Physiological Studies on the Recovery of Salt Tolerance by Staphylococcus aureus After Sublethal Heating

Cultures of S. aureus in 100 mM potassium phosphate buffer heated at 52 C for 15 min lost their tolerance to 7.5% NaCl. After incubation in a complex growth medium or in a diluted dialyzed medium in which unheated cells were unable to grow, salt tolerance was regained. Heat injury caused 30% loss of lipid. During recovery, the concentration of C(15) and C(17) fatty acids returned to normal, and there appeared to be an oversynthesis of C(16) and C(18) unsaturated acids. Penicillin abolished the latter reaction without affecting recovery; chloramphenicol did not affect fatty acid oversynthesis but reduced recovery. The K/Na ratio was 12.6 in control cells and 3.4 in injured cells, where it remained during the recovery of salt tolerance. Aspartate uptake was about 10% of the control level after injury and about 35% at recovery. Control cells grew without a lag on subculture, but injured cells which had regained their salt tolerance needed about 2 more h of incubation. Cells recovering with penicillin needed 6 more h, and cells recovering with chloramphenicol did not grow without a prolonged lag. Cells of S. aureus, therefore, may recover their salt tolerance while various membrane functions are still damaged.     

Effects of heat shock and thermoadaptation on the ultrastructure of cowpea (Vigna unguiculata) cells

Summary Suspension culture cells of cowpea (Vigna unguiculata) were examined using transmission electron microscopy to characterize morphological changes associated with hyperthermal stress. Cultures maintained at 26°C (unadapted cells) and 38°C (thermoadapted cells) were examined before and after exposure to elevated temperatures of 34°C and 45°C, respectively. Observations indicate that while there were significant ultrastructural differences between unadapted and thermoadapted cells, the following structural modifications in response to heat stress were observed in cells of both cultures: (a) almost a complete loss of polyribosomes, rough ER, and dictyosomes, (b) migration of intracellular waste material (presumably proteinaceous in composition) into the cell vacuole, (c) swelling of the nucleolus with assumed accumulation of preribosomal RNP granules, and (d) retraction of the tonoplast from the cytoplasm into the vacuoles of some cells. Heat shock granules (two size classes) were observed in the cytoplasm of stressed thermoadapted cells along with hollow-cored granules within the leucoplasts. Apart from a few minor differences, the morphological modifications that were made in apparent response to hyperthermal stress were remarkably similar in both cultures.     

High frequency conjugative mobilization in natural strains of Bacillus subtilis, bearing a large plasmid]

Conjugative properties of the strain Bacillus subtilis that carrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis 168 strain with a frequency of approximately 10(-2). The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugal mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37 degrees C, but conjugation also proceeded at lower temperatures, up to 21 degrees C.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Genetic Regulation of Cell Division Initiation in Bacillus subtilis

The growth and division properties of a temperature-sensitive mutant of Bacillus subtilis defective in the initiation of cell division have been studied. Log-phase cells transferred from 30 to 45 C continue to increase in length but fail to initiate new divisions. Deoxyribonucleic acid synthesis continues at 45 C, and genomes are segregated along the filament length. When filaments are returned to 30 C, division initiation resumes, and the long multinucleate clones are partitioned into normal-size cells. Occasionally, multiple cross walls initiate in close proximity, resulting in tiny cells, some of which are anucleate. Division resumption is sensitive to protein synthesis inhibitors, suggesting there is a new protein required for the initiation of division in filaments.     

Aging affects expression of 70-kDa heat shock proteins in Drosophila

We examined the effect of cellular aging on adult mortality and hsp70 gene expression in Drosophila melanogaster under thermal stress. The results showed that flies exposed to 37 degrees C for various time intervals had reduced survival rate with age. The level of hsp70 mRNA increases in flies up to 23-28 days of age, but then declines as they get older. When flies are shifted to 25 degrees C after 30 min of heat stress, the time-dependent decrease in hsp70 mRNA levels occurs more rapidly in young flies than in old ones. The hsp70 mRNA present during this recovery period is translated into protein, and senescent flies continue to synthesize this protein for up to 5 h after heat shock. The prolonged expression of hsp70 RNA during recovery from heat shock was also observed in young flies fed canavanine, an arginine analogue. These data suggest that in old insects, the accumulation of conformationally altered proteins plays a role in the regulation of hsp70 RNA expression. These results are discussed in relation to the finding that old flies are more sensitive to thermal stress than young ones.     

