Characterization of glucose oxidase-negative mutants of a lignin degrading basidiomycete Phanerochaete chrysosporium

The isolation and characterization of glucose oxidase-negative (gox ^-) mutants of Phanerochaete chrysosporium, is described. These mutants are deficient not only in their ability to produce hydrogen peroxide (H₂O₂) but also in lignin degradation (2-¹⁴C-synthetic lignin¹⁴CO₂), ligninase and peroxidase activities, decolorization of the dye poly-R 481, and production of ethylene from -oxo--methylthiobutyric acid (KTBA). The gox ^- mutants retained, albeit at a lower level, the capacity to produce veratryl alcohol, a typical secondary metabolite, and produced conidia at a level comparable to that of the wild type. The addition of ligninase and/or glucose oxidase to a gox ^- mutant (GOX-10) did not enhance its capacity to degrade lignin. The Gox⁺ revertant strains regained glucose oxidase activity, the ability to degrade lignin, as well as the other characteristics that were missing in the gox ^- mutants. The results suggest that the genetic lesion in these mutants affects the regulation of a set of secondary metabolic characteristics.Abbreviations Gox glucose oxidase - KTBA -oxo--methylthiobutyric acid Journal article no. 11740 from the Michigan Agricultural Experiment Station     

Biogenesis of mitochondria 49 identification and mapping of a new mitochondrial locus (tsr1) which maps within polar region of yeast mitochondrial genome.

Summary An enrichment procedure which facilitates the isolation of conditional respiratory-deficient mutants of Saccharomyces cerevisiae is reported. Detailed genetic analysis of one mutant which exhibits a respiratory deficient phenotype at low temperature (18°C) is also presented. The phenotype is due to a single lesion at a new locus, tsr1, located on the mitochondrial DNA. By analysis of locus retention patterns in a set of physically characterized petite strains, the tsr1 mutation has been mapped within the segment 0–5 map units on the physical map of the yeast mitochondrial genome. This segment of the mitochondrial DNA also contains the cap1 and ery1 loci and the cistron for the mitochondrial 21S rRNA. Studies of the frequencies of co-retention of markers in petite populations, and of the frequencies of recombination of markers in non-polar crosses (⁺ × ⁺), demonstrate linkage of the tsr1 locus to both the cap1 and ery1 loci. The degree of linkage indicates that tsr1 is closer to the ery1 locus. Comparison of pairwise recombination frequencies for these three markers indicate the order cap1-tsr1-ery1. The tsr1 locus lies within the segment of the mitochondrial genome which is influenced by the polarity locus , and analysis of transmission and recombination frequencies and polarities in a polar (⁺ × ^-) cross show that the behaviour of the tsr1 locus is similar to that of ery1. However striking features of this cross are that the recombination frequency between tsr1 and ery1 is comparable to that observed in non-polar crosses, and that the polarity for recombination between tsr1 and cap1 or ery1 is extremely low.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts

A systematic investigation covering a wide diversity of yeast species was made on the appearance of respiratory deficient (petite) mutants after treatment with acriflavine. Petite mutants were obtained from certain species only, but in these species all strains were found to have in common the property of giving rise to petite mutants; such species were designated as petite positive. Species failing to give rise to petite mutants were accordingly called petite negative. The primary action of acriflavine, namely the inhibition of the synthesis of the respiratory system, was shown to occur not only in petite positive yeasts, but also in petite negative ones. Some implications of the results are discussed.     

