Phenotypic characterization of a trifluoperazine-resistant mutant ofMucor rouxii

A trifluoperazine-resistant (TFP¹) mutant (strain G5) ofMucor rouxii was isolated and some biochemical and physiological parameters were studied. It resisted up to 250 μM TFP compared to 100 μM observed for the wild-type strain. At this drug concentration the mutant strain G5 germinated, grew, exhibited yeast-mycelium transition, and chitin synthesisin vivo. The mutant strain presentedin vitro levels of calmodulin activity similar to those of the wild-type, but with less sensitivity to inhibition by TFP. Also, with regard to spore germination and cell growth, mutant G5 presented cross-resistance to calmidazolium, another potent anticalmodulin drug. Partially purified chitin synthetase preparations of mutant G5 exhibited a diminished enzymatic activity, compared to the wild-type. The results presented in this work suggest the participation of a Ca²⁺-calmodulin complex in growth and differentiative processes ofMucor and substantiate the role of this complex in chitin synthesis.     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea,is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^? mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^? transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^? transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

Properties of trehalose-6-phosphate synthase fromSaccharomycopsis fibuligera

In this paper, we investigated the properties of trehalose-6- phosphate synthase (SfTps1) inSaccharomycopsis fibuligera sdu a high-trehalose-accumulating strain. The purified SfTps1 showed a band on Native-PAGE and SDS-PAGE of about 66 kDa. The optimal pH and temperature of the purified enzyme were 6.6 and 37 °C, respectively. The enzyme was activated by Ca²⁺, K⁺ and Mg²⁺, inhibited by Mn²⁺, Cu²⁺, Fe³⁺, Hg²⁺ and Co²⁺. Iodoacetic acid, EDTA and PMSF had inhibitory effect on the enzyme activity. K_m values of the enzyme for glucose-6-phosphate and UDP-glucose were 38.6 mM and 9.3 mM, respectively. The effects of various stress conditions on SfTps1 activity and trehalose content in this strain were also studied. Neither the activation of SfTps1 nor the change in trehalose content was observed under stress exposure ofSaccharomycopsis fibuligera cells. Our results indicate that the SfTps1 protein and trehalose metabolism in response to stress conditions inSaccharomycopsis fibuligera clearly differ from that ofSaccharomyces cerevisiae and most of other eukaryotes.     

Construction of a trivalent candidateShigella vaccine strain with host-vector balanced-lethal system

A trivalent liveShigella vaccine candidate FSD01 against S.flexneri 2a, S.sonnei and S.dysenteriae I was constructed. This candidate strain was based on the S.flexneri 2a vaccine T32. By homologous recombination exchange, the chromosomalasd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while anotherasd gene of S.mutans was employed to construct an Asd⁺ complementary vector. This combination ofasd ^- host/Asd⁺ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S.sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the challenges of the above threeShigella strains.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

H-2 and Background influences on tissue grafts across the H-Y barrier

Male liver was grafted to kidney beds in syngeneic female mice. Relative influences ofH-2 haplotype, genetic background or interaction ofH-2 haplotype with genetic background on anti-H-Y response were evaluated using 27 inbred strains carrying eightH-2 haplotypes of independent origin and three naturally occurring recombinants. Females ofH-2 ^b haplotype acutely rejected the male graft as is reported for other tissue graft systems. AnH-2 haplotype influence was found for all haplotypes studied, with a greater variation of immunologic response revealed by histological analysis of liver grafts than is demonstrated by skin grafts. Strains carryingH-2 ^k ,H-2 ^j andH-2 ^p haplotypes expressed the greatest range of immunological variability with responses ranging from graft proliferation to graft rejection. Strains carrying theH-2 ^d haplotype had the most consistent responses with little reaction to the graft. The strong immune response by SJL/J (H-2 ^s ) female mice to the H-Y antigen is not typical of otherH-2 ^s strains, but is compatible with the reported hyperresponsiveness of this strain to alloantigens.     

