Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

Photorespiratory 2-phosphoglycolate metabolism and photoreduction of O<Subscript>2</Subscript> cooperate in high-light acclimation of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. strain PCC 6803

In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O₂ (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O₂ in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Δflv1, Δflv3, and ΔgcvT, as well as the double mutant Δflv1/ΔgcvT, were completely segregated but not the double mutant Δflv3/ΔgcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption is supported by the following results: (1) The mutant Δflv3/ΔgcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO₂) to rapid (standard light, 5% CO₂) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Δflv3/ΔgcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant. Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O₂, particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photoinduction of electron transport on the acceptor side of PSI in Synechocystis PCC 6803 mutant deficient in flavodiiron proteins Flv1 and Flv3

After transferring the dark-acclimated cyanobacteria to light, flavodiiron proteins Flv1/Flv3 serve as a main electron acceptor for PSI within the first seconds because Calvin cycle enzymes are inactive in the dark. Synechocystis PCC 6803 mutant Δflv1/Δflv3 devoid of Flv1 and Flv3 retained the PSI chlorophyll P700 in the reduced state over 10?s (Helman et al., 2003; Allahverdiyeva et al., 2013). Study of P700 oxidoreduction transients in dark-acclimated Δflv1/Δflv3 mutant under the action of successive white light pulses separated by dark intervals of various durations indicated that the delayed oxidation of P700 was determined by light activation of electron transport on the acceptor side of PSI. We show that the light-induced redox transients of chlorophyll P700 in dark-acclimated Δflv1/Δflv3 proceed within 2?min, as opposed to 1–3?s in the wild type, and comprise a series of kinetic stages. The release of rate-limiting steps was eliminated by iodoacetamide, an inhibitor of Calvin cycle enzymes. Conversely, the creation with methyl viologen of a bypass electron flow to O₂ accelerated P700 oxidation and made its extent comparable to that in the wild-type cells. The lack of major sinks for linear electron flow in iodoacetamide-treated Δflv1/Δflv3 mutant, in which O₂- and CO₂-dependent electron flows were impaired, facilitated cyclic electron flow, which was evident from the decreased steady-state oxidation of P700 and from rapid dark reduction of P700 during and after illumination with far-red light. The results show that the photosynthetic induction in wild-type Synechocystis PCC 6803 is largely hidden due to the flavodiiron proteins whose operation circumvents the rate-limiting electron transport steps controlled by Calvin cycle reactions.     

Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.     

Operon flv4-flv2 provides cyanobacterial photosystem II with flexibility of electron transfer

Synechocystis sp PCC 6803 has four genes encoding flavodiiron proteins (FDPs; Flv1 to Flv4). Here, we investigated the flv4-flv2 operon encoding the Flv4, Sll0218, and Flv2 proteins, which are strongly expressed under low inorganic carbon conditions (i.e., air level of CO(2)) but become repressed at elevated CO(2) conditions. Different from FDP homodimers in anaerobic microbes, Synechocystis Flv2 and Flv4 form a heterodimer. It is located in cytoplasm but also has a high affinity to membrane in the presence of cations. Sll0218, on the contrary, resides in the thylakoid membrane in association with a high molecular mass protein complex. Sll0218 operates partially independently of Flv2/Flv4. It stabilizes the photosystem II (PSII) dimers, and according to biophysical measurements opens up a novel electron transfer pathway to the Flv2/Flv4 heterodimer from PSII. Constructed homology models suggest efficient electron transfer in heterodimeric Flv2/Flv4. It is suggested that Flv2/Flv4 binds to thylakoids in light, mediates electron transfer from PSII, and concomitantly regulates the association of phycobilisomes with PSII. The function of the flv4-flv2 operon provides many β-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving PSII.     

Functional redundancy between flavodiiron proteins and NDH‐1 in Synechocystis sp. PCC 6803

In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light‐dependent reduction of O₂ to H₂O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero‐oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase‐like complex (NDH‐1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH‐1 types have been characterized in cyanobacteria: NDH‐1₁ and NDH‐1₂, which function in respiration; and NDH‐1₃ and NDH‐1₄, which function in CO₂ uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (?flv1 and Δflv3) and the double NDH‐1 mutants (?d1d2, which is deficient in NDH‐1_1,2 and ?d3d4, which is deficient in NDH‐1_3,4), we studied triple mutants lacking one of Flv1 or Flv3, and NDH‐1_1,2 or NDH‐1_3,4. We show that the presence of either Flv1/3 or NDH‐1_1,2, but not NDH‐1_3,4, is indispensable for survival during changes in growth conditions from high CO₂/moderate light to low CO₂/high light. Our results show functional redundancy between FDPs and NDH‐1_1,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH‐1_1,2, allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO₂ and light availability.     

Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO₂, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (F_AF_B), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s⁻¹). These data unambiguously show that reduced ferredoxin, or possibly reduced F_AF_B, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (F_AF_B) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.     

