Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Biochemical genetic studies of cycloheximide resistance in Neurospora crassa

Genetic analysis of a number of cycloheximide-resistant mutants of Neurospora crassa has shown that resistance is controlled by several genes. Two of these appear to be located on linkage group V. Resistance to the antibiotic is dominant in wild-type-mutant heterokaryons. Two types of cycloheximide-resistant mutants were isolated: one type exhibited colonial morphology only when grown in the presence of cycloheximide and the other type maintained normal morphology even at high concentrations of the antibiotic. Reconstitution experiments with supernatant solutions and 80S monosomes prepared from wild-type and resistant mutant strains indicated that the property of cycloheximide resistance most likely is associated with the ribosomes. No electrophoretic or serological differences were found between the ribosomal proteins of the wild-type and resistant mutants.     

Interactions of bacteria with cadmium

Cadmium pollution arises mainly from contamination of minerals used in agriculture and from industrial processes. The usual situation is of large volumes of soil and water that are contaminated with low — but significant — concentrations of cadmium. Therefore, detoxification of the polluted water and soil involves the concentration of the metal, or binding it in a way that makes it biologically inert.Cadmium is one of the more toxic metals, that is also carcinogenic and teratogenic. Its effects are short term, even acute (diseases like Itai-itai), or long term. The long term effects are intensified due to the fact that cadmium accumulates in the body.This paper describes a study involving several hundred cadmium-resistant bacterial isolates. These bacteria could be divided into three groups—the largest group consisted of bacteria resistant to cadmium by effluxing it from the cells. The bacteria of the other two groups were capable of binding cadmium or of detoxifying it. We concentrated on one strain that could bind cadmium very efficiently, depending on the bacterial biomass and on the pH. This strain could effectively remove cadmium from contaminated water and soil.     

Genetic and Physiological Characteristics of a Slow-Growing Circadian Clock Mutant of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa with a period length of about 25.8 hours (4 hr longer than wild type) has been isolated after mutagenesis of the band strain. This mutant, called frq-5, segregates as a single nuclear gene, maps near the centromere on linkage group III, and is unlinked to four previously described clock mutants clustered on linkage group VII R (Feldman and Hoyle 1973, 1976). frq-5 differs from the other clock mutants in at least two other respects: (1) it is recessive in heterokaryons, and (2) it grows at about 60% the rate of the parent band strain on both minimal and complete media. Double mutants between frq-5 and each of the other clock mutants show additivity of period length--two long period mutants produce a double mutant whose period length is longer than either of the two single mutants, while a long and a short period double mutant has an intermediate period length. Although slow growth and long periodicity of frq-5 have segregated together among more than 300 progeny, slow growth per se is not responsible for the long period, since all the double mutants have the slow growth characteristic of frq-5, but have period lengths both shorter and longer than wild type.     

Nuclear mutations conferring oligomycin resistance in Neurospora crassa.

Mutants of Neurospora crassa have been isolated that are highly resistant to inhibition by oligomycin, an inhibitor of mitochondrial ATPase activity. Dixon plots (Dixon, M., and Webb, E.C. (1964) Enzymes, 2nd Ed, pp. 328-330, Academic Press, New York) of oligomycin inhibition curves of the parent strain and the resistant mutants are linear, indicating that oligomycin interacts at a single site within the ATPase complex. The Ki values obtained from the mutants vary from 150 to 900 times greater than the Ki obtained for the parent strain. The parent strain and the oligomycin-resistant mutants are also inhibited by bathophenanthroline, a lipophilic chelating agent that inhibits F1 ATPase activity. Dixon plots of bathophenanthroline inhibition curves are also linear and Ki values obtained are all approximately equal. Crosses of the oligomycin-resistant mutants to the oligomycin-sensitive parent strain show a mendelian segregation of the resistance characteristic. These data show that mutations leading to oligomycin resistance in Neurospora are due to alterations in nuclear genes.     