β-nitropropionic acid and nitrite in relation to nitrate formation by Aspergillus flavus

Summary -nitropropionic acid (BNP) was converted to nitrate in media inoculated with A. flavus spores or with replacement cultures of mycelium pregrown in glucose-peptone medium. Conversion by replacement cultures was rapid: 8–30% in 2 days; influenced by pH: most rapid at pH 3.5; and extensive: as much as 80% BNP nitrogen appeared as nitrate after 14 days. Nitrite was detectable in BNP replacement cultures at low levels or not at all, and nitrate was formed in BNP replacement media with or without glucose. Nitrite was not oxidized in growing cultures inoculated with spores, but replacement cultures oxidized over 50% of added nitrite to nitrate in 8 days. No nitrite or nitrate appeared in replacement systems with pyruvic oxime, oxalacetic acid oxime, acetoxime, ketoglutaric acid oxime, or hydroxylamine.Of the three non-nitrifying mutants of A. flavus obtained, all formed nitrate from BNP in replacement but only one oxidized nitrite to nitrate. No accumulation of free or bound hydroxylamine or of nitrite could be detected in the mutants. BNP was detected by qualitative test in cultures of the wild type but not the mutants. Evidence indicates that the pathway in A. flavus is BNPNO₃ ^- rather than BNPNO₂ ^-NO₃ ^-.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

A GC cluster repeat is a hotspot for mit- macro-deletions in yeast mitochondrial DNA.

Summary In a random collection of mit^– mutations of the yeast strain 777-3A we find that deletions are exceptionally frequent in the OXI3 gene, a large mosaic gene coding for subunit I of cytochrome oxidase. About 10% of all oxi3 ^– mutants carry the same macro-deletion, del-A, extending from the 5 non-translated leader of OXI3 to intron 5b of this gene. Determination of the respective wild-type sequences and of the del-A junction sequence revealed that the end-points of the deletion are in two GC clusters with 31 by sequence identity which are located at a distance of 11.3 kb. We speculate that not only the sequence identity of the two GC clusters but also the palindromic structure of these putatively mobile elements of yeast mitochondrial DNA (mtDNA) plays a role in deletion formation.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency

Summary The uvrD gene of Escherichia coli that control UV sensitivity and spontaneous mutation frequency has been cloned with phage as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and sup126 mutations were all suppressed by lysogenization of the mutant cells with uvrD ⁺. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvD gene, uvrD ⁺ carried the corA gene that controls transport of Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD^- as well as CorA^- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Microwave Hall mobility measurements on heavy beef heart mitochondria

The observed initial microwave Hall mobility values at 1·21 tesla of heavy beef heart mitochondria is at least six times greater than that observed for bovine serum albumin at similar resistivity values. The respiratory inhibitor cyanide significantly reduces the initial Hall mobility values for HBHM and for a preparation of HBHM cytochrome oxidase.The four enzymic complexes of the respiratory chain were partially or completely separated. Of these complexes cytochrome oxidase exhibits the largest microwave Hall mobility.The maximum hydration content of loosely bound water for freezedried preparations of cytochrome oxidase is 5% by weight; 60% of this hydration content is driven off by microwave power. Since the effective ac resistivity of the samples of cytochrome oxidase did not appreciably vary with changes in hydration content, the true resistivity of cytochrome oxidase has a value of the order 5×10³ ohm cm and possibly much lower.The electron transport pathway (as measured by Hall signal) of cytochrome oxidase is irreversibly damaged by prolonged exposure to microwave irradiation at 9·2 GHz. This is accompanied by the complete loss of capacity to oxidise ferrocytochromec. Such changes do not occur with HBHM or with the other respiratory complexes.There appears to be a direct relationship between observed Hall signals and the capacity of cytochrome oxidase to oxidize ferrocytochromec. There is a background signal which is not directly related to electron transport but which is dependent on the conformation of the cytochrome oxidase.The observed electronic parameters of cytochrome oxidase do not depend appreciably on its redox state.Acid denaturation of cytochrome oxidase drastically reduces the Hall signal, to include almost complete removal of the background signal. It also more than doubles ac resistivity.An electron tunnelling model is outlined.     

