The effect of non-mutagenic stress on liquid holding recovery in Escherichia coli AB2463.

Previous work has shown that exposure to many non-mutagenic stresses causes greater UV resistance in Escherichia coli K12 via an error-free, excision repair-dependent process. Induction of the latter should enhance liquid holding recovery in the bacteria. The results in this paper show that this is the case and that the increased UV-resistance is due entirely to an increase in the capacity of the cells for DNA excision repair. The latter arises wholly or in part from an increase in the intracellular level of the key enzyme of the pathway, UvrABC endonuclease.

Starvation as an inducer of error-free DNA repair in Escherichia coli.

Previous work in this laboratory has shown that heat shock or vitamin B1 deprivation induces an error-free DNA-repair process in Escherichia coli. The system is absolutely dependent on excision repair, while its induction is delayed in lon- or recA- cells. We have now shown that starvation of E. coli for amino acids, glucose or phosphate, conditions known to induce the stringent response or the glu and pho regulons, respectively, leads to a similar uvrA-dependent increase in UV resistance and decrease in UV-induced mutation frequency. These results support the hypothesis that the effect is a general response to non-mutagenic stress that may play an important role in the survival of cells exposed to harsh environments.

Induction of error-free DNA repair in Escherichia coli by thiamine deprivation.

Incubation of Escherichia coli AB1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine prototrophy which are abolished in its uvrA mutant, but only delayed in lon- and recA- cells. The response of the lexA3 mutant resembles that of the parental cells. These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process. They may represent a general response to non-mutagenic stress in these cells.

Studies of adenosine incorporation in Langendorff rat heart and rat heart mitochondria.

The acid-insoluble product isolated from well-oxygenated Langendorff rat heart after perfusion with [14C]adenosine was purified by phenol extraction and subjected to specific phosphorolysis by pure polynucleotide phosphorylase. TLC analysis of the reaction mixture showed that ADP was the only radioactive product, proving that the original substance was a polyribonucleotide. Studies of the time course of labelling and of the distribution of the acid-insoluble product between the mitochondrial and nuclear fractions showed that both are labelled even after 1 min at 25 degrees C, but at short times and low temperature more radioactivity is found in the mitochondria. The kinetics of adenosine incorporation resemble those expected for the labelling of hnRNA and mRNA. Isolated, respiring mitochondria incorporate adenosine and adenine nucleotides into acid insoluble form by a process dependent on oxidative phosphorylation and the adenine nucleotide translocase that is specific for adenine derivatives. The results are discussed in terms of the hypothesis that the polyribonucleotide might be a storage form of adenine nucleotides: it is concluded that the bulk of the labelled product is unlikely to play a major role in energy metabolism.

The fate of thymine-containing dimers in ultraviolet-irradiated Halobacterium cutirubrum.

Extremely halophilic bacteria do not recover in the dark from the effects of ultraviolet radiation, although they can be completely photoreactivated, thus proving that pyrimidine dimers are the cytotoxic photoproducts. We have now shown, by studying the fate of thymine-containing dimers during dark liquid-holding of Halobacterium cutirubrum, that the lack of dark repair in these bacteria is due to their inability to excise such lesions, in contrast to Escherichia coli B. Nevertheless, extensive nonspecific degradation of DNA occurs in H. cutirubrum following ultraviolet irradiation, so the lack of dimer excision is not due to a generalised inability to degrade DNA after such treatment. These findings raise interesting questions concerning the basis of the resistance of halophiles to ultraviolet radiation.

Further studies on the presumed adenine nucleotide storage compound of rat heart.

Further studies on the acid-precipitable radioactive substance formed during perfusion of Langendorff rat hearts with [14C]adenosine have shown that very brief (30 s) ischaemia causes a sudden rise (20-35%) in its level in the tissue which is followed by the steady fall we have previously described. Analysis of the products of alkaline hydrolysis of this compound shows that at least 96% of the radioactivity appears in the form of a mixture of 2'- and 3'-AMP as would be expected for RNA while its relatively high resistance to dilute alkali suggests that it is poly A. Subcellular localization studies indicate that radioactivity enters all compartments of the cell, with maximum label in the nucleus. However, a significant proportion is present in the mitochondria and may be poly A acting as the mitochondrial storage form of adenine nucleotides whose existence we have proposed.

Preparation and properties of highly-purified Vibrio costicola polynucleotide phosphorylase.

Vibrio costicola polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) has been purified to electrophoretic homogeneity. It has an approximate molecular weight of 220 000 and consists of identical subunits with an approximate molecular weight of 72 000. The enzyme appears to be a fairly typical polynucleotide phosphorylase with respect to its pH optima, substrate specificity and requirement for a divalent cation cofactor. However, the effect of salt concentration on its physiologically important phosphorolysis activity suggests that it is a moderately halophilic enzyme, able to function at the intracellular ionic strength of the bacterium. In addition, its ADP polymerization activity is remarkably stimulated by polylysine.

A possible adenine nucleotide storage form in normal and ischaemic rat heart.

