Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.     

Genetic analyses of reddish-brown polyoxin-resistant mutants of Bipolaris maydis

In the southern corn blight fungus, Bipolaris maydis, five polyoxin-resistance genes (Pol1 to Pol5) have been reported. Pol2 and Pol5 are pleiotropic for not only polyoxin resistance but also reddish brown colonies. Here, we used a comparative genomics approach to identify Pol2 and Pol5 at a molecular genetics level. Our analysis revealed that nucleotide sequence variations in the genes for hydroxymethylbilane synthase (HMBS) and ferrochelatase (FECH) were linked to the phenotypes of Pol2 and Pol5, respectively. Further variations in the nucleotide sequences of these genes were also found in other strains of Pol2 and Pol5. Complementation tests with the wild type genes confirmed that these mutations at Hmbs and Fech were responsible for the polyoxin resistance in the Pol2 and Pol5 mutants. The deletion mutants of these genes (ΔHMBS and ΔFECH) were conditionally lethal without exogenous heme. The heme contents of Pol2 and Pol5 mutants were lower than that in the wild type, suggesting that the mutations in hmbs and fech reduced the functions of HMBS and FECH, although neither was completely inactivated. These results suggested Pol2 and Pol5 encode HMBS and FECH, members of enzymes in the heme-biosynthetic pathway of this fungus.     

Isolation,Characterization, and Antifungal Susceptibility of Melanin-Deficient Mutants of <Emphasis Type="Italic">Scedosporium prolificans</Emphasis>

Scedosporium prolificans mutants lacking the ability to synthesize melanin were selected after ultraviolet light (UV) irradiation. UV exposure of S. prolificans conidia resulted in a high frequency of melanin-deficient (mel⁻) mutants. Stable and non-stable morphological variants were found in the population: reversion of the mutant phenotype was always to the wild-type phenotype. Based on their morphological differences, these variants were classified into five different groups that were phenotypically characterized. The mel⁻ mutants plus the wild-type strain were examined for in vitro susceptibility to antifungal agents with different and/or the same mechanism of action. There was no apparent difference in minimum inhibitory concentrations when comparing the wild-type and the mel⁻ mutants. Therefore, melanin does not appear to confer protection against the more important antifungal agents in S. prolificans. Received: 30 April 2002 / Accepted: 10 July 2002     

The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage

Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants’ viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants’ responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

Variability in Sensitivity to Polyoxin B of Isolates of Alternaria mali and Decreased Fitness of Polyoxin resistant Isolates

The 540 monoconidial isolates of Altemaria mali were obtained in 1983 from apple orchards at Seoul, Suweon, Cheongju, Kochang, Daegu, and Jinju in Korea. The sensitivity of A. mali to polyoxin B greatly varied among isolates and locations. Most of isolates were sensitive to polyoxin B, but 11 isolates showed a high level of resistance, particularly from Kochang and Daegu where polyoxin B had been applied frequently. Conidial germination and mycelial growth of resistant isolates were not inhibited at higher concentrations of polyoxin B compared to the sensitive ones. The polyoxin-resistant isolates were not resistant to the fungicides iprodione and polydong and showed a reduced activity for conidial formation and mycelial growth. The isolates of A. mali with resistance to polyoxin B appear to have decreased fitness in nature.     

On the identity of dnaP and dnaF genes of Bacillus subtilis

Summary The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.Abbreviations and symbols HPUra 6-(p-hydroxyphenylazo)-uracil; mic, minimum inhibitory concentration - MMS methyl-methanesufonate - Pol I Pol II and Pol III: DNA polymerase I, II and III respectively - PCMB parachloro-mercuri-benzoate     

Chitin synthetase ofNeurospora crassa: Inhibition by nikkomycin,polyoxin B,and UDP

The inhibitory effects of nikkomycin, polyoxin B, and UDP were tested on particulate chitin synthetase activity (UDP-2-acetamido-2-deoxy-D-glucose: chitin-4-B-acetamidodeoxy-D-glucosyltransferase, E.C.2.4.1.16) fromNeurospora crassa. Two approaches were used: (a) inhibitors were tested for their individual effects on chitin synthetase activity; (b) paired combinations of inhibitors were examined to establish whether the compounds affected inhibition by binding at the same enzyme site. The first method showed that the three compounds are linear competitive inhibitors, i.e. each competes directly with the substrate for binding. K_{i app} values were: UDP, 0.8 mM; polyoxin B, 32 M; and nikkomycin, 2 M. The second method showed that the inhibitors compete with each other for binding; thus they bind at the same site. The pyrimidine nucleoside moiety of these inhibitors is an essential component for effective inhibition, but the potency of inhibition is critically dependent on the conformation of the side group attached to carbon 5 of the ribose sugar.     

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IV^R) or the cleft (Pol IV^C) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IV^R and Pol IV^C, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.     

