Yuu Hirose

Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein

Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from Synechocystis as well as from phycocyanobilin-producing Escherichia coli. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, λmax = 535 nm) and a red-absorbing form (Pr, λmax = 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-Z and C15-E, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light (330–450 nm) via a “two-Cys photocycle” that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin–cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state (15ZPg) and a red-absorbing 15E state (15EPr). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as 15ZPg but protonated as 15EPr. This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a “protochromic photocycle,” in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter

Responding to green and red light, certain cyanobacteria change the composition of their light-harvesting pigments, phycoerythrin (PE) and phycocyanin (PC). Although this phenomenon—complementary chromatic adaptation—is well known, the green light–sensing mechanism for PE accumulation is unclear. The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 (N. punctiforme) regulates PE synthesis in response to green and red light (group II chromatic adaptation). We disrupted the green/red-perceiving histidine-kinase gene (ccaS) or the cognate response regulator gene (ccaR), which are clustered with several PE and PC genes (cpeC-cpcG2-cpeR1 operon) in N. punctiforme. Under green light, wild-type cells accumulated a significant amount of PE upon induction of cpeC-cpcG2-cpeR1 expression, whereas they accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression under red light. Under both green and red light, the ccaS mutant constitutively accumulated some PE with constitutively low cpeC-cpcG2-cpeR1 expression, whereas the ccaR mutant accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression. The results of an electrophoretic mobility shift assay suggest that CcaR binds to the promoter region of cpeC-cpcG2-cpeR1, which contains a conserved direct-repeat motif. Taken together, the results suggest that CcaS phosphorylates CcaR under green light and that phosphorylated CcaR then induces cpeC-cpcG2-cpeR1 expression, leading to PE accumulation. In contrast, CcaS probably represses cpeC-cpcG2-cpeR1 expression by dephosphorylation of CcaR under red light. We also found that the cpeB-cpeA operon is partially regulated by green and red light, suggesting that the green light-induced regulatory protein CpeR1 activates cpeB-cpeA expression together with constitutively induced CpeR2.

The gut microbiome of healthy Japanese and its microbial and functional uniqueness

The human gut microbiome has profound influences on the hosts health largely through its interference with various intestinal functions. As recent studies have suggested diversity in the human gut microbiome among human populations, it will be interesting to analyse how gut microbiome is correlated with geographical, cultural, and traditional differences. The Japanese people are known to have several characteristic features such as eating a variety of traditional foods and exhibiting a low BMI and long life span. In this study, we analysed gut microbiomes of the Japanese by comparing the metagenomic data obtained from 106 Japanese individuals with those from 11 other nations. We found that the composition of the Japanese gut microbiome showed more abundant in the phylum Actinobacteria, in particular in the genus Bifidobacterium, than other nations. Regarding the microbial functions, those of carbohydrate metabolism were overrepresented with a concurrent decrease in those for replication and repair, and cell motility. The remarkable low prevalence of genes for methanogenesis with a significant depletion of the archaeon Methanobrevibacter smithii and enrichment of acetogenesis genes in the Japanese gut microbiome compared with others suggested a difference in the hydrogen metabolism pathway in the gut between them. It thus seems that the gut microbiome of the Japanese is considerably different from those of other populations, which cannot be simply explained by diet alone. We postulate possible existence of hitherto unknown factors contributing to the population-level diversity in human gut microbiomes.

Thiol-Based Photocycle of the Blue and Teal Light-Sensing Cyanobacteriochrome Tlr1999

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.

Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4

Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used “dynamic” metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

Loss of Cytochrome cM Stimulates Cyanobacterial Heterotrophic Growth in the Dark

Although cyanobacteria are photoautotrophs, they have the capability for heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and the WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp) and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from the WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding Cyt c(M). The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of the WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of the WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of the WT. These results suggest a suppressive role(s) for Cyt cM in regulation of heterotrophic activity.

Complete Genome Sequence of Enterococcus mundtii QU 25, an Efficient l-(+)-Lactic Acid-Producing Bacterium

Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified—one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

Primary photodynamics of the green/red-absorbing photoswitching regulator of the chromatic adaptation E domain from Fremyella diplosiphon.

Phytochromes are red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the red to far-red light ratio. Cyanobacteriochromes (CBCRs) are distantly related cyanobacterial photosensors with homologous bilin-binding GAF domains, but they exhibit greater spectral diversity. Different CBCR subfamilies have been described, with spectral sensitivity varying across the near-ultraviolet and throughout the visible spectrum, but all known CBCRs utilize photoisomerization of the bilin 15,16-double bond as the primary photochemical event. The first CBCR discovered was RcaE, responsible for tuning light harvesting to the incident color environment (complementary chromatic adaptation) in Fremyella diplosiphon. The green/red RcaE photocycle has recently been described in detail. We now extend this analysis by examining femtosecond photodynamics using ultrafast transient absorption techniques with broadband detection and multicomponent global analysis. Excited-state dynamics in both directions are significantly slower than those recently published for the red/green CBCR NpR6012g4. In the forward reaction, the primary Lumi-G photoproduct arises from the longer-lived excited-state populations, leading to a low photoproduct quantum yield. Using dual-excitation wavelength interleaved pump-probe spectroscopy, we observe multiphasic excited-state dynamics in the forward reaction ((15Z)Pg → (15E)Pr), which we interpret as arising from ground-state inhomogeneity with different tautomers of the PCB chromophore. The reverse reaction ((15E)Pr → (15Z)Pg) is characterized via pump-probe spectroscopy and also exhibits slow excited-state decay dynamics and a low photoproduct yield. These results provide the first description of excited-state dynamics for a green/red CBCR.

Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES-2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes

The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.