Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima "Pink Banana."     

Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate,WBG1022

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, p WBG620 and pWBG637, that also lack resistance phenotypes.     

Comparative studies of the quality of fresh and stored mushrooms of Agaricus bisporus with two tropical Agaricus bitorquis strains

Three white strains of mushroom were grown for quality assessment tests, a commercial Agaricus bisporus strain SOMYCEL U3 currently popular in most major mushroom producing countries and two tropical Agaricus bitorquis strains, ATCC 32675 and AGC W20. Mushrooms were harvested as stage 2 mushrooms (closed buttons with universal veil intact) and stored at 18°C (± 0.5°C) for 5 days during which time colour development, the rate of fruitbody maturation and weight loss were assessed. Throughout the storage period, a reflectance colormeter was used to monitor colour changes on the tops and sides of mushroom fruitbodies. The tops of both A. bitorquis strains were significantly more yellow than the A. bisporus strain, whereas the sides were significantly less yellow. Overall, the A. birorquis strain AGC W20 was clearly the least discoloured and least yellow at the time of harvest. Although all the three strains tested gave similar fresh weight losses during storage, i.e. approximately 10% per day, ATCC 32675 exhibited a very slow maturation rate. Both U3 and AGC W20 matured at a similar much faster rate forming open cups within the 5 day storage period. ATCC 32675 also showed the least increase in the degree of discolouration with time, whether readings were taken from the sides or tops of mushrooms. A breeding programme to combine the most salient features of AGC W20 (an intensely white mushroom at harvest, high yielding with distinct flush pattern) and ATCC 32675 (very slow maturation rate during storage) is suggested.     

Restriction Fragment Length Polymorphisms in the Mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML3, and M18

Lactose- and proteinase-negative (Lac⁻ Prt⁻) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac⁻ Prt⁻ mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

A mitochondrial plasmid from the plant pathogenic fungus Cochliobolus heterostrophus

Summary Twenty-three strains of Cochliobolus heterostrophus were examined for the presence of plasmid DNA. One isolate, T40, contained a 1.9 kb sequence which occurred as a series of circular head-to-tail multimers with from 1 to 17 or more monomers per plasmid molecule. The plasmid was cloned in pBR322 to facilitate analysis. It was homologous to the mitochondrial chromosome of isolate T40 as well as to the mitochondrial DNAs of C. heterostrophus isolates that did not contain the plasmid; each isolate, including T40, had only one copy of the plasmid sequence integrated into the mitochondrial chromosome and the sequence mapped at the same location in all isolates tested. In the T40 isolate there were about 30 excised copies per chromosome in addition to the single integrated sequence. Presence of the plasmid had no apparent effect on the structural integrity of the mitochondrial chromosome. There was no detectable homology between the plasmid and either C. heterostrophus nuclear DNA or plasmids that have been isolated from mitochondria of Neurospora or Podospora. A circular map was constructed which has 6 sites for hexan-ucleotide-recognizing enzymes and the region of the splice site; no sites were detected in the plasmid for an additional 17 restriction enzymes. The plasmid functioned as an ARS (autonomously replicating sequence) in yeast, although it was highly unstable compared to other ARSs.     

Structural characterization of a water-soluble beta-(1-->6)-linked D-glucan isolated from the hot water extract of an edible mushroom, Agaricus bitorquis

A water-soluble polysaccharide, isolated from the hot aqueous extract of an edible mushroom, Agaricus bitorquis, was found to consist of d-glucose only. On the basis of total hydrolysis, methylation analysis, and NMR studies (¹H, ¹³C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit was established as→6)-β-d-Glcp-(1→     

Direct action of ethidium bromide upon mitochondrial oxidative phosphorylation and morphology.

Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount P_i released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of P_i during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [¹⁴C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure.     