Flavodiiron Protein Flv2/Flv4-Related Photoprotective Mechanism Dissipates Excitation Pressure of PSII in Cooperation with Phycobilisomes in Cyanobacteria

Oxygenic photosynthesis evolved with cyanobacteria, the ancestors of plant chloroplasts. The highly oxidizing chemistry of water splitting required concomitant evolution of efficient photoprotection mechanisms to safeguard the photosynthetic machinery. The role of flavodiiron proteins (FDPs), originally called A-type flavoproteins or Flvs, in this context has only recently been appreciated. Cyanobacterial FDPs constitute a specific protein group that evolved to protect oxygenic photosynthesis. There are four FDPs in Synechocystis sp. PCC 6803 (Flv1 to Flv4). Two of them, Flv2 and Flv4, are encoded by an operon together with a Sll0218 protein. Their expression, tightly regulated by CO₂ levels, is also influenced by changes in light intensity. Here we describe the overexpression of the flv4-2 operon in Synechocystis sp. PCC 6803 and demonstrate that it results in improved photochemistry of PSII. The flv4-2/OE mutant is more resistant to photoinhibition of PSII and exhibits a more oxidized state of the plastoquinone pool and reduced production of singlet oxygen compared with control strains. Results of biophysical measurements indicate that the flv4-2 operon functions in an alternative electron transfer pathway from PSII, and thus alleviates PSII excitation pressure by channeling up to 30% of PSII-originated electrons. Furthermore, intact phycobilisomes are required for stable expression of the flv4-2 operon genes and for the Flv2/Flv4 heterodimer-mediated electron transfer mechanism. The latter operates in photoprotection in a complementary way with the orange carotenoid protein-related nonphotochemical quenching. Expression of the flv4-2 operon and exchange of the D1 forms in PSII centers upon light stress, on the contrary, are mutually exclusive photoprotection strategies among cyanobacteria.Photosynthetic light reactions are evolutionarily highly conserved among oxygenic photosynthetic organisms from cyanobacteria to higher plants. Because of dangerous chemistry of the water splitting reactions, oxygenic photosynthesis produces reactive oxygen species (ROS) and other radicals that potentially could destroy the photosynthetic machinery. To avoid permanent damage, all oxygenic photosynthetic organisms are equipped with an array of various photoprotective and regulatory mechanisms. Accumulating evidence on these regulatory mechanisms has revealed vast evolutionary differences between organisms performing oxygenic photosynthesis.Photosynthetic organisms have a capacity to adjust to different light intensities and to changes in the availability of electron sinks, which depends largely on metabolic cues. When light or metabolic conditions change, photosystems can dissipate excess energy as heat in nonphotochemical energy dissipation processes in the light-harvesting antenna systems (for review, see Horton et al., 1996; Müller et al., 2001). Cyanobacteria have phycobilisomes (PBs) as light-harvesting antenna, which also participate in state transitions (for review, see van Thor et al., 1998; Mullineaux and Emlyn-Jones, 2005) and nonphotochemical quenching (NPQ) of excitation energy (for review, see Bailey and Grossman, 2008). Both of these processes are involved in short-term regulation of light-harvesting processes and concomitantly function as photoprotective mechanisms. These nonphotochemical energy quenching mechanisms, however, have only limited capacity, and it often occurs that more electrons are excited than can be safely used in photochemistry for reduction of natural metabolic electron acceptors, particularly under stress conditions. In such situations, electrons can be directed, for example, to molecular oxygen resulting in production of ROS. To avoid harmful reactions by ROS that threaten the cell viability, a repertoire of different photoprotection mechanisms have evolved in cyanobacteria as well as in all other oxygenic photosynthetic organisms.Flavodiiron proteins (FDPs), originally called A-type flavoproteins or Flvs (Wasserfallen et al., 1998), were recently demonstrated to have an important role in photoprotection of the photosynthetic machinery (Zhang et al., 2009, 2012; Allahverdiyeva et al., 2011, 2013; Ermakova et al., 2013). FDPs in general are most widespread among strict and facultative anaerobic bacteria. Many of their FDPs have been characterized structurally and functionally, showing homodimeric or homotetrameric forms (Vicente et al., 2008b, 2009). A typical FDP consists of a core composed of a metallo-β-lactamase-like domain and a C-terminal flavodoxin domain. The former domain contains a nonheme diiron center, whereas the latter harbors a FMN moiety. It has been shown that FDPs in anaerobic bacteria are involved in O₂ and/or NO detoxification (Vicente et al., 2008a). Completely unique FDPs are, however, found in specific groups of oxygenic photosynthetic organisms. FDPs found in cyanobacteria and some photosynthetic eukaryotes possess an extra C-terminal flavin reductase domain (Zhang et al., 2009). This particular domain composition theoretically allows NAD(P)H oxidation to be coupled with O₂ reduction in the same enzyme.Synechocystis sp. PCC 6803 (hereafter Synechocystis), a widely used model organism among cyanobacteria in photosynthesis research, contains four FDPs encoded by the sll1521 (flv1), sll0219 (flv2), sll0550 (flv3), and sll0217 (flv4) genes. In vivo, Flv1 and Flv3 acquire electrons after PSI and deliver them further to molecular oxygen, reducing it to water. We have denominated this process as a Mehler-like reaction (Allahverdiyeva et al., 2013) because the excess of electrons is donated to O₂, similarly to the genuine plant-type Mehler reaction, but there is no production of ROS in the FDP-mediated reaction (Helman et al., 2003). Up to 60% of the electrons produced by the oxygen splitting activity of PSII are redirected to Flv1- and Flv3-mediated Mehler-like reactions in severe inorganic carbon starvation conditions (Allahverdiyeva et al., 2011). Flv1 and Flv3 proteins form a very important electron sink that protects PSI against oxidative damage under fluctuating light conditions (Allahverdiyeva et al., 2013).Flv2 and Flv4 can be found only in cyanobacteria and they have been assigned a role in photoprotection of PSII (Zhang et al., 2009, 2012). PSII is historically known to be extremely vulnerable to oxidative damage upon illumination, with the severity of damage being dependent on light intensity and on the availability of electron acceptors. At air-level CO₂ concentrations (low CO₂ or LC) and/or high light (HL) irradiances, terminal acceptors are consistently limiting the electron flow, making PSII particularly sensitive to these conditions, widely exceeding the repair capacity of damaged PSII centers (Aro et al., 1993).Flv2 and Flv4 proteins are encoded in an operon including a small Sll0218 protein. Importantly, the flv4-2 operon is strongly induced in LC and HL conditions (Zhang et al., 2009). Flv4 and Flv2 proteins form a heterodimer that localizes in cytoplasm but also has a high affinity to membrane in the presence of cations (Zhang et al., 2012). Sll0218, the 19-kD protein encoded by the flv4-2 operon, locates in the thylakoid membrane and forms a high molecular mass complex in association with unknown partners. In the model proposed by Zhang et al. (2012), Sll0218 stabilizes PSII dimers and facilitates the opening of a novel electron transfer pathway through the Flv2/Flv4 heterodimer, which associates with the thylakoid membrane in light. The Flv2/Flv4 complex is also important for proper energy transfer from PBs to PSII as evidenced by a high emission peak at 685 nm in the 77K fluorescence spectra. This effect is caused by uncoupled PB terminal emitters, as deduced from detailed examination of the deconvoluted emission spectra (Zhang et al., 2012). This strongly suggested a distorted energy transfer from PB terminal emitters to the PSII reaction centers in flv4-2 operon deletion mutants. However, the photoprotection mechanism induced by the flv4-2 operon is not yet clearly understood. Here, with an overexpression approach, we provide evidence that Flv2/Flv4 acts as an important electron sink at the PSII acceptor side, allowing the maintenance of the plastoquinone (PQ) pool in an oxidized state and preventing the production of singlet oxygen in PSII. Furthermore, regular PBs are required for the Flv2/Flv4-related mechanism to be expressed. Genome mining of sequenced cyanobacteria strains provided evidence for the loss of the flv4-2 operon in the genomes of cyanobacteria that have acquired a stress-inducible D1 copy.     