The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.     

A psychrophilic strainActinoplanes sp. 220

The strain designated Actinoplanes sp. 220 differed in its characteristics from other strains of the genus Actinoplanes listed in Bergey's Manual (1974). The strain belongs to psychrophilic culture growing within the range of 0-30 degrees C. The optimal temperature for growth on yeast--malt agar is 10-23 degrees C. Cultures transferred at 23 and 28 degrees C differed in morphological and physiological properties, enzyme activity and pigmentation in standard media. Submerged culture transferred at 28 degrees C inhibited growth of Bacillus subtilis ATCC 6633 and ATCC 9945. LL-2,6-Diaminopimelic acid was chromatographically detected in the submerged mycelium of this culture. This compound was not found in the mycelium of the original culture transferred at 23 degrees C. The cultures did not substantially differ in the composition of other amino acids contained in larger quantities in the mycelium.     

Thermal Injury and Recovery of Bacillus subtilis

Exposure of Bacillus subtilis NCTC 8236 to sublethal temperatures produced a change in the sensitivity of the organism to salt and polymyxin. After 30 min at 47 C, 90% of the population was unable to grow on a modified sulfite polymyxin sulfadiazine agar containing an added 1% NaCl, 1% glucose, and 1% asparagine. The data presented demonstrate that thermal injury results in degradation of both 16S and 23S ribonucleic acid (RNA) and in damage to the cell membrane, suggested by leakage into the heating mestruum of material absorbing at 260 nm. When the cells were placed in a recovery medium (Trypticase soy broth), complete recovery, indicated by a returned tolerance to salt and polymyxin, occurred within 2 hr. The presence of a protein inhibitor (chloramphenicol) and cell wall inhibitors (vancomycin and penicillin) during recovery had no effect, whereas the presence of an RNA inhibitor (actinomycin D) effectively inhibited recovery. Further data demonstrated that the injured cells were able to resynthesize both species of ribosomal RNA during recovery by using the fragments which resulted from the injury process. Also, precursor 16S and precursor 23S particles accumulated during recovery. The maturation of the precursor particles during recovery was not affected by the presence of chloramphenicol in the recovery medium.     

Nucleolar localization of Aspergillus fumigatus CgrA is temperature-dependent

Pathogenic fungi must adapt to multiple adverse environmental conditions during the transition from the environment to a mammalian host, one of which is temperature. The ability of Aspergillus fumigatus to grow optimally under conditions of thermal stress requires the nucleolar protein CgrA. In this study, we have determined how temperature affects the intracellular localization of CgrA in A. fumigatus using a green fluorescent protein (GFP)-tagging approach. At 22 degrees C, CgrA was almost exclusively in the nucleolus, with a ratio of nucleolar to cytoplasmic fluorescence of 10:1. At 37 degrees C, the ratio of nucleolar to cytoplasmic fluorescence was reduced fivefold, and increased correspondingly in the cytoplasm. This effect was not seen with the nucleolar protein NopA in wild-type A. fumigatus. However, in a DeltacgrA mutant NopA was delocalized from the nucleolus at 37 degrees C but not at 22 degrees C. These results provide evidence for a temperature-dependent mechanism of intracellular localization for CgrA, and suggest that CgrA facilitates nucleolar compartmentalization of NopA at higher temperature.     

Morphological Changes and Antibiotic-Induced Thermal Resistance in Vegetative Cells of Bacillus subtilis

