DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Properties of cell-free extracts fromBacillus subtilis and its thermophilic mutant

After being heated at 65°C for 10 min, 51% of the protein in a cell-free extract fromBacillus subtilis BR151 was denatured, whereas the comparable value was 8% for the S-30 of a spontaneously occurring, temperature-resistant (T/r) mutant. Although ribosomes isolated from the T/r mutant retained 97% of their initial protein synthetic activity when preincubated at 60°C for 30 min, ribosomes prepared from the mesophilic parent were completely inactivated under these conditions. The optimum temperature for poly U-directed phenylalanine incorporation was 45°C for both parental and mutant extracts assayed in the absence of polyamines. The addition of spermidine to the S-30 from the mesophilic parent inhibited protein synthesis at each temperature tested, whereas this polyamine stimulated polyphenylalanine synthesis in the T/r extract at both 55°C and 65°C.     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary The mutant T44() of Escherichia coli K12, grown in the presence of adenine, develops an increased tolerance to streptomycin. In cultures grown on streptomycin, the ts character (tif) may temporarily be suppressed but, on further transfer, both the temperature-sensitive phenotype and streptomycin tolerance disappear. In a cell-free system, the relative efficiency of translation of MS2 and poly U messenger RNAs was, respectively, 75 and 50% lower in extracts from cultures grown at 37° with adenine than in extracts from 30° cultures. Similar results were obtained when adenine was added in vitro to an extract from a culture grown at 37° in the absence of adenine, using MS2 RNA as messenger. Moreover, the 37° extracts showed a much lower misincorporation of isoleucine into polyphenylalanine in the poly U system. In addition, the Mg⁺⁺ concentration required for optimal translational activity was higher for the 37° than for the 30° extracts. Extracts from a culture grown in L medium at 37° or from a tif ^-/F tif ⁺ merodiploid grown at 37° with adenine behaved similarly to that from the 30° culture when poly U was used as messenger RNA. It is suggested that the tif ⁺ gene product may play a regulatory role in ribosomal function and the pleiotropic nature of the tif-1 mutation could be due to impairment of translational activity augmented by elevated temperature or by adenine.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Transfer of a benomyl resistance marker by heat-inactivated Trichoderma reesei protoplasts

Summary Protoplasts from a benomyl resistant Trichoderma reesei mutant were heat inactivated at 60°C for 8 min and fused with viable protoplasts from an osmosensitive, non-sporulating T. reesei strain. Fusants recovered on 50 g/ml benomyl containing potato dextrose agar plates grew and sporulated well. Cellulolytic enzyme activities produced in liquid culture by selected fusants were higher than those produced by parental strains.     

Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues

Summary To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [³H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37°C and 45°C were similarly stable. At 37°C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45°C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37°C. Shift back to 37°C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue - methylene ATP during 45°C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45°C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or P_i had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).     

Plasmid pKM101-mediated mutagenesis in Escherichia coli is inducible

Summary A tif-1 umuC36 double mutant of Escherichia coli was constructed. It has been found that the umuC36 mutation prevents both increased spontaneous mutagenesis and enhanced reactivation of UV-irradiated , phenomena normally observed in the tif-1 strain grown at 42°C. When the plasmid pKM101 was introduced into tif-1 umuC36, an elevated spontaneous reversion rate of the his-4 mutation observed at 30°C was further increased 6-fold at 42°C. This was accompanied by a 10-fold increase in the ability of tif-1umuC36 containing pKM101 and grown before infection at 42°C to reactivate UV-irradiated .     

Temperature-sensitive mutants ofCoprinus cinereus defective in hyphal growth and stipe elongation

Temperature-sensitive variants defective in hyphal growth of the basidiomyceteCoprinus cinereus were isolated from oidia treated by UV orN-methyl-N-nitro-N-nitrosoguanidine. In 23 out of 27 variant strains isolated, the temperature-sensitive variation was shown to be due to recessive single-gene mutation. All of the 23 mutants exhibited apparent interallelic complementation with one another, with the exception of one pair. The mutants were mated with their respective progeny to construct temperature-sensitive dikaryons carrying the mutant genes homozygously. Fruiting test of the respective dikaryons showed that eight mutations reduce stipe elongation during basidiocarp maturation at a restrictive temperature (37°C).     