Crossing analysis of sorbose resistant mutants of Neurospora crassa

Summary After treatment of the conidia of Neurospora crassa with nitrous acid 19 of the resulting mutants were selected for their resistance to sorbose, an agent which has a toxic effect on the wild type. The selected sorbose resistant mutants were analyzed genetically by crossing them with tester strains containing known markers. Mutational sites were thus found to be located on at least 5 of the full complement of 7 linkage groups. 9 sites were found to be located on the left arm of linkage group VI near the ylo-marker. These were so far not separable by recombinational analysis and therefore seem to represent a single gene-locus (A). Other sorbose resistant sites were mapped on linkage groups I, III (gene C), V and VII (gene B). Another site which has not yet been definitely located is not closely linked to any of the aforementioned. One site each on linkage groups I and VI were mapped more precisely by means of 3-point crosses. These crosses permitted the location of these sites of sorbose resistance in regard to neighbouring markers. From these crosses it is concluded that the mutation of any 1 of at least 6 different chromosomal genes can cause the formation of a sorbose resistant phenotype. The mutation of any 1 of 4 of these genes could result in a mutant with an altered sorbose permease system or containing a suppressor which decreases the efficiency of the permease system.

---

---

I. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.     

Cadmium carcinogenesis

Cadmium is a heavy metal of considerable environmental and occupational concern. Cadmium compounds are classified as human carcinogens by several regulatory agencies. The most convincing data that cadmium is carcinogenic in humans comes from studies indicating occupational cadmium exposure is associated with lung cancer. Cadmium exposure has also been linked to human prostate and renal cancer, although this linkage is weaker than for lung cancer. Other target sites of cadmium carcinogenesis in humans, such as liver, pancreas and stomach, are considered equivocal. In animals, cadmium effectively induces cancers at multiple sites and by various routes. Cadmium inhalation in rats induces pulmonary adenocarcinomas, in accord with its role in human lung cancer. Cadmium can induce tumors and/or preneoplastic lesions within the rat prostate after ingestion or injection. At relatively high doses, cadmium induces benign testicular tumors in rats, but these appear to be due to early toxic lesions and loss of testicular function, rather than from a specific carcinogenic effect of cadmium. Like many other metals, cadmium salts will induce mesenchymal tumors at the site of subcutaneous (s.c.) or intramuscular (i.m.) injections, but the human relevance of these is dubious. Other targets of cadmium in rodents include the liver, adrenal, pancreas, pituitary, and hematopoietic system. With the exception of testicular tumors in rodents, the mechanisms of cadmium carcinogenesis are poorly defined. Cadmium can cause any number of molecular lesions that would be relevant to oncogenesis in various cellular model systems. Most studies indicate cadmium is poorly mutagenic and probably acts through indirect or epigenetic mechanisms, potentially including aberrant activation of oncogenes and suppression of apoptosis.     

Cellular uptake of cadmium and zinc

CHO mutants, resistant to over 100-fold of a normally toxic level of extracellular cadmium have been used to examine the mutually antagonistic effect of Cd and Zn on their uptake. Cadmium uptake in these mutants is only 7–10% that of the parental cells. Zinc uptake in these mutants is equal to or greater than that in the wild-type cells. Results of kinetic studies on uptake indicated that the two metals interact by competitive inhibition. TheK _m andK _i values for Cd and/or Zn were different in some of the mutants and indicate multiple carriers may be involved in the transport of these metals. The reduction in Cd uptake and concomitant increase in Zn uptake contribute to the increased Cd resistance in these mutants.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea)

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.     

A Mutant Affecting Meiosis in Neurospora

Many mutants affecting meiosis increase the occurrence of aneuploid meiotic products. In Neurospora, mutants of this type cause ascospore abortion which is reflected by an increase in the proportion of ascospores failing to develop black pigment. The usefulness of the criterion white-ascospore-production as a signal for the presence of a mutant affecting meiosis is demonstrated by the recovery of several such mutants. One of these is mei-1 (meiotic-1), a recessive mutant on linkage group IV. Crosses homozygous for mei-1 produce 90% white ascospores (vs. 5% in wild-type crosses). Viable ascospores, invariably black, are always disomic for one or more linkage groups; the chromatids assorted into viable ascospores do not engage in crossing over in meiosis. The distribution of viable ascospores in individual asci suggests that all meioses are defective in the first meiotic division, and that most meioses are defective in both divisions.     

