Cyanobacteriochrome TePixJ of Thermosynechococcus elongatus harbors phycoviolobilin as a chromophore

Cyanobacteria have several putative photoreceptors (designated cyanobacteriochromes) that are related to but distinct from the established phytochromes. The GAF domain of the phototaxis regulator, PixJ, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixJ_GAF) is a cyanobacteriochrome which exhibits reversible photoconversion between a blue light-absorbing form (max = 433 nm) and a green light-absorbing form (max = 531 nm). To study the chromophore, we prepared TePixJ_GAF chromoprotein from heterologously expressed Synechocystis and performed spectral analysis after denaturation by comparing it with the cyanobacterial phytochrome Cph1 which harbors phycocyanobilin (PCB) as a chromophore. The results indicated that the chromophore of TePixJ is not PCB, but its isomer, phycoviolobilin (PVB). It is suggested that the GAF domain of TePixJ has auto-lyase and auto-isomerase activities.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.     

Reconstitution of blue-green reversible photoconversion of a cyanobacterial photoreceptor, PixJ1, in phycocyanobilin-producing Escherichia coli

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediates positive phototactic motility and contains two GAF domains, the latter of which binds a bilin chromophore. Full-length PixJ1 expressed and purified from Synechocystis showed unique reversible photoconversion between a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to the reversible phototransformation between the red light-absorbing form and far-red light-absorbing form of the other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin of the blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformation by synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis of four different bilins, biliverdin (BV), bilirubin (BR), phycocyanobilin (PCB), and phycocyanorubin (PCR), as final products. The three expression systems, the BR system being the exception, produced a GAF polypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibited an irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversion between Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible for the blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producing system exhibited almost the same reversible spectral change, possibly coming from the PCB accumulated in the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealed that the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif for phytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorption spectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in a twisted conformation that disrupts the pi-electron conjugation between the A and B rings, possibly being held in position through a second covalent linkage.     

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.     

嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

通过蛋白质序列同源性比对分析,在嗜热藻（Thermosynechococcus elongatus BP-1）里面找到了与已知的Pb/Pg型蓝细菌光敏色素TePixJ和TeTlr0924同源的3个基因tlr0911、tlr1215和tlr1999。通过分子克隆技术把它们的GAF结构域分别构建在pET30a（＋）表达载体上,与可生成藻蓝胆素（PCB）的质粒pACYCDuet-ho1-pcyA在大肠杆菌BL21（DE3）体内重组,生成重组蛋白,利用亲和层析柱分离纯化,纯化后的蛋白质经过锌荧光和蛋白质酸性尿素变性以及荧光光谱和吸收光谱等实验分析鉴定,结果表明,Tlr0911-GAF存在蓝光吸收态Pb406 nm和绿光吸收态Pg527 nm之间的可逆光转换,它可共价结合两种藻胆色素,即藻紫胆素（PVB）和藻蓝胆素（PCB）,Tlr1999-GAF则存在蓝光吸收态Pb417 nm和青光吸收态Pt496 nm之间的可逆光转换,它同样共价结合PVB和PCB,而Tlr1215-GAF1和Tlr1215-GAF2不能自发结合藻胆色素,不具有光活性。     

Uptake of Pb2+ by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).     

Characterization of the photoactive GAF domain of the CikA homolog (SyCikA, Slr1969) of the cyanobacterium Synechocystis sp. PCC 6803

Phytochromes and bacteriophytochromes in plants and some species of bacteria, respectively, are photoreceptors that bind linear tetrapyrroles and can respond to red and far-red light signals in a reversible manner. A related but distinct photoreceptor candidate, CikA (denoted ScCikA), has been reported to reset the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942 after a dark pulse. However, recent studies have indicated that ScCikA does not function as a photoreceptor but as a redox sensor. Moreover, the Cys residue that covalently ligates the chromophore in phytochromes is not conserved in the ScCikA protein. On the other hand, the CikA homolog in Synechocystis sp. PCC 6803 (Slr1969, denoted SyCikA) retains this conserved Cys residue. In our present study, we have isolated the putative chromophore-binding GAF domain of SyCikA from Synechocystis and phycocyanobilin-producing Escherichia coli. Absorption spectra of both preparations showed two peaks in the UV and violet regions. Irradiation of these proteins with violet light yielded a broad peak in a yellow region at the expense of the peaks in the UV and violet regions. Interestingly, successive irradiation with yellow light did not revert these absorption spectra but a partial dark reversion to the original form was detected. These results suggest that SyCikA may function as a violet light sensor in Synechocystis.     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.     

Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket.

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s(-1), is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5-10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.     

Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.     

Dynamic Structural Changes Underpin Photoconversion of a Blue/Green Cyanobacteriochrome between Its Dark and Photoactivated States

The phytochrome superfamily of photoreceptors exploits reversible light-driven changes in the bilin chromophore to initiate a variety of signaling cascades. The nature of these alterations and how they impact the protein moiety remain poorly resolved and might include several species-specific routes. Here, we provide a detailed picture of photoconversion for the photosensing cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain from Thermosynechococcus elongatus (Te) PixJ, a member of the cyanobacteriochrome clade. Solution NMR structures of the blue light-absorbing dark state Pb and green light-absorbing photoactivated state Pg, combined with paired crystallographic models, revealed that the bilin and GAF domain dynamically transition via breakage of the C10/Cys-494 thioether bond, opposite rotations of the A and D pyrrole rings, sliding of the bilin in the GAF pocket, and the appearance of an extended region of disorder that includes Cys-494. Changes in GAF domain backbone dynamics were also observed that are likely important for inter-domain signal propagation. Taken together, photoconversion of T. elongatus PixJ from Pb to Pg involves complex structural changes within the GAF domain pocket that transduce light into a mechanical signal, many aspects of which should be relevant to others within the extended phytochrome superfamily.     

Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process.

A glucose-tolerant strain of Synechocystis sp. strain 6803 will not grow on glucose under complete darkness unless given a daily pulse of white light, typically 5 min of 40 mumol m-2 s-1 (light-pulsed conditions). The light pulse is insufficient for photoautotrophy, as glucose is required and growth yield is dependent on glucose concentration. Growth rate is independent of fluence, but growth yield is dependent on fluence, saturating at 40 to 75 mumol m-2 s-1. A Synechocystis strain 6803 psbA mutant strain grows under light-pulsed conditions at rates similar to those for the glucose-tolerant strain, indicating that photosystem II is not required for growth. The relative spectral sensitivity of the growth of light-pulsed cultures (growth only in blue light, 400 to 500 nm, maximum at 450 nm) precludes energetic contribution from cyclic electron transport around photosystem I. Pulses of long-wavelength light (i.e., 550 and 650 nm) did not support the growth of Synechocystis strain 6803 and, when supplied before or after a blue-light pulse, did not inhibit blue-light-stimulated growth of Synechocystis strain 6803. We conclude that the required blue-light pulse does not support growth via photosynthetic electron transport but appears instead to function as an environmental signal regulating heterotrophic metabolism, cell division, or other photomorphogenic processes. We have termed the growth of Synechocystis strain 6803 pulsed with light and kept otherwise in complete darkness light-activated heterotrophic growth. This observation of a blue-light requirement for the growth of Synechocystis strain 6803 represents a novel blue light effect on the growth of a cyanobacterium.     

Fluorescence from low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at physiological temperatures

Fluorescence spectra from Photosystem I (PS I) are measured from 25 to –5 °C on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Emission from antenna chlorophylls (Chls) with energy levels below that of the reaction center, or low-energy Chls (LE Chls), is resolved verifying their presence at physiological temperatures. The 25°C spectrum is characterized by peaks at 688 and 715 nm. As temperature decreases, fluorescence at 688 nm decreases while at 715 nm it increases. The total fluorescence yield does not change. The temperature dependent spectra are fit to a sum of two basis spectra. At 25°C, the first basis spectrum has a major peak at 686 nm and a minor peak at 740 nm. This is attributed to fluorescence from the majority or bulk antenna Chls. The second basis spectrum has a major peak at 712 nm, with shoulders at 722 and 770 nm. It characterizes fluorescence from a small number of LE Chls. A progressive shift to the red in the fluorescence spectra occurs as the temperature is decreased. The temperature dependence in the relative amount of fluorescence from the bulk and LE Chls is fit using a two-component energy transfer model at thermal equilibrium.     

