SummaryWhile bacteria such as Escherichia coli and Bacillus subtilis harbour a single circular chromosome, some freshwater cyanobacteria have multiple chromosomes per cell. The detailed mechanism(s) of cyanobacterial replication remains unclear. To elucidate the replication origin (ori ), form and synchrony of the multi-copy genome in freshwater cyanobacteria Synechococcus elongatus PCC 7942 we constructed strain S. 7942 TK that can incorporate 5-bromo-2'-deoxyuridine (BrdU) into genomic DNA and analysed its de novo DNA synthesis. The uptake of BrdU was blocked under dark and resumed after transfer of the culture to light conditions. Mapping analysis of nascent DNA fragments using a next-generation sequencer indicated that replication starts bidirectionally from a single ori, which locates in the upstream region of the dnaN gene. Quantitative analysis of BrdU-labelled DNA and whole-genome sequence analysis indicated that the peak timing of replication precedes that of cell division and that replication is initiated asynchronously not only among cell populations but also among the multi-copy chromosomes. Our findings suggest that replication initiation is regulated less stringently in S. 7942 than in E. coli and B. subtilis.

We have shown by Si nuclease mapping with in vivo transcripts that the differential expression of a sporulation-regulatory gene, spoOA, is regulated by switching of two discrete promoters during the initiation of sporulation in Bacillus subtilis; vegetative mRNA was transcribed from an upstream promoter (Pv, vegetative promoter), and sporulation-specific mRNA was transcribed from the other promoter (Ps, sporulation-specific promoter) about 150 bp downstream of the P, promoter. Transcription from the P, promoter was at a low level and shut off at To05. On the other hand, transcription from the P, promoter was strongly induced at To.5 and increased until T2 5. In the presence of 2% glucose, Pv-directed transcription was not shut off and was observed even at T1.5, whereas the induction of Ps-directed transcription was completely repressed. A mutant in which the spoOA gene was transcribed only from the P,s promoter could sporulate normally in the presence of 0.1% glucose but could not sporulate at all in the presence of 2% glucose. In a catabolite-resistant sporulation mutant carrying crsA47 (sigA47), a mutation within the gene encoding rA, normal promoter switching from P, to P.was observed in the presence of 2% glucose. Sporulation in Bacillus subtilis, which responds to nutritional starvation in the environment, proceeds through an ordered series of morphological changes leading from a vegetative cell to a dormant spore. This differential process is recognized as a result of the sequential expression of a set of spo genes, whose mutations arrest the process at characteristic intermediate morphological stages. Recently, a number of spo genes have been cloned, and some functions of their products have been characterized, especially for those defined as sporulation-specific sigma factors (for a review, see reference 19). The functions of many of the other spo gene products, however, are still obscure.The transition from the vegetative to the sporulation phase requires at least seven genes known as spoO, since spoO mutants cannot form an asymmetric septum, which is the first morphological change in the sporulation process. Among these genes, the spoOA gene is thought to play a central role in the initiation of sporulation. First, a spoOA mutant exerts the most-pleiotropic effects on sporulationassociated phenotypes such as antibiotic formation, extracellular protease synthesis, and competence development for transformation (22). Second, the sof-l mutation, a missense mutation in the 12th codon of the spoOA gene, suppresses the sporulation-deficient phenotype of spoOB, spoOE, and spoOF mutants (10). Third, the spoOA gene product shares homology with a group of proteins called receivers, which are elements of prokaryotic two-component signal-transducing systems (24,28), and has been shown to be a phosphoacceptor like the other receiver proteins (21). In addition, we have recently found that sporulation in a spoOA temperature-sensitive mutant could be normally induced by a shift-down of the culture temper-* Correspon...

We have isolated a new mutant of Saccharomyces cerevisiae that exhibits a glucose-derepression resistant (and sucrose-non-fermentor) phenotype. This mutant was obtained by screening for overproduction of alpha-amylase in a strain containing the mouse alpha-amylase gene under the control of the PGK promoter. The mutation designated pop2 (PGK promoter directed over production). The pop2 mutant overproduced amylase 5-6 fold and displayed several other pleiotropic defects: (1) resistance to glucose derepression, (2) temperature-sensitive growth, (3) failure of homozygous diploid cells to sporulate and (4) reduced amount of reserve carbohydrates. We mapped pop2 to chromosome XIV, distal to lys9 and SUP28, indicating that POP2 is a newly-identified locus. We isolated the POP2 gene from two yeast strains of different genetic backgrounds, S288C and A364A, and determined their nucleotide sequences. The predicted amino acid sequence of the POP2 protein contains three glutamine-rich region, a proline-rich region and a serine/threonine-rich region, characteristic of many transcription factors. Steady state levels of RNA transcribed from the PGK-amylase fusion gene and from endogenous PGK gene in stationary-phase pop2 cells were 5- to 10-fold higher than those observed in wild-type cells, showing that the pop2 mutation affects transcription of the PGK gene transcription.

An RGRI gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgrl cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wail of the daughter cell remained attached to that of mother cell. We cloned the RGRI gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGRI locus. The cloned RGRI gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)' RNA. The RGRI gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgrl between gal2 and pep3 by genetic analysis. rgrl was shown to be a new locus. We also determined the nucleotide sequence of RGRI, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGRI gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgrl cells. Possible functions of the RGRI product are discussed.Glucose regulates the expression of many genes in Saccharomyces cerevisiae (for a review, see reference 5). One of them, the SUC2 gene, which encodes invertase, is repressed by glucose (2, 3). Recently, we reported the isolation of a new mutation, rgrl, which affects expression of the SUC2 gene (21). A recessive rgrl-l mutation which caused overexpression of mouse a-amylase under the control of the SUC2 promoter was isolated, and the RGRI gene was found to be required for glucose repression. The rgrl mutation affected several cellular functions. Cells were resistant to glucose repression, temperature sensitive for cell growth, and sporulation deficient and showed abnormal cell morphology. Expression of the SUC2 gene in rgrl strains was resistant to glucose repression, and SUC2 expression was increased under glucose-derepressing conditions. In this report, we describe studies of the morphology of rgrl cells, the cloning and molecular analysis of the RGRI gene, and meiotic linkage analysis of rgrl. We constructed deletion mutations to determine the phenotypes of strains lacking a functional RGRI gene product and determined the nucleotide sequence of the gene. The RGRI gene affected accumulation of reserve carbohydrates. MATERIALS AND METHODSStrains and genetic methods. The strains of S. cerevisiae used in this study are listed in Table 1. All strains were derived from S288C. Crossing, sporulation, and tetrad analysis were carried out by standard genetic methods (23). The permissive and restrictive temperatures were 24 and 37°C, respectively. The phenotype of pep3 strains was scored as described elsewhere (12). The transformation of yeast was performed by the LiOAc-method of Ito et al. (11). Escherichia coli HB101 and JM109 were employed as hosts for * Corresponding author.constructing and propagating plasmids. The transform...

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.