The morphology and thermal resistance of vegetative cells of Bacillus subtilis W168 were examined after growth at 37 and 53 C. Vegetative cells grown at 37 C exhibited a typical trilaminar morphology, whereas cells grown at 53 C exhibited a cell wall which was apparently thicker and more loosely organized and had a poorly defined periphery. A concurrent increase in thermal resistance to a heat shock of 60 C occurs with the change in cell wall morphology. The change to the aberrant cell wall form, or its reversal to the normal form, is always accompanied by the gain or the loss of thermal resistance, respectively. The inhibition of protein synthesis by chloramphenicol has little effect upon the acquisition of thermal resistance at 53 C. Addition of the disaccharide pentapeptide subunit to the cell wall peptidoglycan is apparently essential to growth at 53 C and the acquisition of thermal resistance, since both growth and thermal resistance are inhibited by bacitracin. Two antibiotics, penicillin and cycloserine, which inhibit the final cross-linking of the cell wall peptidoglycan at two separate points, do not affect the acquisition of thermal resistance at 53 C. These same antibiotics induce a high degree of thermal resistance at 37 C. It is proposed that a change in the cell wall structure is related to an increased thermal resistance.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15 degrees C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees C membranes, while 15 degrees C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15 degrees C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15 degrees C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37 degrees C, but only 50% at 15 degrees C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15 degrees C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Poly(epsilon-caprolactone) polyurethane and its shape-memory property

A series of segmented poly(epsilon-caprolactone) polyurethanes (PCLUs) were prepared from poly(epsilon-caprolactone) (PCL) diol, 2,4-toluene diisocyanate and ethylene glycol. The molecular weight (M(n)) of PCL was 500-10,000, and the soft-to-hard molar ratio was 1:2 to 1:6. Their shape-memory behaviors were investigated as a function of PCL molecular weight, PCLU composition, and thermal/mechanical history. When a deformation temperature 15-20 degrees C below T(m) was chosen, the lowest recovery temperature (LRT) was 15-18 degrees C below T(m), and the recovery ratio was 94-100% for tensile deformation of 300% and for compression of 2.7-fold. The reasons for this deformation-recovery procedure and the mechanism for this shape recovery below T(m) were discussed. The shape recovery was associated with the premelting of the crystals formed during the deformation and fixation, and, thus, it could be accomplished in the solid state. Its driving force was the inner stress stored in the deformed specimen during deformation and crystallization. Therefore, the LRT was a more practical temperature for shape-memory PCLU than T(m). It could be conveniently measured by means of thermal mechanical analysis. By adjusting the molecular weight of the PCL diol and the hard-to-soft ratio, the LRT of PCLU could be adjusted to the range of 37-42 degrees C, and reasonable rigidity could be retained after shape recovery, fulfilling the essential requirements of medical implantations.     

Thermosensitive Export of Amylases in the Yeast Schwanniomyces alluvius

Cultures of Schwanniomyces alluvius were grown aerobically at30?C in a phosphate-buffered defined medium containing solublestarch as sole carbon source. Extracellular amylolytic activity,minimal when cells were most actively dividing, increased dramaticallyat the end of exponential growth and became maximal during stationaryphase. When cultures were grown at 37?G, no increase in extracellularamylolytic activity was detectable. This lack was only partlyascribable to thermal inactivation of the amylases. When culturesgrowing at 30?C were shifted to 37?C, excretion was arrested.Cultures growing at the restrictive temperature could be madeto excrete full amylolytic activity at the permissive temperature,only if the shift was made before the end of exponential growth. ¹NRCC contribution No. 23556. (Received December 10, 1983; Accepted April 20, 1984)     

Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death. Thermotolerance measured in terms of cell survival was paralleled by thermal resistance of protein synthesis and the cellular ability to refrain from rounding up under heat stress.     

GENETIC TRANSFER OF THE ABILITY TO GROW AT 55 C IN BACILLUS SUBTILIS

McDonald, William C. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.) and Thomas S. Matney. Genetic transfer of the ability to grow at 55 C in Bacillus subtilis. J. Bacteriol. 85:218-220. 1962.-Two strains of Bacillus subtilis, 168 and P1, were found to grow at 55 C (55(+)) on complete media; strain 168S(r) failed to grow at temperatures above 50 C (55(-)). When 168S(r) bacteria were grown in the presence of deoxyribonucleic acid extracted from 168, the ability to grow at 55 C was transformed at a frequency of 10(-4). An incubation period of 3 to 4 hr at 37 C was necessary for phenotypic expression of the 55(+) character. Only 10 to 20% of the 55(+) transformants retained the high-level streptomycin resistance (S(r)) of the recipient, indicating close linkage between the S(s) and 55(+) loci.     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.