Analysis of L-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: Complementation for intracellular degradation of the mutant polypeptide

Summary Null and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (-GPDH, NAD⁺ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to providing information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh ⁺. CRM levels appear to be the result of a differential sensitivity to the normal intracellular degradative process and the CRM^- mutants represent hypersensitive alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.This investigation was supported in part by NIH Research Grants No. GM-23617, AG-01739, and by NIH Training Grant No. GM 296. Paper No. 6192 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Synthetic multifunctional proteins

Summary Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and -galactosidase. The mutants were isolated as TonB^- Lac⁺ derivatives of a phenotypically Lac^- TrpR^- strain carrying a lac I ⁺-Z⁺ fusion on a 80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA^- and 3 Trp^- strains produce triple chimeras. The chimeras from the two TrpA^- strains were further examined. They consist of tryptophan synthetase -subunit, lac repressor and -galactosidase. In crude extracts of these strains the tryptophan synthetase -subunit part can be identified by its ability to aggregate with the -subunit since some of the -subunit activity can be precipitated with antiserum against -galactosidase. Furthermore -galactosidase precipitates with antiserum against tryptophan synthetase -subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The -galactosidase part is as unaffected as in the original lac repressor--galactosidase chimera. The molecular weigths of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.     

α2 and β-adrenoceptor agonists modulate [3H]dopamine release from rat nucleus accumbens slices: Implications for research into depression

The modulatory effects of noradrenergic agonists on the 25 mM K⁺-induced release of [³H]dopamine (³H-DA) from rat brain nucleus accumbens slices was investigated, using a superfusion technique. The K⁺-induced release of³H-DA was Ca²⁺ dependent, significantly enhanced (25–32%;p<0.02) by the -adrenoceptor agonist isoproterenol (10 M), and significantly decreased (13–25%;p<0.05) by the ₂-adrenoceptor agonist clonidine (10 M). At these concentrations neither drug affected basal release of³H-DA. Clonidine (100 M) increased the basal release of³H-DA, while decreasing the K⁺-induced release by 19% (p<0.01). The inclusion of desipramine in the incubation medium, to prevent accumulation of³H-DA into noradrenergic neurons, did not alter the inhibitory effect of clonidine (10 M) on³H-DA release. This study provides direct evidence that noradrenergic neurons can modulate dopaminergic neurotransmission in the mesolimbic system.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

Mitochondrial mutations restricting spontaneous translational frameshift suppression in the yeast Saccharomyces cerevisiae.

Summary The +1 frameshift mutation, M5631, which is located in the gene (oxi1) for cytochrome c oxidase II (COXII) of the yeast mitochondrial genome, is suppressed spontaneously to a remarkably high extent (20%–30%). The full-length wild-type COXII produced as a result of suppression allows the mutant strain to grow with a leaky phenotype on non-fermentable medium. In order to elucidate the factors and interactions involved in this translational suppression, the strain with the frameshift mutation was mutated by MnCl₂ treatment and a large number of mutants showing restriction of the suppression were isolated. Of 20 mutants exhibiting a strong, restricted, respiration-deficient (RD) phenotype, 6 were identified as having mutations in the mitochondrial genome. Furthermore, genetic analyses mapped one mutation to the vicinity of the gene for tRNA^Pro and two others to a region of the tRNA cluster where two-thirds of all mitochondrial tRNA genes are encoded. The degree of restriction of the spontaneous frameshift suppression was characterized at the translational level by in vivo ³⁵S-labeling of the mitochondrial translational products and immunoblotting. These results showed that in some of these mutant strains the frameshift suppression product is synthesized to the same extent as in the leaky parent strain. It is suggested that more than one +1 frame-shifted product is made as a result of suppression in these strains: one is as functional as the wild-type COXII, the other(s) is (are) non-functional and prevent leaky growth on non-fermentable medium. A possible mechanism for this heterogenous frameshift suppression is discussed.     

The bI4 RNA mitochondrial maturase of Saccharomyces cerevisiae can stimulate intra-chromosomal recombination in Escherichia coli

Summary When the bI4 RNA maturase, encoded by the fourth intron of the mitochondrial cytochrome b gene of Saccharomyces cerevisiae, was expressed in Escherichia coli, formation of intra-chromosomal Lac⁺ recombinants was stimulated threefold. This hyper-rec phenotype was recA as well as recBCD dependent. The most active form of the bI4 maturase stimulated homologous recombination whereas splicing deficient mutants of bI4 maturase were either deficient in or unable to stimulate homologous recombination.