Mechanism of Mitochondrial Mutation in Yeast

THE yeast Saccharomyces cerevisiae can mutate to the respiratory-incompetent petite colony form. The mutation is probably caused by damage to, or loss of, the yeast's mitochondrial DNA, for petite mutants often lack mitochondrial DNA, possess it in abnormal amounts or with abnormal buoyant density¹. Some of the agents, such as acrifiavine or ethidium bromide, which induce the petite mutation interfere with mitochondrial DNA synthesis^2,3 whereas ethidium bromide also causes or permits degradation of Saccharomyces cerevisiae mitochondrial DNA^2,3. We have observed that nalidixate (50 µg/ml.), an inhibitor of DNA synthesis, can prevent or delay petite mutation induced by ethidium bromide⁴. A similar effect has been observed by Hollenberg and Borst using a higher nalidixate concentration⁵. We have investigated the mechanism of this effect. A diploid prototrophic strain of Saccharomyces cerevisiae (NCYC 239) was used throughout.     

Isozyme characterization of dikaryotic strains of the edible basidiomyceteAgaricus bitorquis (Quel.) Sacc. (syn.Agaricus edulis)

Seven isozyme activities were studied by isoelectric focusing and blotting of total protein extracts of dikaryotic strains ofAgaricus bitorquis (Quel.) Sacc. (syn.Agaricus edulis) to characterize strains and varieties and to provide information for subsequent protection of new putative commercial varieties. Isoelectric focusing gave reproducible patterns and blotting onto nitrocellulose membrane increased the sensitivity of enzyme detection. Five activities showed high variability: alcohol dehydrogenases, dopa-reacting enzyme phenoloxidases, tolidine-reacting enzyme phenoloxidases, esterases, and peroxidases. Two activities, diaphorases and acid phosphatases, presented low variability. Compilation of patterns obtained for the seven isozyme activities allowed the distribution of the closely genetically related varieties into separate groups. The five enzyme activities with high variability were sufficient to discriminate all theA. bitorquis varieties tested and to define a method for characterization and protection of new strains. Analysis of these patterns and comparison with other higher fungi showed that the variability inA. bitorquis is comparable with those described forPleurotus eryngii, Coprinus congregatus, and·Lentinus edodes, and higher than the variability found forAgaricus bisporus.     

Supercoiling, knotting and replication fork reversal in partially replicated plasmids

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter–TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive ΔLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.     

Relationship between plasmid occurrence and antibiotic resistance in Myroides odoratimimus SKS05-GRD isolated from raw chicken meat

Fifteen flavobacterium strains were isolated from raw chicken meat, raw goat meat and poultry soil in Coimbatore, Tamil Nadu. Most of the isolates developed yellow pigmented colonies with mucoid-spreading edges on food flavobacterium medium. The flavobacteria were Gram-negative rods and failed to produce indole and were non-fermentative. Moreover, they produced a rich array of enzymes such as amylase, lipase, catalase, urease, gelatinase, DNase, and oxidase. Phylogenetic analyses of the strain SKS05-GRD based on 16S rRNA gene sequences revealed the bacterium as Myroides odoratimimus (nucleotide sequence accession number JQ178355). Antimicrobial susceptibility test for M. odoratimimus SKS05-GRD and other strains were assessed by disc diffusion method. M. odoratimimus SKS05-GRD showed wide resistance to the antibiotics such as amikacin, ampicillin, cefadroxil, cefoperazone, ceftazidine, ceftriaxone, netillin and gentamicin. M. odoratimimus was subjected to plasmid isolation and plasmid curing to seek the relationship between plasmid and antibiotic resistance. Plasmid curing was done by using ethidium bromide and was found to be effective at 300 and 500 μg/ml. Assessment of antibiotic sensitivity of M. odoratimimus SKS05-GRD showed sensitivity to amikacin, gentamicin and kanamycin confirming that resistance to these three antibiotics is plasmid mediated and other antibiotic resistance are chromosomal mediated.     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.Abbreviations Ap ampicillin - ARS autonomously replicating sequence - EtBr ethidium bromide - kb kilobase(s) - Mc. Methanococcus - R resistance - RE replication enhancer - RS replication sequence - Tc tetracycline     

Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18

Lactose- and proteinase-negative (Lac Prt) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac Prt mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.