Shizue Yoshihara

Phototactic motility in the unicellular cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a unicellular motile cyanobacterium that shows positive and negative phototaxis on agar plates under lateral illumination. Recent studies on the molecular mechanisms of the phototactic motility of Synechocystis have revealed that a number of genes are responsible for its pilus-dependent motility and phototaxis. Here we describe what is known about these genes. We also discuss the novel spectral properties of the phytochrome-like photoreceptor PixJ1 in Synechocystis, that is essential for positive phototaxis and which has revealed the existence of a new group of chromophore-binding proteins in cyanobacteria.

Novel Photosensory Two-Component System (PixA–NixB–NixC) Involved in the Regulation of Positive and Negative Phototaxis of Cyanobacterium Synechocystis sp. PCC 6803

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet- (Pv, λ(peak) = 396 nm) and green-absorbing (Pg, λ(peak) = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation.

Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp. PCC 6803.

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily (spkA ∼ spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni2+ affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg2+ and Mn2+, but not in Ca2+. Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.

Oligomeric structure of LOV domains in Arabidopsis phototropin

PHOT2 (uniprotkb:P93025) and PHOT2 (uniprotkb:P93025) bind (MI:0407) by cross‐linking studies (MI:0030)

CccS and CccP are Involved in Construction of Cell Surface Components in the Cyanobacterium Synechocystis sp. strain PCC 6803

We have previously identified two target genes (slr1667 and slr1668) for transcriptional regulation by a cAMP receptor protein, SYCRP1, in a cAMP-dependent manner. For this study we investigated the localizations of products of slr1667 and slr1668 (designated cccS and cccP, respectively) biochemically and immunocytochemically, and examined the phenotypes of their disruptants. CccS protein was detected in the culture medium and the acid-soluble fraction containing proteins derived from outside the outer membrane. Disruptants of cccS and cccP showed a more or less similar pleiotropic phenotype. Several proteins secreted into the culture medium or retained on the outside of the outer membrane were greatly reduced in both disruptants compared with the wild type. Electron microscopy revealed that the cccS disruptant lacked the thick pili responsible for motility and that the cccP disruptant had almost no discernible thick pili on its cell surface. Both disruptants largely secreted far greater amounts of yellow pigments into the culture medium than did the wild type. Furthermore, the disruptions reduced the amount of UV-absorbing compound(s) extractable from the exopolysaccharide layer. These results suggest that the cccS and cccP genes are involved in the construction of cell surface components in Synechocystis sp. strain PCC 6803.

The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor.

Phototropin (phot), a blue light receptor in plants, is composed of several domains: LOV1, LOV2, and a serine/threonine kinase (STK). LOV2 is the main regulator of light activation of STK. However, the detailed mechanism remains unclear. In this report, we focused on the linker region between LOV2 and STK excluding the Jα‐helix. Spectroscopy and a kinase assay for the substituents in the linker region of Arabidopsis phot1 LOV2‐STK indicated that the linker is involved in the activation of STK. A putative module in the middle of the linker would be critical for intramolecular signaling and/or regulation of STK.

Cross-Reconstitution of Four Extrinsic Proteins From a Red Alga with Higher Plant and Cyanobacterial PSII

Oxygen-evolving PSII complex contains a number of intrinsic membrane proteins commonly found from prokaryotic cyanobacteria to eukaryotic higher plants (1). There is, however, a remarkable difference in the extrinsic proteins associated with and functioning in the oxygen-evolving PSII complex among cyanobacteria, red algae and higher plants: In green algal and hi gher plant PSII, three proteins of 33, 23 and 17 kDa are present as extrinsic proteins functioning in maintaining the stability and activity of the oxygen-evolving complex (2). Of these three proteins, only the 33 kDa protein is found in red algal and cyanobacterialPSII but the other two proteins are absent. In contrast, red algal PSII contains three different extrinsic proteins, 20 and 12 kDa proteins and cyt c550 (3), while cyanobacterial PSII contains two proteins, cyt c550 and 12 kDa protein (4).