Plasmid diversity in senescent and nonsenescent strains of Neurospora

Summary A sample of 171 natural isolates of Neurospora crassa and Neurospora intermedia was tested for senescence. Of these, 28 strains senesced within the duration of the experiment. These senescent strains, together with a selection of nonsenescent strains, were examined for the presence of mitochondrial plasmids. This was done by digesting mitochondrial DNA preparations with proteinase K, and running these samples on agarose gels. Most of the strains examined, both senescent and nonsenescent, contained plasmids, many of them new. Some new plasmids were linear, as inferred from their resistance to 5 exonuclease and sensitivity to 3 exonuclease. New circular plasmids were also found. Some strains carry several plasmids, and mixtures of circular and linear elements were common. A cross-homology study was performed on a sample of plasmid-bearing strains, and several cases of apparent relatedness were found, some between strains from distant geographical locations. Linear plasmids homologous to the maranhar linear senescence plasmid were quite common. A new member of the LaBelle circular plasmid homology group was found. In the sample tested for homology, no strains contained elements related to the kalilo linear senescence plasmid. The relationship of the new plasmids to senescence is not known. In addition to plasmid monomers, several different types of derivatives were found. The kalilo linear plasmid was found to occur in linear and circular forms of low mobility, presumed to be giant concatamers, and, in some strains, variant sibling structures and ladders of short derivatives were found. Circular plasmids also gave rise to extensive ladders on electrophoresis, probably representing different relaxation states and head-to-tail concatameric series. Some such forms migrated more slowly than mitochondrial DNA. One unique type of plasmid modification observed was a pair of linear elements that had apparently arisen de novo which showed homology to a circular plasmid.     

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements.     

Lack of mutagenicity of vinyl chloride in two strains of Neurospora crassa.

No detectable induction of mutations could be found in two strains of Neurospora crassa after their conidia were treated with vinyl chloride, in ethanol solution and in its gaseous form. The results suggest that although N. crassa seems to lativating systems does not increase mutagenic activity of vinyl chloride in the two strains tested. At the same time these strains were mutated easily by UV and methyl methanesulfonate.     

Genetic Determinants of Circadian Rhythmicity in Neurospora

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.     

Plasmid Suppressors Active in the Sexual Cycle of Neurospora Intermedia

We have discovered that, in certain crosses of natural isolates of Neurospora intermedia, linear and circular mitochondrial plasmids of the maternal parent are not transmitted to the progeny. This contrasts with the maternal transmission of organellar genetic elements generally observed in crosses between laboratory strains and between other natural isolates. Formally, failure of plasmid transmission is a type of plasmid suppression. The present cases represent the first report of plasmid suppressors in natural populations of fungi. Strains used as female parents can transmit or not transmit plasmids depending on the strain used as male parent. Males that act to suppress in one cross fail to suppress in others. Therefore, the suppression of plasmids depends on a strain-specific interaction and is not determined exclusively by the males. Since suppression is a specific interaction we inferred that it must be genetically based and tested this hypothesis by seeking segregation of suppressed and nonsuppressed phenotypes in octads. Segregation of the original full suppression of all plasmids was indeed observed in each of the three sets of testcrosses examined. The interaction type of suppression must be initiated in ascogenous tissue during the sexual cycle. It is a nonautonomous type of suppression, affecting all descendent cells. In any one case of suppression, either one, several, or all plasmids can be lost. Both linear and circular plasmids can be eliminated by the same suppressor genotype. In addition, several strains were found to contain suppressors that act after ascospore delineation. This autonomous type of suppression has been observed previously in laboratory strains, but not in natural isolates. All the cases of plasmid suppression identified in this study involved a range of apparently neutral circular and linear plasmids. Using one senescent Kalilo strain of N. intermedia, we did not detect any case of suppression of the senescence-determining linear plasmid kalDNA.     

Cloning of the structural gene for orotidine 5'-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli

A Neurospora gene bank in plasmid pRK9 was used to complement pyrimidine auxotrophs in E. coli. Two plasmids were obtained that complement a pyrF mutant of E. coli. These plasmids hybridise to Neurospora DNA and transform a pyr-4 strain of Neurospora. The promoter used in expressing the orotidine 5'-monophosphate carboxylase in E. coli is within the Neurospora sequence.     

Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid.

We previously described two plasmids that replicate autonomously in both Neurospora and E. coli (Stohl and Lambowitz, Proc. Natl. Acad. Sci., U.S.A. 80, 1058-1062, 1983). One plasmid, pALS1, consists of the Neurospora ga-2+ gene (3 kb Hind III-fragment), the mitochondrial plasmid from N. intermedia strain P405-Labelle, and E. coli plasmid pBR325. The other, pALS2, is a putative deletion derivative of pALS1 that lacks most or all of the Labelle insert and that was repeatedly recovered from Neurospora transformants. We have now sequenced the region encompassing the deletion in five pALS2 plasmids isolated independently in two different laboratories. All five plasmids are identical in this region and completely lack the Labelle insert. We have also characterized an additional deletion derivative that retains a small (approximately 0.5 kb) segment of the Labelle insert. The results for pALS2 suggest that pBR325 plus the ga-2+ segment constitute a Neurospora replicon.     

Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans

We have cloned two different beta-tubulin sequences from the filamentous fungus Aspergillus nidulans. Each was used in the construction of transforming plasmids that carry the pyr4 gene of Neurospora crassa. We used these plasmids to transform a pyrG-strain of Aspergillus to uridine prototrophy. Both plasmids were shown to integrate site specifically into the homologous chromosomal sequences. We then used transformant strains in genetic crosses to demonstrate that one of the cloned beta-tubulin sequences was the benA beta-tubulin gene, which codes for the beta 1-and beta 2-tubulins. The other cloned beta-tubulin sequence was shown to be the structural gene for beta 3-tubulin by gene disruption and to participate in conidial development. This is the first report of a gene disruption by site specific, integrative recombination in Aspergillus nidulans.     

Aminocyclitol resistance in Staphylococcus aureus: presence of plasmids and aminocyclitol-modifying enzymes

Aminocyclitol resistance in Staphylococcus aureus has been investigated by the analysis of the plasmids and aminocyclitol-modifying enzymes present in several clinical staphylococcal isolates. All of the strains tested were resistant to a broad range of aminocyclitols and contained large plasmids which encoded a variety of aminocyclitol-modifying enzymes in addition to other antibiotic resistances. All strains expressed multiple aminocyclitol-modifying enzymes. The plasmids present in these strains appear to be related by virtue of their similar restriction endonuclease digestion patterns. The plasmids are related and differ by the gain or loss of small DNA segments, one of which encodes erythromycin and kanamycin resistance.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

Senescence in fungi: the view from Neurospora

Some naturally occurring strains of fungi cease growing through successive subculturing, i.e., they senesce. In Neurospora, senescing strains usually contain intramitochondrial linear or circular plasmids. An entire plasmid or its part(s) integrates into the mtDNA, causing insertional mutagenesis. The functionally defective mitochondria replicate faster than the wild-type mitochondria and spread through interconnected hyphal cells. Senescence could also be due to spontaneous lethal nuclear gene mutations arising in the multinucleated mycelium. However, their phenotypic effects remain masked until the nuclei segregate into a homokaryotic spore, and the spore germinates to form a mycelium that is incapable of extended culturing. Ultimately the growth of a fungal colony ceases due to dysfunctional oxidative phosphorylation. Results with senescing nuclear mutants or growth-impaired cytoplasmic mutants suggest that mtDNA is inherently unstable, requiring protection by as yet unidentified nuclear-gene-encoded factors for normal functioning. Interestingly, these results are in accord with the endosymbiotic theory of origin of eukaryotic cells.     

Plasmids in Vibrio parahemolyticus Strains Isolated in Japan and Bangladesh with Special Reference to Different Distributions

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30–40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.     

