Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

Genetic analysis of mutagenesis in aging Escherichia coli colonies

Bacteria live in unstructured and structured environments, experiencing feast and famine lifestyles. Bacterial colonies can be viewed as model structured environments. SOS induction and mutagenesis have been observed in aging Escherichia coli colonies, in the absence of exogenous sources of DNA damage. This cAMP-dependent mutagenesis occurring in Resting Organisms in a Structured Environment (ROSE) is unaffected by a umuC mutation and therefore differs from both targeted UV mutagenesis and recA730 (SOS constitutive) untargeted mutagenesis. As a recB mutation has only a minor effect on ROSE mutagenesis it also differs from both adaptive reversion of the lacI33 allele and from iSDR (inducible Stable DNA Replication) mutagenesis. Besides its recA and lexA dependence, ROSE mutagenesis is also uvrB and polA dependent. These genetic requirements are reminiscent of the untargeted mutagenesis in λ phage observed when unirradiated λ infects UV-irradiated E. coli. These mutations, which are not observed in aging liquid cultures, accumulate linearly with the age of the colonies. ROSE mutagenesis might offer a good model for bacterial mutagenesis in structured environments such as biofilms and for mutagenesis of quiescent eukaryotic cells. Received: 30 April 1997 / Accepted: 1 July 1997     

Use of the somatic fusion method to introduce the flocculation property into Saccharomyces diastaticus

Summary Spheroplasts of a petite mutant of the amylolitic Saccharomyces diastaticus 1376 yeast strain were successfully fused with spheroplasts of a flocculent and respiratory competent Saccharomyces cerevisiae 1161 yeast strain.Flocculent and non-flocculent stable recombinants were recovered after regeneration of the cell walls all of which formed halos around their colonies in media containing starch/dextrin as carbon source. The sporulation ability varied in some of the fusion products and the possible influence of genetic instability is discussed.     

Mutagenesis resulting from depurination is an SOS process

When bacteriophage phi X174 am3 DNA depurinated in vitro is transfected into E. coli spheroplasts prepared from bacteria previously exposed to UV light, a strong mutagenic response is observed. This mutagenic response does not occur in spheroplasts derived from pre-irradiated bacteria carrying defective recA, recF or umuC genes. These findings indicate that mutagenesis at apurinic sites is an SOS-dependent process. The mutagenic response is not dependent on the multiplicity of transfection. This suggests that mutagenesis is not mediated by recombination.     

Parasexual genetics of Torulopsis glabrata.

Prototrophic hybrids were generated in the asexual yeast Torulopsis glabrata by the fusion of spheroplasts derived from parent strains which bore complementing auxotrophic markers. The DNA content (per cell) of two hybrids was essentially that predicted by summing the corresponding parental values. UV irradiation of these two hybrids resulted in the formation of sectored colonies with genetic properties consistent with their origin by either mitotic recombination or chromosomal nondisjunction.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

<Emphasis Type="Italic">IXR1</Emphasis> and <Emphasis Type="Italic">HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

Transformation of Vinca protoplasts mediated by Agrobacterium spheroplasts

Summary Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

Creating auxotrophic mutants in Methylophilus methylotrophus AS1 by combining electroporation and chemical mutagenesis

Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) across the cell membrane. By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone. MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%). This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen. Received: 9 December 1996 / Received revision: 31 March 1997 / Accepted: 13 April 1997     

Genetic analysis of mutagenesis in aging Escherichia coli colonies

Bacteria live in unstructured and structured environments, experiencing feast and famine lifestyles. Bacterial colonies can be viewed as model structured environments. SOS induction and mutagenesis have been observed in aging Escherichia coli colonies, in the absence of exogenous sources of DNA damage. This cAMP-dependent mutagenesis occurring in Resting Organisms in a Structured Environment (ROSE) is unaffected by a umuC mutation and therefore differs from both targeted UV mutagenesis and recA730 (SOS constitutive) untargeted mutagenesis. As a recB mutation has only a minor effect on ROSE mutagenesis it also differs from both adaptive reversion of the lacI33 allele and from iSDR (inducible Stable DNA Replication) mutagenesis. Besides its recA and lexA dependence, ROSE mutagenesis is also uvrB and polA dependent. These genetic requirements are reminiscent of the untargeted mutagenesis in λ phage observed when unirradiated λ infects UV-irradiated E. coli. These mutations, which are not observed in aging liquid cultures, accumulate linearly with the age of the colonies. ROSE mutagenesis might offer a good model for bacterial mutagenesis in structured environments such as biofilms and for mutagenesis of quiescent eukaryotic cells.     

Characterization and sequence determination of the replicator region in the hairy-root-inducing plasmid pRiA 4b

Summary By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene. When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC ⁺ and recF ⁺ genes. When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis. This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative. Thus, we conclude that the induced (unuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers. glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9. This uninduced mutagenesis is independent of the umuC ⁺ , recF ⁺ and recA ⁺ genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides. The role of recF in UV mutagenesis was tested in two ways. First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent. Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play. Second, UV mutagenesis of E. coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent. We conclude that the mechanism of induced UV mutagenesis of the E. coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Spring “bleaching” among <Emphasis Type="Italic">Pocillopora</Emphasis> in the Sea of Cortez,Eastern Pacific

A mild bleaching event was observed among Pocillopora spp. in the southern Gulf of California in the spring of 2006. Uniform bleaching occurred in numerous colonies on the upper portions of their branches. Most (∼90%) colonies that exhibited bleaching contained a species of endosymbiotic dinoflagellate, Symbiodinium C1b-c, which differed from the Symbiodinium D1 found inhabiting most unbleached colonies. Analysis of chlorophyll fluorescence, indicated a decline in photosystem II photochemical activity, especially among colonies populated with C1b-c. By early August, most affected colonies had recovered their normal pigmentation and fluorescence values were once again high for all colonies. No mortality was observed among tagged bleached colonies nor did symbiont species composition change during recovery. This unusual episode of bleaching did not appear to be a response to thermal stress, but may have been triggered by high levels of solar radiation during a period of unseasonally high water clarity in the early spring.     

