Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

Thermostability of an alkaline protease, AprP, is enhanced by replacements of Ser307 and Ser331 at the cleavage sites

The thermostability of an alkaline protease, AprP from Pseudomonas sp. KFCC 10818, was improved by replacing Ser307 and Ser331 at the autoproteolytic cleavage sites with various amino acids. Six mutant enzymes were purified and characterized. Two of these had half-lives four and three times longer than the wild-type protease at 55 °C in the presence of 1 mM CaCl₂. Three mutant enzymes had half-lives twice as long as the wild-type under the same condition.     

Phenomenon of Temperature-Dependent Chlorophyll Deficiency in Festuca pratensis

The composition and amount of pigments were studied in temperature-dependent chlorophyll-deficient seedlings of wild type (control) and several mutant lines of Festuca pratensis Huds. at room (25°C) and high (35°C) temperatures. In seedlings of all mutant lines grown at 25°C, chlorophyll b content was lower and the concentration of carotenoids was higher than in control seedlings. At 35°C, the concentration of all pigments decreased in a row from dark-green to xantha phenotypes, and this trend was retained when the temperature was lowered to 25°C. The phenotype xantha completely lacked violaxanthin and neoxanthin. The observed effects are related to the protective dissipative function of the xanthophyll cycle.     

Effects of temperature and medium composition on the ethanol tolerance ofBacillus stearothermophilus LLD-15

The effects of temperature (60°–70°C) and medium composition (complex and defined) on ethanol tolerance ofBacillus stearothermophillus LLD-15, an L-lactate dehydrogenase mutant have been determined in shake flasks under aerobic conditions. In all cases, there was complete inhibition of growth in the presence of 6%v/v ethanol.B. stearothermophillus LLD-15 was found to be less tolerant to ethanol at 70°C than at 60°C and also less tolerant to ethanol in a defined medium than in a complex medium.     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

Summary A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35°C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40°C indicated that the maximal growth rate occurs at 35°C in the wild type, and at 25°C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35°C than at 25°C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20°C is not sufficient to allow germination at 37°C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37°C. The reciprocal temperature shift-down experiments showed that the molecular events at 37°C may be qualitatively useful for germination after shifting to 20°C. Studies of macromolecular synthesis showed that the biochemical defect in the heat-sensitive strain appears to affect RNA synthesis before protein synthesis, although there were differences in the relative effects depending on the age of the germinating conidia and the inhibition of the two processes was never complete. Messenger RNA synthesis is normal in the mutant at 37°C. Previous work has shown that the rip-1 mutant strain has a conditional defect in the accumulation of 25S rRNA and, hence, in 60S ribosomal subunit production (Loo et al. 1981). There are also indications from those studies that the 60S ribosomal subunit may be functionally impaired at the higher temperature. Thus, the growth and macromolecular synthesis phenotypes may result as a consequence of a conditional, ribosome function defect and leads to the hypothesis that the mutation in the rip-1 strain may be in a gene for a 60S ribosomal subunit component, perhaps a ribosomal protein.     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

Temperature and salinity regulation of growth and gas exchange of Salicornia fruticosa (L.) L.

Summary Salicornia fruticosa was collected from a salt marsh on the Mediterranean sea coast in Libya. Growth and gas exchange of this C₃ species were monitered in plants pretreated at various NaCl concentrations (0, 171, 342, 513 and 855 mM). Maximum growth was at 171 mM NaCl under cool growth conditions (20/10° C) and at 342 mM NaCl under warm growth conditions (30/15° C) with minimum growth at 0 mM NaCl (control). Net photosynthesis (Pn) was greatest in plants grown in 171 mM NaCl with plants grown at 513 and 855 mM having lowest rates. Maximum Pn was at 20–25° C shoot temperatures with statistically significant reductions at 30° C in control plants while salt treated plants showed such reductions at 35° C. Salt treatments increased dark respiration over the control at 171 and 342 mM but reduced it at higher concentrations. Photorespiration was reduced by salt treatment and increased by increasing shoot temperature. Greatest transpiration was in 171 mM NaCl treated plants and increasing shoot temperature increased transpiration in all treatments. Stomatal resistance to CO₂ influx was influenced only moderately by temperature while increasing salinity resulted in increased stomatal resistance. In general both temperature and salinity increased the mesophyll resistance to CO₂ influx. The species seems adapted to the warm saline habitat along the Mediterranean sea coast, at least partially, by its ability to maintain relatively high Pn at moderate NaCl concentrations over a broad range of shoot temperatures.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis.