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Homogeneity of the Morphological Groups of Bacteriophages Infecting <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> Strain HSP40 and Strain RYC2056

Bacteriophages infecting Bacteroides fragilis strains RYC2056 and HSP40 have been proposed as indicators of water quality. To accomplish this function, homogeneity of the group of phages detected by these strains is necessary to ensure that the final results are not due to the different kinetics of inactivation of the phages. To evaluate homogeneity, we observed by electron microscopy bacteriophages isolated from sewage with two Bacteroides fragilis strains (HSP40 and RYC2056). A predominant group of phages was observed, Siphoviridae with slightly curved tails. Detection of other minority groups, which could be present in the sample, was done with neutralization experiments by using antiserum against the majority group and with host mutants resistant to infection with the predominant phage. Although two other minority groups were observed, results showed that bacteriophages infecting B. fragilis strain HSP40 and strain RYC2056 form a homogeneous group, Siphoviridae with slightly curved tails being the most predominant in sewage. Received: 7 March 2002 / Accepted: 5 August 2002     

Growth inhibition effect of pyroligneous acid on pathogenic fungus,<Emphasis Type="Italic">Alternaria mali</Emphasis>, the agent of Alternaria blotch of apple

We investigated the growth inhibition effect of pyroligneous acid on the pathogenic fungus,Alternaria mali, which is known to be the agent of Alternaria blotch of apple plants. Chemical control ofA. mali could be achieved through the use of agrochemical fungicides, while the substitute for agrochemical control is gradually increasing. It was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi. More specifically, the growth ofA. mali was completely inhibited in pyroligneous acid at a dilution of 1∶32. When its antifungal activity was compared to that of polyoxin B, which is used for the chemical control of Alternaria blotch of apple, it was observed that the antifungal activity of pyroligneous acid diluted at 1∶32 corresponded to 2.0 mg/mL of polyoxin B. Consequently, it is concluded that the diluted pyroligneous acid can substitute for polyoxin B, thereby reducing the use of the agrochemical for the control of Alternaria blotch of apple.     

Growth,differentiation and secondary metabolites of colour mutants ofTrichoderma viride

The parental strainTrichoderma viride and 3 colour mutants (milk white, yellow and brown) blocked at various stages of colony pigmentation derived from it were characterized. The parental strain and the mutants exhibited different growth rates. The identical type of induced fluorescence was observed in all the strains. Hyphae and septa lighted first, whereas reproduction structures did not; after treatment with fluorescein-isothiocyanate and Blankophor RKH the growing hyphal apices were accentuated. In the mutants conidiation was induced at 1-, 2- and 3-d intervals, similarly to the parent strain. Pigmentation of conidiation rings depended on their type and age. The yellow and brown mutants excreted chromatographically different pigments, extractable with ethylacetate, into the medium. Two anthraquinone pigments,viz. 1,3,6,8-tetrahydroxyanthraquinone (1) and 1-acetyl-2,4,5,7-tetrahydroxyanthraquinone (2) were isolated from the brown mutant (Betinaet al. 1986).     

Design,synthesis and evaluation of novel quinazoline-2,4-dione derivatives as chitin synthase inhibitors and antifungal agents

A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized, and characterized by ¹H NMR, ¹³C NMR and MS spectral data. Their inhibition against chitin synthase (CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j, 5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency among these compounds, had a half-inhibition concentration (IC₅₀) of 0.08 mmol/L, while polyoxin B as positive drug had IC₅₀ of 0.18 mmol/L. These IC₅₀ values of compounds 5i, 5m, 5n, and 5s were greater than 0.75 mmol/L, which revealed that those compounds had weak inhibition activity against CHS. Moreover, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the minimum-inhibition-concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to their IC₅₀ values against CHS. Furthermore, these compounds had negligible actions to bacteria. Therefore, these compounds were promising selective antifungal agents.     

Isolation and characterization of pigment mutants from a filamentous cyanobacterium

Five strains of a pigment mutant were isolated following UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis from a non-nitrogen fixing mutant of the cyanobacteriumGloeotrichia ghosei. Two of them (B-1 and V-1) were isolated by UV mutagenesis and other three (B-3, B-7 and Br-6) by MNNG mutagenesis. Among the five strains cultures of three strains (B-1, B-3 and B-7) were typically blue-green in colour. Culture of strain V-1 was found to be violet-pink and of Br-6 was brownish in colour. The parent strain of these mutants was dark-blue in colour. Blue-green mutants showed the predominance of phycocyanin (610 nm) whereas violet-pink and brown strains showed the predominance of phycoerythrin (550 nm) in the absorption spectra of water-soluble pigments. In contrast to these strains their parent strain showed both the absorption peaks (at 550 and 610 nm). Occurrence of stable pigment mutants of a filamentous cyanobacterium indicates that the synthesis of water-soluble pigments is genetically controlled in these mutant strains.     

Mutants of the Phytopathogenic FungusMagnaporthe griseaDeficient in Alternative, Cyanide-Resistant, Respiration

The phytopathogenic fungusMagnaporthe griseahas a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport ofM. griseaand induces the alternative respiratory pathway. Twelve mutants ofM. griseamore susceptible to SSF-126 than wild type were identified afterN-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein ofM. grisea,indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.