Relationship between the composition of phospholipids and respiratory activity of choline-deficient mutants ofNeurospora crassa

Phosphatidylcholine is one of the most frequent phospholipid components of the inner mitochondrial membrane ofNeurospora crassa. Quantitative analysis of phospholipids of the wild strain ofNeurospora crassa and of its twocho mutants showed that these strains did not significantly differ in the content of phosphatidylcholine. Mutants cultivated in a medium without choline contained, as compared with the wild strain, an increased amount of phosphatidylserine and a decreased quantity of phosphatidic acid. Respiratory activity increased and sensitivity to inhibitors of respiration changed. It is likely that the presence of choline in the growth medium exerts a regulatory effect on the cell metabolism of these mutants.     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: the role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 muM CdSO(4), showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

Effects of acute exposure to cadmium (II) chloride and lead (II) chloride on the haematological profile of Oreochromis aureus (Steindachner)

1. Oreochromis aureus (Steindachner) was exposed to two concentrations of lead and cadmium for 24 hr and 1 week to assess the effects of these pollutants on haematological parameters.2. Plasma osmolality was found to be the most sensitive blood parameter, affected before other parameters changed.3. Cadmium appears to be more toxic to O. aureus than lead, having an adverse effect on blood parameters earlier than lead.4. In the earlier stages of toxicity cadmium appears to have a more pronounced effect on haemoglobin concentration than lead.5. Cadmium does not depress erythrocyte counts but lead does.     

Neurospora crassa Cytoplasmic Ribosomes: Isolation and Characterization of a Cold-Sensitive Mutant Defective in Ribosome Biosynthesis

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.     

Senescent: a new Neurospora crassa nuclear gene mutant derived from nature exhibits mitochondrial abnormalities and a "death" phenotype

Fungi are capable of potentially unlimited growth. We resolved nuclear types from multinuclear mycelium of a phenotypically normal wild isolate of the fungus Neurospora intermedia by plating its uninucleate microconidia and obtained a strain which, unlike the "parent" strain, exhibited clonal senescence in subcultures. The mutant gene, senescent, was introgressed into N. crassa and mapped four map units to the right of the his-1 locus on linkage group VR. senescent is the first nuclear gene mutant of Neurospora derived from nature that shows the death phenotype. Death of the sen mutant occurred faster at 34 degrees C than at 22 or 26 degrees C. Measurements of oxygen uptake of conidia using respiratory inhibitors and the spectrophotometric analyses of mitochondrial cytochromes showed that in sen cultures grown at 34 degrees C, cytochromes b and aa(3) were present but cytochrome c was absent. By contrast at 26 degrees C, cytochromes b and c were present but cytochrome aa(3) was diminished in the late subcultures. This suggested that the sen mutation does not affect the potential to produce functional cytochromes. The deficiency of the respiratory chain cytochromes may not be the cause of death of the sen mutant because the cytochrome c and aa(3) mutants of N. crassa are capable of sustained growth whereas sen is not. Possible explanations for the observations are discussed.     

P22-Mediated Transduction Analysis of the Rough A (rfa) Region of the Chromosome of Salmonella typhimurium

Seven temperature-sensitive rough mutants of Salmonella typhimurium were found to be sensitive to smooth-specific phages at low temperature (25 C, 30 C) and resistant or partially resistant to rough-specific phages, whereas at high temperatures (37 C, 45 C) they were resistant or partially resistant to smooth-specific phages but sensitive to rough-specific phages. These data indicate that at low temperature each strain makes lipopolysaccharide which is relatively normal, but at high temperatures O-specific side chains are not added to the lipopolysaccharide. At 45 C, these strains have the R-res-1 or R-res-2 phage sensitivity phenotype, and their genetic lesions map by P22-mediated transduction in the rfa gene cluster between cysE-pyrE, suggesting a mutation in genes with transferase functions. P22-mediated joint transduction with temperature-sensitive rfa mutants, leaky rfa mutants, and rfa P22 lysogens have shown the following order of genes in the S. typhimurium linkage map: xyl-mtlA-mtlB-cysE-rfaF-rfaG-pyrE. An rfaE allele was not jointly transduced in the cysE-pyrE segment.