A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

HtpG Plays a Role in Cold Acclimation in Cyanobacteria

The heat shock protein HtpG is homologous to members of the Hsp90 protein family of eukaryotes and is essential for basal and acquired thermotolerances in cyanobacteria. In this study we have examined the role of HtpG in the cyanobacterium, Synechococcus sp. PCC 7942, in the acclimation to low temperatures. The inactivation of the htpG gene resulted in severe inhibition of cell growth and of the photosynthetic activity when the htpG mutant was shifted to 16°C from 30°C. Wild-type cells were able to resume growth without a lag period when shifted to 30°C after 5 days at 16°C, while the mutant displayed a detectable lag. The HtpG protein was induced in the wild-type cells at 16°C. Electrophoresis in the absence of sodium dodecyl sulfate (SDS) showed that a novel, high-molecular-weight complex containing GroEL and DnaK accumulated at 16°C, but the accumulation was strongly inhibited in the htpG mutant. Our results demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures. Received: 16 July 2001/Accepted: 15 August 2001     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22° C, the un-18 mutation did not affect the period length, but at temperatures between 22° C and 32° C, the period length of the un-18 mutant was ∼2 h longer than that of the wild-type strain. The un-18 ⁺ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora. Received: 17 October 1997 / Accepted: 26 April 1998     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

Flight pattern induced by temperature in a single-gene mutant of Drosophila melanogaster

Simultaneous intracellular recordings were made from the six dorsal longitudinal flight muscle (DLM) fibers in the temperature-sensitive mutant of Drosophila melanogaster, shibire^{ts 1} (shi). The DLM firing pattern induced by temperature in shi was compared with the DLM pattern of a wild-type fly in stationary flight. The firing pattern, induced at temperatures between about 26 and 27°C, was very similar to that of the wild-type pattern. At higher temperatures, the pattern became abnormal, characterzed by many synchronous firings among the fibers and occasional bursting.     

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38°C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38°C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication.

Mutants of simian virus 40 (SV40) with base substitutions at or near the origin of replication of the viral genome have been constructed by bisulfite mutagenesis at the BglI restriction site of SV40 DNA, followed by transfection of cells with the BglI-resistant (BglI^r) DNA so generated. Based on plaque morphology at different temperatures, the resulting BglI^r mutants could be classified into four-groups. Class I mutants (designated ar for “altered restriction”) were indistinguishable from wild-type SV40; class II mutants (designated shp for “sharp plaque”) produced small, sharp-edged plaques; class III mutants (designated sp for “small plaque”) produced small plaques at 32 °C, 37 °C and 40 °C; and class IV mutants (designated cs for “cold sensitive”) produced small plaques at 32 °C and wild-type plaques at 37 °C and 40 °C. That the altered plaque morphology of sp and cs mutants was related to mutation at the BglI restriction site was demonstrated by co-reversion to wild-type of the plaque phenotype and BglI sensitivity. The nucleotide sequence around the original BglI site was determined in the DNA from one mutant of each class. In each case a different base-pair substitution was found, at a site outside sequences coding for SV40 proteins. When rates of replication of mutant DNAs were measured during productive infection, ar mutant DNA was synthesized at a rate comparable to that of wild-type SV40 DNA, shp mutant DNA was made at a rate exceeding that of wild-type, sp mutant DNA was synthesized at a lower rate than that of wild type. and cs mutant DNA synthesis was reduced at 32 °C, but about the same as the wild-type rate at 40 °C. These patterns of mutant DNA synthesis were unaltered in cells co-infected with mutant and wild-type virus, i.e. the defects in DNA synthesis were not trans-complementable. We conclude that the defective mutants have single base-pair changes in a cis element that determines the rate of viral DNA replication, presumably within the origin signal itself.     

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12°C and 25°C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS^*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS^* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.     

Gln54Leu of the conserved polar residue inthe interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

Summary The assembly domain of cartilage oligomeric matrix protein (COMP) forms an α-helical coiled coil homopentamer with a conserved polar glutamine in the interior (d) position. We substituted Gln⁵⁴ for apolar Leu in the recombinant fragment of the rat COMP domain. Biochemical studies and circular dichroism (CD) spectroscopy showed that the mutant, similarly to the wild-type (w.t.) peptide, forms spontaneously an α-helical pentamer. Thermal transitions of the w.t. and mutant pentamers were analyzed by CD spectroscopy and differential scanning calorimetry. The Gln⁵⁴Leu mutation increased the thermal stability of the pentamer with reduced disulfide bonds from 73°C to 104°C. The denaturation of the disulfide bonded w.t. pentamer was observed at 108°C while the mutant pentamer cannot be denatured up to 120°C (the apparatus limit). Thus, by Gln⁵⁴Leu mutation we found a way to significantly stabilize the coiled coil pentamer, making this peptide even more attractive as an oligomerization tool for various biotechnological applications.     

Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation

Cyanobacteria acclimate to low temperature by desaturating their membrane lipids. Mutant strains of Synechococcus sp. PCC 7002 containing insertionally inactivated desA (Δ12 acyl-lipid desaturase) and desB (ω3 acyl-lipid desaturase) genes were produced, and their low-temperature susceptibility was characterized. The desA mutant synthesized no linoleic acid or α-linolenic acid, and the desB mutant did not produce α-linolenic acid. The desA mutant grew more slowly than the wild-type at 22° C and could not grow at 15° C. The desB mutant could not continuously grow at 15° C, although no observable phenotype appeared at higher temperatures. It has been shown that expression of the desA gene occurs at 38° C and is up-regulated at 22° C, and that the desB gene is only expressed at 22° C. These results indicate that the expression of the desA and desB genes occurs at higher temperatures than those at which a significant decline in physiological activities is caused by the absence of their products. The temperature dependency of photosynthesis was not affected by these mutations. Since chlorosis and inability to grow at 15° C with nitrate was suppressed by the substitution of urea as a nitrogen source, it is very likely that the chilling susceptibility of the desaturase mutants is attributable to nutrient limitation. Received: 24 April 1997 / Accepted: 5 August 1997     

HSP16.6 Is Involved in the Development of Thermotolerance and Thylakoid Stability in the Unicellular Cyanobacterium, Synechocystis sp. PCC 6803

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress. Received: 14 October 1999 / Accepted: 16 November 1999     

The fatty acid constitution and ordering state of membranes in dominant temperature-sensitive lethal mutation and wild-typeDrosophila melanogaster larvae

The ordering state and changes in fatty acid composition of microsomal (MS) and mitochondrial (MC) membranes of two dominant temperature-sensitive (DTS) lethal mutations and the wild-type Oregon-R strain larvae ofDrosophila melanogaster have been studied at 18 and 29°C and after temperature-shift experiments. The membranes of wild-type larvae have a stable ordering state, with “S” values between 0.6 (18°C) and 0.5 (29°C) in both membranes which remained unchanged in shift experiments, although the ratios of saturated/unsaturated fatty acids were changed as expected. The stronglyDTS mutation1(2)10 ^DTS forms very rigid membranes at the restrictive temperature (29°C) which cannot be normalized after shift down, while shift up or development at the permissive temperature results in normal ordering state. This mutant is less able to adjust MS and MC fatty acid composition in response to the growth temperature than the wild type. The less temperature-sensitive1(2)2 ^DTS allele occupies an intermediate state between Oregon-R and1(2)10 ^DTS in both respects. We assume and the genetical data suggest that the DTS mutant gene product is in competition with the wild-type product, resulting in a membrane structure which is not able to accommodate to the restrictive temperature.     

Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli

Two ssb mutants of Escherichia coli, whic carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation. We have investigated the influence of SSB on the “SOS” repair pathway by examining the levels of recA protein synthesis. These strains fail to induced normal levels of recA protein after treatment with nalidixic acid or ultraviolet light. The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells. This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30–41.5°). In contrast, the ssb-1 mutant has no effect on temperature-induced recA induction in a recA441 (tif-1) strain. Cells carrying ssb⁺ plasmids and overproducing normal DNA-binding protein surprisingly are moderated UV-sensitive and have reduced levels of recA protein synthesis. Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensivitiy of ssb mutant. Three possible mechanisms to explain the role of SSB are discussed.     

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: II. Active mutant proteins matured at 30 °C

Little is known of the molecular mechanisms by which temperature-sensitive mutations interfere with the formation of biologically active proteins. We have studied the effects of such mutations at 13 different sites on the properties of the multifunctional tail spike protein of bacteriophage P22, a thermostable structural protein composed of 76,000 M_r chains.Using multiple mutant strains blocked in capsid assembly, we have examined the free mutant tail spikes that accumulate in active form at permissive temperature. When assayed for the ability to bind to phage heads at the restrictive temperature, the mutant proteins were as active as the wild type. Similarly, when assayed for the ability to adsorb to bacteria at restrictive temperature, the mutant proteins were as active as the wild type. Thus the temperature-sensitive phenotypes of the mutants are not due to the thermolability of these functions in the mature mutant protein.The wild-type protein is heat-resistant, requiring incubation at 90 °C, to give a half-time of inactivation of ten minutes. The 13 ts mutant proteins, once matured at 30 °C, were as resistant as the wild-type protein to inactivation at elevated temperatures.Though the mature wild-type protein is heat stable, its maturation is heat-sensitive; the number of polypeptide chains synthesized at 30 °C and 39 °C is the same, but the yield of active tail spikes at 39 °C is only 25% of the yield at 30 °C.The results show that the amino acid substitutions in the mutant proteins, though lethal for the formation of the virus at 39 °C, do not affect the thermostability of the mature tail spike protein formed at 30 °C. They may act by destabilizing thermolabile intermediates in the folding or subunit assembly of the tail spike protein.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.