis in Myxococcus xanthus and Myxococcus virescens (Myxobacterales)

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.     

The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock

Nuclear pore complexes (NPCs) play an essential role in RNA export. Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC. The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs. Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase. Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions. We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C. Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells. Export following ethanol shock was less dependent on the presence of Rip1p. Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells. Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells. We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p. This motif is not required under normal growth conditions or following ethanol shock. These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs. These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.