Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC .

Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/∼gisljh/kinome .

The evolutionally conserved MAPK Sty1 and bZIP transcriptional activator Atf1 are known to play a pivotal role in response to the reactive oxygen species in S. pombe. However, it is unclear whether all of the H₂O₂-induced genes are directly regulated by the Sty1-Atf1 pathway and involved in growth fitness under H₂O₂-induced stress conditions. Here we present the study on ChIP-chip mapping of the genomic binding sites for Sty1, Atf1, and the Atf1's binding partner Pcr1; the genome-wide transcriptional profiling of the atf1 and pcr1 strains in response to H₂O₂; and the phenotypic assessment of ∼90 Atf1/Pcr1-bound or unbound genes for growth fitness under H₂O₂ conditions. ChIP-chip analysis shows that Atf1 and Pcr1 binding sites are overlapped in the genome and constitutively present before H₂O₂ stress. On the other hand, Sty1 recruitment primarily occurs at the Atf1/Pcr1 binding sites and is induced by H₂O₂. We found that Atf1/Pcr1 is clearly responsible for the high-level transcriptional response to H₂O₂. Furthermore, phenotypic assessment indicates that among the H₂O₂-induced genes, Atf1/Pcr1-bound genes exhibit a higher likelihood of functional requirement for growth fitness under the stress condition than the Atf1/Pcr1-unbound genes do. Notably, we found that the Atf1/Pcr1-bound genes regardless of their responsiveness to H₂O₂ show a high probability of requirement for growth fitness. Together, our analyses on global mapping of protein binding sites, genome-wide transcriptional profiling, and phenotypic assessment provide insight into mechanisms for global transcriptional regulation by the Sty1-Atf1 pathway in response to H₂O₂-induced reactive oxygen species.

Petroleum pollutants emulsified by biosurfactants facilitate uptake and biodegradation by environmental microbes. In this report, we show the characterization of an indigenous surfactant-producing crude-oil-eating microbe isolated from petroleum-sludge in Zhoushan islands, China, where one of the national strategic petroleum reservoirs is located. We examined biosurfactant activities using surface tension analysis on mixed culture originated from oil-sludge microbes. In parallel, dynamics of individual microbial populations in cultures were monitored using the terminal fluorescence labeled (TFL)-RFLP method. Biosurfactant activity was found to be associated with a dominant microbial species designated as ZS1 (Zhou-Shan isolate 1). Cell-free supernatant from the ZS1 culture exhibited 100% emulsification index against crude oil and reduces surface tension to 26.5 mN/m. Sequence-based analysis suggested that the ZS1 belongs to the group of Pseudomonas aeruginosa. LC–MS/MS analysis indicated that the rhamnolipids produced by the ZS1 consisted of 7 monorhamnolipid and 11 dirhamnolipid homologues (RL7-11), two of which were novel. Maximum yield of rhamnolipids in shake-flask ZS1 culture could reach 44 g/l. Furthermore, we showed that ZS1 was capable of growing in MS medium supplemented with 1% crude oil as sole carbon source, in which cell mass increase coincided with the formation of crude-oil emulsion. Half of the crude oil was consumed by the ZS1 in 12 days. Taken together, our results show that ZS1 produces high level of rhamnolipids that effectively emulsify crude oil accelerating its uptake and degradation. Hence, it is suitable for bioremediation of petroleum pollutants in Zhoushan coastal areas.

To understand genes involved in neutral lipid accumulation upon nitrogen deprivation (ND) in a novel isolate of Nannochloropsis sp. PJ12, we performed comparative transcriptomic and lipidomic analyses of cells under ND and NR (nitrogen replete) conditions. Transcriptomic profiling indicated that, while enzymes involved in TCA cycle in PJ12 under ND condition were upregulated compared to that under NR condition, those involved in Calvin cycle and glycolysis under ND condition were downregulated. Furthermore, we showed that enzymes involved in fatty acid synthesis and glycerolipid synthesis were downregulated but not β-oxidation. Lipidomic profiling indicated that, while the level of neutral lipids in ND cells was increased compared to that of NR cells, level of photosynthetic membrane-lipids DGDG and PG was decreased. Taken together, our analysis indicated that TAG accumulation is attributed to the modification of membrane lipids derived primarily from “prokaryotic” pathway and secondarily from “eukaryotic” pathway based on the 16:X or 18:X fatty acid at the sn2 position of the glycerol backbone. We propose that two-phase (NR-ND) growth is ideal for biomass and biofuel production because ND reduces cell growth rate due to the loss of photosynthetic membrane and decreased quantum yield.