Summary The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division. Markers in the combined mutant were verified by transduction. DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated. Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30° C compared to 8.6% after 2 h at 45° C). The probability of a minicell producing division in double mutant clones is essentially the same at 30° C and during the initial 2–3 h growth at 45° C at which time lysis begins. Residual division at 45° C is attributable to processes initiated at 30° C. The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature. This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA.     

Degradation of phenol and toxicity of phenolic compounds: a comparison of cold-tolerant<Emphasis Type="Italic"> Arthrobacter</Emphasis> sp. and mesophilic<Emphasis Type="Italic"> Pseudomonas putida</Emphasis>

Phenol degradation efficiency of cold-tolerant Arthrobacter sp. AG31 and mesophilic Pseudomonas putida DSM6414 was compared. The cold-tolerant strain was cultivated at 10°C, while the mesophile was grown at 25°C. Both strains degraded 200 mg and 400 mg phenol/l within 48–72 h of cultivation, but the cold-tolerant strain produced more biomass than the mesophile. Both strains oxidized catechol by the ortho type of ring fission. Catechol 1,2 dioxygenase (C1,2D) activity was found intra- and extracellularly in the absence and in the presence of phenol. In the presence of 200 mg phenol/l, C1,2D activity of the mesophile was about 1.5- to 2-fold higher than that of the cold-tolerant strain. However, an initial phenol concentration of 400 mg/l resulted in a comparable enzyme activity of the cold-tolerant and the mesophilic strain. The two strains differed significantly in their toxicity pattern towards 12 aromatic (mostly phenolic) compounds at different growth temperatures, which was determined via growth inhibition in the presence of nutrients and toxicants. For the cold-tolerant strain, toxicity was significantly lower at 10°C than at 25°C. The mesophile showed a significantly lower susceptibility to high hydrocarbon concentrations when grown at 25°C compared to 10°C.Communicated by K. Horikoshi     

Effect of Combined Salt and Heat Treatments on Germination and Heat-Shock Protein Synthesis in Lentil Seeds

The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 –12 h imbibition. [³⁵S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.     

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli

Summary The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the perimissive growth temperature of 30°C and only one per cell at the higher permissive growth temperature of 38°C. When the growth temperature of this mutant is changed from 30° to 38°C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.     

A mutant rho ATPase from Escherichia coli that is temperature-sensitive in the presence of RNA

Summary The Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40°C, in contrast to 45°C for rho ⁺. At higher temperatures (50°C, 55°C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho ⁺. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37°C but at 45°C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho ⁺ were linear at both 37°C and 45°C. The rho-115 and rho ⁺ ATPase activities were equally heat-stable during preincubation at 45°C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho ^- activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 g/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45°C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45°C to 35°C, indicating that polyC-induced destabilization of rho-115 was irreversible.     

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

Summary Sporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.     

Thermokinetic and energetic profiles of the yeast Debaryomyces hansenii in the presence of sodium chloride

Summary Sodium chloride decreased the growth yield with respect to glucose by 60% (at 17% NaCl, w/v), and narrowed the temperature span of growth of Debaryomyces hansenii from 6.8–40.5°C without the salt to 13.2–34.5°C with 16% NaCl. The thermokinetic profile (conjunct display of the Arrhenius plots of the specific rates of growth and thermal death) was dissociative either without or with NaCl, the minimum temperature of thermal death increasing from 42 to 45°C with 10% NaCl.     

Effect of glycine betaine on the sucrose catabolism of an l-lysine producing mutant of Brevibacterium lactofermentum

Summary Effect of exogenous betaine on the growth of an l-lysine-producing mutant of Brevibacterium lactofermentum was examined in a medium containing different carbon sources such as glucose, fructose, or sucrose. The growth rate decreased significantly with a rise in temperature when sucrose was the carbon source. Both the specific sucrose consumption rate and the invertase activity of the mutant decreased with the culture period when the cultivation temperature was 35°C. The addition of betaine restored both growth and invertase activity on medium containing sucrose as the carbon source at 35°C. Betaine protected the invertase activity against the inactivating effects of high temperature in vitro. Furthermore, the addition of exogenous invertase into production medium at 35°C restored the growth rate to that at 32°C. These results indicated that growth decreased on medium containing sucrose at 35°C due to a decrease in invertase activity, and that addition of betaine was an effective way to enhance growth on this medium at a higher temperature. Offprint requests to: Y. Kawahara     

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

Summary A temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu ^-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.