A promoter-trap vector for clock-controlled genes in the cyanobacterium Synechocystis sp. PCC 6803

We constructed a promoter-trap vector pPT6803-1 to isolate circadian clock-controlled promoters in the cyanobacterium Synechocystis sp. strain PCC 6803. The vector contains a promoterless luciferase gene set (luxAB) from Vibrio harveyi that is targeted to a specific site of the Synechocystis genome as a reporter for gene expression. A library was constructed in pPT6803-1 by introducing the genomic DNA fragments upstream of luxAB to transform Synechocystis cells. Of approximately 10,000 Synechocystis transformants, at least 55 (#1-55) showed circadian rhythms of bioluminescence under continuous illumination. Clones #19, #22, and #26 exhibited obviously different waveforms of bioluminescence from each other. Deletion analysis and primer extension experiments mapped the promoters for the clpP, slr1634, and rbpP genes that are responsible for bioluminescence from #19, #22, and #26, respectively.     

Ultrafast primary processes in photosystem I of the cyanobacterium Synechocystis sp. PCC 6803.

Ultrafast primary processes in the trimeric photosystem I core antenna-reaction center complex of the cyanobacterium Synechocystis sp. PCC 6803 have been examined in pump-probe experiments with approximately 100 fs resolution. A global analysis of two-color profiles, excited at 660 nm and probed at 5 nm intervals from 650 to 730 nm, reveals 430 fs kinetics for spectral equilibration among bulk antenna chlorophylls. At least two lifetime components (2.0 and 6.5 ps in our analysis) are required to describe equilibration of bulk chlorophylls with far red-absorbing chlorophylls (>700 nm). Trapping at P700 occurs with 24-ps kinetics. The multiphasic bulk left arrow over right arrow red equilibration kinetics are intriguing, because prior steady-state spectral studies have suggested that the core antenna in Synechocystis sp. contains only one red-absorbing chlorophyll species (C708). The disperse kinetics may arise from inhomogeneous broadening in C708. The one-color optical anisotropy at 680 nm (near the red edge of the bulk antenna) decays with 590 fs kinetics; the corresponding anisotropy at 710 nm shows approximately 3.1 ps kinetics. The latter may signal equilibration among symmetry-equivalent red chlorophylls, bound to different monomers within trimeric photosystem I.     

Role of a NifS-like protein from the cyanobacterium Synechocystis PCC 6803 in the maturation of FeS proteins

In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.     

Blue light stimulates cyanobacterial motility via a cAMP signal transduction system

The participation of cAMP in photosignal transduction in cyanobacteria was investigated. When cells of the cyanobacterium Synechocystis sp. PCC 6803 were exposed to light, cellular cAMP contents increased within a few minutes. Among incident monochromatic lights, blue light (450 nm) markedly increased cellular cAMP content, while red (630 nm) and far-red (720 nm) lights did not. Disruption of the cya1 gene encoding an adenylate cyclase caused the insensitivity of cellular cAMP level to blue light. Treatment of wild-type cells with the flavin antagonist phenylacetic acid inhibited this blue light effect. The motility of wild-type cells was enhanced by blue light, whereas that of cya1 mutant cells was not. Based on these results, we concluded that a blue light-cAMP signal transduction system stimulates the motility of Synechocystis sp. PCC 6803.     

An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402.

The unicellular diazotrophic cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, contains a novel insertion sequence, IS8402, in the apcA gene encoding a pigmented protein of phycobilisomes. IS8402 comprises 1,322 bp, flanked by two inverted repeats of 15 bp. Upon insertion in the target DNA, direct duplications of 8 nucleotides were generated. One open reading frame, potentially coding for a protein of 399 amino acids, was found. The deduced amino acid sequence shows homology to putative transposases of the IS4 family. Precise excision of the insertion element resulted in a spontaneous revertant, Synechocystis sp. strain BO 9201, that had regained the ability to form hemidiscoidal phycobilisomes. Apart from the unique insertion of IS8402 into apcA in strain BO 8402 both strains contain at least 12 further homologous insertion elements at corresponding sites in the genomes. The unique insertion in strain BO 8402 prevents the expression of apcABC operon and hence abolishes the formation of intact phycobilisomes. This decreases the quantum efficiency of photosystem II and promotes anaerobic N2 fixation in a unicellular cyanobacterium with a highly oxygen-sensitive nitrogenase.