Conjugal transferability of multiple resistance in Shigella strains

Shigella strains isolated in Japan between 1971 and 1979 were surveyed for drug resistance and distribution of R plasmids. Of 2,510 strains, 89.3% were resistant to either one or various combinations of four drugs, tetracycline, chloramphenicol, streptomycin, and sulfanilamide. About 66% of the Shigella isolates were quadruply resistant. The frequency of isolation of R plasmids from quadruply resistant Shigella strains was the highest when compared with other strains resistant to various combinations of the four drugs. The conjugal transferability of 204 quadruply resistant strains isolated between 1977 and 1979 was tested by various mixed-culture methods. Among the total strains examined, 70.6% carried transferable resistance when tested by the conventional broth culture method, 90.2% transferred their resistance when, in addition the replica-plating method was used and 97.5% could transfer their resistance when the membrane filter method was also used. Although the remaining five strains could not transfer their resistance by any of the mixed culture methods, the drug resistance of four of the five strains was mobilized by the concomitant presence of F-tet or T-kan plasmid. These results indicate that almost all of the quadruple resistance in Shigella isolates was mediated by plasmid.     

Plasmid occurrence and diversity in the genus Paracoccus

The results of screening for the occurrence of plasmids in several strains representing 11 out of 13 species of the genus Paracoccus are presented. We show that plasmids (ranging in size from 2.7 to above 450 kb) are widely distributed in this genus. Only one tested strain (P. alkenifer) appears to be plasmid-free. The majority of the strains harbour at least two plasmids, one of which usually fits into the class of megaplasmids.     

Ecological behavior of plasmids and resistance to lead of Enterobacter agglomerans isolated from soil

Soil samples taken monthly in 1990 from five parks outside Tokyo were examined for Enterobacter agglomerans. A total of 348 strains were isolated and 250 of them were tested for the presence of plasmids by DNA agarose gel electrophoresis. All the isolates carried at least two kinds of plasmids. Those isolated from July to September showed four or five kinds of plasmids (group I) and those isolated from January to June and from October to December showed two or three kinds of plasmids (group II). A majority of the plasmids detected were of 1,500 or fewer base pairs. The isolates were tested for the Pb2+ resistance; group I strains were more resistant to lead than group II strains. It is presumed that bacterial plasmids are related with the ecosystem of soil and the resistance to lead in E. agglomerans.     

62-kb Plasmids Harboring <Emphasis Type="Italic">rulAB</Emphasis> Homologues Confer UV-tolerance and Epiphytic Fitness to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.     

A Demonstration of the Role of het Genes in Heterokaryon Formation in Neurospora under Simulated Field Conditions

An experimental system was developed for assessing the role of het genes in heterokaryon formation in Neurospora in nature. Burned sugar cane segments planted in soil were infected using a mixture of mutant ascospores of two genotypes. Neurospora ramified in the cane and erupted as distinct pustules of conidia. When ascospores carried identical het alleles, the (macro) conidial pustules which formed were heterokaryotic. On the other hand, when ascospores carried dissimilar het alleles, the pustules were homokaryotic. These results showed that stable heterokaryons between compatible strains can form in nature. When two strains are growing together on a natural substrate, heterozygosity at het loci serves to maintain their individuality.     

Elimination of active tad elements during the sexual phase of the Neurospora crassa life cycle.

Tad is an active LINE-like retrotransposon isolated from the Adiopodoumé strain of Neurospora crassa. Extensive analysis of other Neurospora strains has revealed no other strain with active Tad, but all strains tested have multiple copies of defective Tad elements. We have examined the ability of Tad to survive during the sexual cycle of Neurospora and find that active Tad is rapidly eliminated. The characteristics of this elimination suggest that the repeat-induced point mutation (RIP) mechanism was responsible. By the use of transformation to switch the mating type of the Adiopodoumé strain we concluded that this strain is not defective in the RIP process. Analysis of defective Tad elements isolated from a variety of strains indicates that the major difference between these elements and active Tad is due to the presence of a large number of G-C to A-T transition mutations. This would be expected if the changes were due primarily to the RIP process. Mapping of a selection of defective Tad elements reveals that they are present on all of the chromosomes; however, many of the elements are not widely shared among strains. This suggests that repeated introduction and elimination of Tad elements has occurred. Mechanisms that might be responsible for this repeated introduction are discussed.     

Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the east European plain

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.