Resistance to Insecticides in Insect Vectors of Disease:estα3, a Novel Amplified Esterase Associated with Amplifiedestβ1from Insecticide Resistant Strains of the MosquitoCulex quinquesfasciatus

Vector control programmes in many countries face the dual problems of parasite drug resistance and insecticide resistance in the insect vectors of the disease. Here we report for the first time a new esterase-based insecticide resistance mechanism in the filariasis vectorCulex quinquefasciatus.The field collected COL strain ofC. quinquefasciatusfrom Columbia was heterogeneous for organophosphorus insecticide resistance. On native polyacrylamide gels it had an elevated β-naphthyl acetate specific esterase with the same R_fas that for the Estβ1s involved in insecticide resistance in other strains of this mosquito species. After five generations of temephos insecticide selection, both the esterase specific activity withp-nitrophenyl acetate and the temephos LC₅₀values were increased, suggesting that elevation of esterase activity was the underlying mechanism of resistance. Western blots with antisera raised to Estα2¹and Estβ2¹fromC. quinquefasciatusindicated that the COL strain had an elevated Estα3 enzyme which co-migrated on native gels with Estβ1. Southern blots indicated that anestα3gene was amplified in the COL strain and a Cuban mosquito strain (MRes), although the restriction digest patterns of theestβ1 genes in these two strains are different. In contrast, the Californian TEMR strain, with the amplifiedestβ1¹gene, had no associated elevated Estα.Restriction digest patterns for COL and TEMR DNA suggest that they contain an identicalestβ1¹gene, but our data suggest that theestα3gene occurs on the same amplicon as anestβ1 gene although the genes are probably >10 kb apart. Hence, either the COL strain has twoestβ1genes or theestβ1¹amplicon in TEMR has been disrupted at some stage during the long colonisation of this strain and the amplifiedestα has been lost.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.     

Production of biosurfactant by a genetically-modified strain ofBacillus subtilis expressing green fluorescent protein

Biosurfactant production was investigated using two strains ofBacillus subtilis, being one a reference strain (B. subtilis 1012) and the other a genetically-modified strain (B. subtilis W1012) made able to produce the green fluorescent protein (GFP). A new method based on oil displacement technique was set up to measure the biosurfactant level in the medium. Although the tested microorganisms showed similar results in terms of cell growth parameters, the recombinant strain, besides expressing GFP, exhibited an average yield of extracellular surfactant on biomass (Y _{B/X, av} =239 mg_B g_x ^?1) more than twice that of the reference strain. The ability of the genetically-modified strain to simultaneously overproduce biosurfactant and GFP even at low cell concentration makes it an interesting candidate for possible use as a biological index-finger to monitor cell viability in bioremediation and oil recovery operations.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^? transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.     

A ketoreductase gene fromStreptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet.     

Pilot testing ofKloeckera apiculata for the biological control of postharvest diseases of citrus

The efficacy of the yeastKloeckera apiculata, strain 34-9, in controlling postharvest decay of citrus fruit was evaluated in small-scale and pilot tests in commercial packinghouse. Kloeckera apiculata grew efficiently on different media and maintained its antagonistic activity against spore germination ofPenicillium italicum. In small-scale experiments with citrus fruits dipped in the yeast cell suspension, the development of decay in citrus was effectively inhibited. The yeast was compatible with a mixture of low concentration of a commonly chemical fungicide. In packinghouse tests, combining the yeast with 40 mg/kg Carbendazim (MBC) resulted a reduction in the incidence of decay to a level equal to that of the commercial treatment of 200 mg/kg MBC. The efficacy of the strain 34-9 could also be maintained under packinghouse conditions at a cell concentration of the yeast antagonist as low as 10⁶ cells/ml. No significant difference in the efficacy ofK. apiculata was found in either the drench or the spray application systems tested in citrus packinghouse. Scanning electron microscopy revealed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of strain 34-9 against citrus blue mould may be related to its capability to compete withPenicillium italicum, for space and nutrients and /or involvement of directly antagonist of the yeast on the fungus.     

Über die ursache der hemmwirkung der humusstoffe auf den populationszuwachs vonoscillatoria sancta

Algologically pure strain ofOscillatoria sancta (KÜTZING) GOMONT was cultivated in a phototermostat using a modified medium of Chu-Gerloff. The inhibiting effect of Na-humate on the numbers of cells and dry weight of this blue-green alga was investigated in dependence on polyvalent cations (Ca²⁺, Mg²⁺, Fe³⁺) concentration, nature of associated anions, and pH of the nutrient solution. Moreover the absorption of light due to humate colour was taken into consideration. Humate restricted the uptake of polyvalent cations (especially calcium); its unfavourable effect on the growth of the investigated organism decreased at the optimum pH.