Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated.

Competitive advantage and tolerance of selected shochu yeast in barley shochu mash.

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the fermentation superiority of BAW-6. Here, we demonstrate the growth advantage of the BAW-6 strain over the Ko strain by competitive cultivation in barley shochu preparation, where alcohol yield and nihonshudo of the former strain were higher than those of the latter strain. In addition, the maximum growth rate of BAW-6 was less affected than that of Ko by high Brix values of barley koji medium, suggesting that BAW-6 is less sensitive to growth inhibitory compounds derived from barley or barley koji. The tolerance of BAW-6 to growth inhibitory compounds, cerulenin and diethylstilbestrol (an H⁺-ATPase inhibitor), was also higher than that of other yeast strains. Consistent with BAW-6's tolerance to diethylstilbestrol in the presence of 8% ethanol (pH 4.5), H⁺-ATPase activity, but not transcription of its gene, was higher in BAW-6 than in Ko. We conclude that the BAW-6 strain is associated with certain gene alterations other than PHO3, such that it can maintain cellular ion homeostasis under conditions of ethanol stress during the latter phase of fermentation.

Genetic instability of constitutive acid phosphatase in shochu and sake yeast.

Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission.

In a dimorphic yeast, Yarrowia lipolytica, replicative plasmids can be established only in the coexistence of the replication origin (ORI) and centromere (CEN) from its own chromosomal DNA. Although six CEN sequences so far isolated from this yeast exhibit no similarity with conventional CEN DNA elements from other budding yeasts, they are confined within short regions (approximately 0.2 kb) and contain various conserved sequence blocks. We surveyed here a CEN1-1 sequence on an ORI-containing plasmid by deletion and site-directed mutagenesis, and found a partial palindrome, CCTAATTTGG designated DS9, to be an essential element for high-efficiency transformation. In particular, point mutations that alter symmetry and/or length of the palindrome abrogated the activity of CEN1-1. Gel mobility shift assay of CEN1-1 DNA fragments incubated with Y. lipolytica nuclear proteins revealed four bands corresponding to protein-DNA complexes, whereas the mutations within DS9 that disabled transformation also abolished the formation of part of these complexes, depending on particular mutations. These results demonstrate that the palindrome is a binding site for specific protein(s) necessary for plasmid transmission in Y. lipolytica.

Derivation of consensus sequence for protein binding site in Yarrowia lipolytica centromere.

The transmission of replicative plasmids in the yeast Yarrowia lipolytica is mediated solely by a part of its centromere DNA, in which an essential protein binding site has been analyzed recently. Here, we extended the analysis to other minimized centromeric regions, revealing a consensus sequence of a 17- to 21-bp imperfect palindrome.

Establishment of a pure culture of the hitherto uncultured unicellular cyanobacterium Aphanothece sacrum, and phylogenetic position of the organism.

Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells approximately 6 microm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.

High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene.

Multiple targeted gene replacements are often required for functional analyses of cyanobacterial genomes. For this purpose, we previously devised a simple genetic method, termed rps12-mediated gene replacement, in a cyanobacterium Synechococcus elongatus PCC 7942 for construction of mutants free from drug resistance markers. Here, we improved the method by employing a heterologous rps12 gene encoding a ribosomal protein S12 from Synechocystis sp. PCC 6803. Dominant streptomycin-sensitive phenotype of the Synechocystis rps12 gene was manifested only when it was expressed under the strong promoter of psbAI gene in S. elongatus PCC 7942 bearing a streptomycin-resistant rps12 allele. Transformation of the rps12 heteroallelic strains with non-replicating template plasmids permitted the selection of recombinants with gene replacement at frequencies up to 50% among streptomycin-resistant progeny.

Construction and analysis of a recombinant cyanobacterium expressing a chromosomally inserted gene for an ethylene-forming enzyme at the psbAI locus.

The coding sequence of a gene for a Pseudomonas syringae ethylene-forming enzyme was inserted at the psbAI locus in a cyanobacterium, Synechococcus elongatus PCC 7942 via rps12-mediated gene replacement. The recombinant strain photoautotrophically produced ethylene at 451 nl ml(-1) h(-1) OD730(-1), but showed a depressed specific growth rate as well as a yellow-green phenotype indicating a severe metabolic stress. The rate of ethylene production in the recombinant culture decreased as a result of competition with faster growing ethylene-non-forming mutants that carried short nucleotide insertions within the coding sequence of the gene for the ethylene-forming enzyme.

Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers.

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile gene replacement system without this restriction is reported in a unicellular cyanobacterium, Synechococcus sp. PCC 7942. The system is based upon the dominance of a streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with a Lys-43-->Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps12 gene and a kan gene conferring kanamycin resistance was integrated into the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite spontaneous gene conversion in these merodiploids to produce streptomycin-resistant progeny at frequencies ranging from 1x10(-5) to 5x10(-5), homologous recombination could be induced by transforming the merodiploids with template plasmids carrying psbAI 5' and 3' non-coding sequences flanking the D1 coding sequence, which was then replaced by either the gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3-16% of streptomycin-resistant progeny of the merodiploids after transformation were homogenote recombinants with concomitant loss of the kan gene, even in these polyploid cyanobacteria. The rps12-mediated gene replacement thus makes it possible to construct mutants free from drug resistance markers and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.