Perfusion of Langendorff rat hearts with [14C]adenosine yields an acid-insoluble, radioactive product whose concentration falls during ischaemia. The properties of the substance show that it is a polyribonucleotide. It is suggested that it may be mitochondrial poly A acting as a storage form of adenine nucleotides.

Photoreactivation in pigmented and non-pigmented extreme halophiles.

The sensitivity to ultraviolet radiation (254 nm) and the photoreactivability of four pigmented and three colourless strains of the extremely halophilic bacteria Halobacterium cutirubrum and Halobacterium salinarium have been studied. The results with three pigmented/non-pigmented pairs show that the pigments play an accessory role in photoreactivation at low visible light intensities and confirm that they do not provide passive protection against ultraviolet light. Evidence is presented that photoreactivation plays an unexpected direct role in the resistance of extreme halophiles to ultraviolet radiation and that colourless mutants of H. cutirubrum NRC 34001 only arise in cultures that have been both ultraviolet-irradiated and photoreactivated. None of these extreme halophiles is capable of excision repair of ultraviolet damage to DNA.

A comparison of liquid-holding recovery and photoreactivation in halophilic and non-halophilic bacteria.

The ability of the extreme halophile Halobacterium cutirubrum to recover from the effects of ultraviolet radiation during liquid holding in the dark in non-nutrient medium has been compared with that of (i) a moderately halophilic bacterium (NRC 41227) and (ii) Escherichia coli B. The photoreactivabilities of all three bacteria have also been studied. The extreme halophile was incapable of liquid-holding recovery in these conditions, in marked contrast to both E. coli B and the moderate halophile, and also failed to recover when held in nutrient medium in the dark. These results strongly support the hypothesis that H. cutirubrum lacks DNA excision repair. It was also found that ultraviolet-irradiated H. cutirubrum could be almost completely photoreactivated from any level of survival in the range 10(-4)-80%, provided exposure to visible light was not delayed, whereas the moderate halophile resembled E. coli B and had a comparatively limited capacity for photoreactivation.

Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum.

1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.

Partial purification and properties of Halobacterium cutirubrum L-alanine dehydrogenase.

1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.

Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase.

1. An alkaline phosphatase was partially purified from extracts of Halobacterium cutirubrum. 2. The enzyme has a mol.wt. of 15 500 and is therefore less than one-quarter of the size of other known bacterial alkaline phosphatases. 3. It is stimulated up to ten-fold by Mn2+, but not by Ca2+ or Mg2+. 4. The activities with and without Mn2+ cannot be separated by gel filtration and have similar restricted substrate specificities. 5. The only substrates for the enzyme that have so far been found are p-nitrophenyl phosphate, 5'-dATP, 5'-dTMP and 5'-dTTP.

Evidence for the lack of deoxyribonucleic acid dark-repair in Halobacterium cutirubrum.

1. Halobacterium cutirubrum does not perform dark-repair of DNA either after u.v. irradiation or during normal growth. 2. Cultures irradiated with u.v. are readily photoreactivated, but do not recover viability in the dark. 3. No increase in the rate of DNA synthesis is observed in the surviving cells after u.v. irradiation. 4. At early times during normal semiconservative replication, newly incorporated thymidine is found only in the hybrid DNA. 5. It is suggested that these bacteria may be useful in the study of DNA replication and photoreactivation.

The relationship between the deoxyribonucleic acid-bound and low-molecular-weight soluble forms of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase.

1. Slow, spontaneous lysis of Halobacterium cutirubrum in 3 M-KCl yields DNA-dependent RNA polymerase as a complex with DNA that sediments completely at 45 000g. 2. Controlled deoxyribonuclease digestion of the complex, with or without subsequent sonication, releases the enzyme quantitatively in a soluble form that passes through ultrafilters with a molecular-weight exclusion limit of 50 000. 3. Purification of the active ultrafiltrate by gel filtration and hydroxyapatite chromatography gives a high yield of the purified alpha and beta subunits. 4. The low mol.wt. (17 800-19 000) of the soluble enzyme was confirmed by gel filtration and is unchanged by sonication of the DNA-enzyme complex. 5. A new assay applicable to both forms of the enzyme was developed. 6. The bivalent-cation requirement of the soluble form depends on the buffer concentration. 7. Both the DNA-enzyme complex and the low-molecular-weight soluble forms of the polymerase catalyse formation of short RNA chains only.

A study of the localization of polynucleotide phosphorylase within rat liver cells and of its distribution among rat tissues and diverse animal species.

1. Rat liver polynucleotide phosphorylase was localized in the mitochondrion, but may also occur in the nucleus. 2. The mitochondrial enzyme was found in rat heart, kidney, liver, muscle and spleen. 3. Mitochondrial polynucleotide phosphorylase is also present in calf, chicken, guinea-pig and rabbit liver and in goldfish muscle. 4. A possible physiological role for the enzyme in the control of the intramitochondrial ADP concentration is suggested.

Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase.

1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.