The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSMl (5,416 base pairs), isolated from Zygosaccharomycesfermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2,um DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSRl, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2,Lm DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSRl are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSRl showed clear homology with that of open reading frame B of pSB2.Since the discovery of 2,um DNA in Saccharomyces species (21; see reference 6 for a review), several yeast plasmids resembling the 2Rm plasmid were isolated and characterized. We call these plasmids 2,um DNA-like plasmids. For unknown reasons, 2ptm DNA-like plasmids were found frequently in Zygosaccharomyces species. We have found five plasmids in Zygosaccharomyces yeasts: pSB1 and pSB2 from Z. bailii (23), pSR1 and pSB3 from Z. rouxii (23,24), and pSM1 from Z. fermentati (unpublished data). Recently, another 2,um DNA-like plasmid, pKD1, was discovered in Kluyveromyces drosophilarum (8). Of these, the nucleotide sequences of 2,um DNA (13), pSR1 (2), pSB3 (25), and pKD1 (8) have been published. These plasmids, along with 2,um DNA from Saccharomyces cerevisiae, have several structural features in common: (i) they are closed circular DNA plasmids whose size is ca. 6 kilobases (kb), (ii) they have a pair of inverted repeats (IRs) consisting of several hundred bases, (iii) they are cryptic and exist in high copy number, and (iv) intramolecular recombination results in two isomeric forms. In spite of resemblances in their global structure, homology at the nucleotide sequence level is poor (23).Plasmid-encoded functions have been most extensively studied in 2,um DNA. The prominent features of genes on 2,um DNA are briefly summarized as follows. It has one replication origin, which is activated once in the cell cycle by the same replication mechanism as that for replication of chromosomes (16,28). It has four open reading frames (ORFs) or genes, A, B, C, and D. The A gene (or FLP gene) encodes a recombination enzyme which functions as a site-specific recombinase (7). The B and C genes produce trans-acting factors that are needed for the stable maintenance of this plasmid. These two genes, along with the cis-acting locus STB, participate in partitioning of plasmid molecules during cell division (14,15 Our...

Five of eight strains of Succharomyces bailii and one of 13 strains of S. bisporus were found to harbour DNA plasmids. pSB I and pSB2 plasmids were isolated from S . hailii strains I F 0 0488 and I F 0 1047, respectively, and pSB3 and pSB4 from S. hisporus strain I F 0 1730. All four plasmids resemble 2-pm DNA of S. cererisiae in that (i) their molecular sizes are about 6 kb, (ii) each molecule possesses a pair of inverted repeats, (iii) they exist as a mixture of two isomers and (iv) their copy numbers in the native host are similar. None of them showed homology with 2-pm DNA or with each other by Southern hybridization under moderately stringent conditions, but pSB4 hybridized with the pSR 1 DNA, which was found previously in a strain of S. ruuxii. Each of the pSB plasmids has D N A sequence(s) effective for autonomous replication in S. cererisiue. In S. cererisiue, pSB3 and pSB4 showed intramolecular recombination but neither supported isomerization of 2-pm DNA.

The plasmid pSB3 of yeast Zygosacharomyces bisporus has been sequenced. It contains 6,615 base pairs, including a pair of inverted repeats (IR) consisting of 391 base pairs and 3 large open reading frames (ORF). One of the ORFs (A gene) participates in the recombination at the IRs and the other two (B and C genes) are necessary for the stable maintenance of this plasmid. The ARS sequence, which functions in a Saccharomyces cerevisiae host, was localized within 168 base pairs consisting of part of one of the IRs and a unique sequence contiguous to it. pSB3 can be maintained as stably in Z. rouxii as in the natural host Z.bisporus. In contrast, pSB3 is maintained fairly unstably in S.cerevisiae. The reason for this instability was found to be inefficient partitioning of pSB3 in S.cerevisiae. The molecular construction of pSB3 resembles that of 2-micron DNA, however, sequence homology at the DNA level was very poor.

Washed cells of the virulent isolates and avirulent or weakly-virulent mutants of Pseudomonas solanacearum suspended in pure water at a concentration of 102-3 cells/ml rapidly multiplied up to a level of 106 cells/ml during four days of incubation at 22C.