Stereochemical Behavior of an NADH Model Compound Carrying l-Prolinamide at 3,5-Positions

Interspecific recombinants have been produced between Streptococcus cremoris H-61 and S. lactis J-1 by polyethylene glycol-induced protoplast fusion. All of the fusants obtained showed mixed physiological properties of the two parents, and possessed plasmids derived from both parents at random. Physiological properties of primary colonies were stably maintained among the progenies after the single-colony isolation procedure. Similarly, in most of the fusants the plasmid profiles of the primary colonies were stably maintained, but one lost 2 out of the 7 plasmid bands. However, there was no indication that plasmids from either one of the parents were preferentially lost. These results showed that interspecific genetic transfer occurred on chromosomal and plasmid DNA on the protoplast fusion and that the fusants obtained were not heterokaryons, but true recombinants.     

Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii

The production of a low-temperature alkalophilic lipase from Acinetobacter johnsonii was improved using genome shuffling. The starting populations, obtained by UV irradiation and diethyl sulfate mutagenesis, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 0.15 mg lysozyme/ml for 45 min at 37°C. The protoplasts were inactivated under UV for 20 min or heated at 60°C for 60 min and a fusant probability of ~98% was observed. The positive colonies were created by fusing the inactivated protoplasts. After two rounds of genome shuffling, one strain, F22, with a lipase activity of 7 U/ml was obtained.     

DISEASE IMPACT AND LOCAL GENETIC DIVERSITY IN THE CLONAL PLANT PODOPHYLLUM PELTATUM

Genotypic diversity is restricted within local colonies of mayapple (Podophyllum peltatum), due to extensive asexual reproduction. Transplant experiments were used to examine whether disease impact from a specialist fungal pathogen (Puccinia podophylli) was affected by the local frequency of host genotypes within colonies. In each of six large mayapple colonies, I measured infection intensity on 1) ramets replanted in their native colony (which were thus surrounded mostly by identical genotypes) and 2) transplants from two foreign colonies (surrounded by different genotypes). Disease incidence during the pathogen's first generation did not vary significantly between native (11% infected) and foreign host genotypes (6% infected). In the pathogen's second generation, significant variation in infection intensity occurred among ramets from different source populations. However, at five of the six transplant sites, mean infection intensity was higher on some nonnative plants (locally rare host genotypes) than on natives (locally common host genotypes). In this system, pathogen attack does not act in a frequency-dependent manner to promote local genetic diversity among hosts.     

Adenine Auxotrophic Mutants of Aspergillus oryzae: Development of a Novel Transformation System with Triple Auxotrophic Hosts

adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD ^?, sC ^?), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.     

Genotypic variability following fusion in the invasive colonial tunicate Didemnum vexillum

Fusion to form a chimera has been documented in many marine invertebrate taxa, including poriferans, cnidarians, bryozoans, and colonial ascidians. Allogenic interactions in chimeric ascidian colonies vary widely across taxonomic groups but are poorly characterized in the invasive colonial ascidian Didemnum vexillum. The moderate level of discrimination expressed in the fusion–rejection response of D. vexillum suggests that there is some integration of cells beyond the fusion line in a chimeric colony. We tracked the shifts in representation of microsatellite alleles between fused colonies of D. vexillum to elucidate the extent of genotypic integration in fused colonies and the patterns of changes to the genotypic composition of colonies immediately following chimera formation. By genotyping colonies before and after fusion, we found that allogeneic fusion in D. vexillum may lead to genotypic changes beyond the visible fusion line. Alleles from one colony were found in multiple tissue samples in the chimera 7–10 days after fusion had occurred. In some instances, alleles that were in a single colony prior to fusion were lost following fusion. We observed multiple patterns of allelic change, including both the unidirectional transfer and reciprocal exchange of alleles between fused colonies. Our findings suggest that tissue or cells are exchanged following allogeneic fusion between colonies of D. vexillum and that the genotypic composition of chimeric colonies may be fluid.     

Mycelium of Alternaria alternata as a potential biological control agent for Eupatorium adenophorum

The fungus, Alternaria alternata (Fr.) Keissler Strain 501, has been evaluated as a bioherbicide for control of Eupatorium adenophorum Spreng., but the biology of the pathogen–host interaction and the optimal environmental conditions for disease development and effective weed control are unknown. Disease development of A. alternata Strain 501 mycelia on E. adenophorum was assessed under several factors including pathogen inoculum concentration, plant age, dew period duration, post-dew temperature, storage temperature and duration. The minimum inoculum concentration required to kill E. adenophorum seedlings was 3.2×10⁶ mycelial fragment mL^?1. E. adenophorum seedlings at the four-leaf-pair stage were more susceptible than the older plants, especially those at the older than seven-leaf-pair stage. With a dew period of at least 14 h, 100% mortality occurred. The optimal post-dew temperature for disease development was 18–25°C. Storage at <4°C maintained the infectivity of A. alternata strain 501 mycelia on E. adenophorum longer. Using light and scanning electron microscopy to examine the infection process of A. alternata Strain 501 mycelia, it was shown that the time from initiation to completion of infection with mycelia was much shorter (14 h) than with conidia (72 h). It was further shown that mycelial infection occurred predominately through direct penetration at intercellular junctions, while conidial infection occurred predominately through stomatal penetration. This suggests that mycelia are more suitable as infection propagules for A. alternata strain 501 in a bioherbicide for the control of E. adenophorum.