Iwane Suzuki

Complete Genomic Structure of the Bloom-forming Toxic Cyanobacterium Microcystis aeruginosa NIES-843

Abstract The nucleotide sequence of the complete genome of a cyanobacterium, Microcystis aeruginosa NIES-843, was determined. The genome of M. aeruginosa is a single, circular chromosome of 5 842 795 base pairs (bp) in length, with an average GC content of 42.3%. The chromosome comprises 6312 putative protein-encoding genes, two sets of rRNA genes, 42 tRNA genes representing 41 tRNA species, and genes for tmRNA, the B subunit of RNase P, SRP RNA, and 6Sa RNA. Forty-five percent of the putative protein-encoding sequences showed sequence similarity to genes of known function, 32% were similar to hypothetical genes, and the remaining 23% had no apparent similarity to reported genes. A total of 688 kb of the genome, equivalent to 11.8% of the entire genome, were composed of both insertion sequences and miniature inverted-repeat transposable elements. This is indicative of a plasticity of the M. aeruginosa genome, through a mechanism that involves homologous recombination mediated by repetitive DNA elements. In addition to known gene clusters related to the synthesis of microcystin and cyanopeptolin, novel gene clusters that may be involved in the synthesis and modification of toxic small polypeptides were identified. Compared with other cyanobacteria, a relatively small number of genes for two component systems and a large number of genes for restriction-modification systems were notable characteristics of the M. aeruginosa genome.

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

Systematic Analysis of the Relation of Electron Transport and ATP Synthesis to the Photodamage and Repair of Photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [35S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.

The Histidine Kinase Hik34 Is Involved in Thermotolerance by Regulating the Expression of Heat Shock Genes in Synechocystis

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (ΔHik34 cells) survived incubation at 48°C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44°C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in ΔHik34 cells after incubation of cells at 42°C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44°C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39.

A filamentous non-N2-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large number of genes for adenylate cyclase and haemolysin-like Ca2+-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis.

Wavelength specificity of growth, photosynthesis, and hydrocarbon production in the oil-producing green alga Botryococcus braunii

The effect of monochromatic light on growth, photosynthesis, and hydrocarbon production was tested in Botryococcus braunii Bot-144 (race B), which produces triterpenoid hydrocarbons. The growth was higher in order of red, blue, and green light. The color of red light-grown cells became more orange-yellow and their shape dominantly changed to grape-like with long branches. Photosynthetic carbon fixation activity was higher in order of blue, red, and green light-grown cells, but photosystem activities showed no difference. In the pulse-chase experiments with (14)CO(2), no major difference was observed in the production of lipids, hydrocarbons, polysaccharides, or proteins among the three kinds of cells, although hydrocarbon production was slightly lower in green light-grown cells. These results indicate that blue and red light were more effective for growth, photosynthetic CO(2) fixation, and hydrocarbon production than green light, and that red light is the most efficient light source when calculated based on photoenergy supplied.

Proteomic Analysis of High-CO2-Inducible Extracellular Proteins in the Unicellular Green Alga, Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.

Cis – trans isomerase gene in psychrophilic Pseudomonas syringae is constitutively expressed during growth and under conditions of temperature and solvent stress

In a recent study, we established that psychrophilic Pseudomonas syringae (Lz4W) requires trans-monounsaturated fatty acid for growth at higher temperatures (Kiran et al. in Extremophiles, 2004). It was also demonstrated that the cti gene was highly conserved and exhibited high sequence identity with cti of other Pseudomonas spp. (Kiran et al. in Extremophiles, 2004). Therefore it would be interesting to understand the expression of the cti gene so as to unravel the molecular basis of adaptation of microorganisms to high temperature. In the present study, the expression of cti was monitored by RT-PCR analysis during different growth stages and under conditions of high temperature and solvent stress in P. syringae. Results indicated that the cti gene is constitutively expressed during different stages of growth and the transcript level is unaltered even under conditions of temperature and solvent stress implying that the observed increase in trans-monounsaturated fatty acids (Kiran et al. in Extremophiles, 2004) is not under transcriptional control. A putative promoter present in the intergenic region of the metH and cti gene has also been characterized. The translation start site ATG, the Shine-Dalgarno sequence AGGA and the transcription start site “C” were also identified. These results provide evidence for the first time that the cti gene is constitutively expressed under normal conditions of growth and under conditions of temperature and solvent stress thus implying that the Cti enzyme is post-transcriptionally regulated.

Optimization of light for growth, photosynthesis, and hydrocarbon production by the colonial microalga Botryococcus braunii BOT-22.

Optimization of the light conditions for biofuel production by the microalga Botryococcus braunii BOT-22 (race B) was performed using monochromatic red light. The lipid and sugar contents were approximately 40% and 20-30% of the cell dry weight, respectively, and about half of the lipids were liquid hydrocarbons. The half-saturation intensities for the production rate of lipids, hydrocarbons, and sugars were 63, 49, and 44μmolm(-2)s(-1), respectively. Fluorescence microscopic images of Nile Red-stained cells showed an increased number of intracellular neutral lipid granules due to increased light intensity. After 16days of incubation in the dark, lipid and sugar, but not hydrocarbon content decreased. Growth, metabolite production, and photosynthesis were saturated at 100, 200 and 1000μmolm(-2)s(-1), respectively. These results indicate that photosynthetically captured energy is not used efficiently for metabolite production; thus, improvements in metabolic regulation may increase hydrocarbon production.

Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.