A comparison of proline, thiol levels and GAPDH activity in cyanobacteria of different origins facing temperature-stress

Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and monitored for levels of proline, thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thermophile Mastigocladus laminosus (growth optimum, 40 °C), raised the proline level 4.2-fold at low temperature (20 °C), for the psychrophile Nostoc 593 (growth optimum, 20 °C), it was raised 8-fold at 40 °C while in the mesophile Nostoc muscorum (growth optimum, 30 °C), the imino acid level increased 2.3-fold during temperature shiftdown to 20 °C or 3.5-fold in sets facing shiftup (40 °C). Alterations in thiol levels in the above strains were in line with proline. It is suggested that such fluctuations reflect metabolic shifts as a response to stress. Interestingly, GAPDH activity was maximum at the respective growth temperature optimum of M. laminosus (122 nmol NADPH oxidized min ^–1 mg ^–1 protein) and Nostoc 593 (141 nmol NADPH oxidized min ^–1 mg ^–1 protein) while in N. muscorum, it increased at 40 °C (101 nmol NADPH oxidized min ^–1 mg ^–1 protein) and to 93.3 nmol NADPH oxidized min ^–1 mg ^–1 protein (20 °C) relative to 86 nmol NADPH oxidized min ^–1 mg ^–1 protein at 30 °C. It seems that extremophiles maintain the GAPDH activity/level during growth at their respective temperatures optimal while the mesophile increases it in order to cope up with temperature-stress.     

Isolation and characterization of an ω3-desaturation-defective mutant of an arachidonic acid-producing fungus,Mortierella alpina 1S-4

A mutant considered to be defective in the conversion of n-6 to n-3 fatty acids (3-desaturation) was derived from a 5-desaturation-defective mutant (Mut44) of Mortierella alpina 1S-4, after treating its spores with N-methyl-N-nitro-N-nitrosoguanidine. This mutant cannot produce 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid or any other n-3 fatty acids, of which about 10% was found in its parental strain upon cultivation at 12°C. The mutant's growth rate was comparable to that of the parental strain when grown at 28°C, but it became much slower when the mutant grew at 12°C, at which the lag phase for Mut44 was about 2 d but 5 d for the mutant.Abbreviations 18:33 9(Z),12(Z),15(Z)-octadecatrienoic acid - 18:43 6(Z),9(Z),12(Z),15(Z)-octadecatetraenoic acid - 20:43 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid - AA arachidonic acid - DHGA dihomo--linolenic acid - EPA 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid - GLC gas-liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PC phosphatidylcholine     

Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

Summary The accumulation of interferon (IFN) -2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon -2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN -2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t _1/2 values for IFN -2 decreased with increasing incubation temperature. Pulse-chase studies using [³⁵S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN -2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN -2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.     

Chlorophyll a Fluorescence Analysis in Response to Excitation Irradiance in Bean Plants (Phaseolus vulgaris L. and Vigna unguiculata L. Walp) Submitted to High Temperature Stress

Bean plants Phaseolus vulgaris L. (cv. Carioca and Negro Huasteco) and Vigna unguiculata L. Walp (cv. Epace-10) were grown in a growth chamber with a photosynthetic photon flux density of 200 mol m^–2 s^–1 at leaf level and air temperature of 25+1 °C. Fully expanded, first pair leaves of 12-d-old plants were submitted for 90 min to high temperature (25, 30, 35, 40, 45, and 48 °C). Chlorophyll a fluorescence parameters (ETR, q_P, q_N, and F₀) were investigated using a modulated fluorimeter at 25 °C during recovery considered here as 48 h after stress induction period. An accentuated decrease in q_P and an increase in q_N at 48 °C in Carioca and Negro Huasteco was not observed in Epace-10. In response to excitation irradiance a great potential for ETR was found in Negro Huasteco at 25 °C, also demonstrated by net photosynthetic rate. At 48 °C ETR was high for Epace-10 while it was equal to zero for Carioca and Negro Huasteco. Tolerance to high temperature observed in Epace-10 provided important information about the adaptative characteristics of Vigna cultivars to warm climates.     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

Bud dormancy and vegetative growth in Salix polaris as affected by temperature and photoperiod

Summary Vegetative growth of two ecotypes (lat. 78° 15N and 69°37N) of Salix polaris L. was studied in phytotron experiments. Dormancy of the winter buds was broken by chilling at 0.5°C for 14 to 42 days. Chilling requirement increased with decreasing growth temperature. The optimum temperature for bud break and shoot growth was about 15°C for both ecotypes. Cessation of apical shoot growth and abscission of shoot tip was not prevented by long photoperiods. However, at high temperature, 15°C or more, and in 18 to 24 h photoperiod, two or three growth flushes occurred frequently in both ecotypes. Leaf abscission in the arctic ecotype from lat. 78°N was not affected by photoperiod when grown at 6°C, but was stimulated by short photoperiod when grown at 15°C. In the ecotype from lat. 69°N leaf abscission was enhanced by short photoperiod even at 6°C.     

Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.     

Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.