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.     

The acyl-acyl carrier protein synthetase from Synechocystis sp. PCC 6803 mediates fatty acid import

The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.     

The S-layer biogenesis system of Synechocystis 6803: Role of Sll1180 and Sll1181 (E. coli HlyB and HlyD analogs) as type-I secretion components for Sll1951 export

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.     

Functional characterization of sll0659 from Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that Sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types and mutants. In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cycilzation. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.     

Novel putative photoreceptor and regulatory genes Required for the positive phototactic movement of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803

Synechocystis: sp. PCC 6803 is a unicellular motile cyanobacterium, which shows positive or negative phototaxis on agar plates under lateral illumination. By gene disruption in a substrain showing of positive phototaxis, it was demonstrated that mutants defective in sll0038, sll0039, sll0041, sll0042 or sll0043 lost positive phototaxis but showed negative phototaxis away from the light source. Mutants of sll0040, which is located within the cluster of these genes, retained the capacity of positive phototaxis but to a lesser extent than the parent cells. These genes are homologous to che genes, which are involved in flagellar switching for bacterial chemotaxis. Interestingly, sll0041 (designated pisJ1) is predicted to have a chromophore-binding motif of phytochrome-like proteins and a signaling motif of chemoreceptors for bacterial chemotaxis. It is strongly suggested that the positive phototactic response was mediated by a phytochrome-like photoreceptor and CheA/CheY-type signal transduction system.     

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak (<0.5 micromol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 micromol m(-2) s(-1) or above, but no growth at 0.5 micromol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence.     

The role of novel genes rrp1+ and rrp2+ in the repair of DNA damage in Schizosaccharomyces pombe

We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants. Single Δrrp1 and Δrrp2 mutants were not sensitive to CPT, 4NQO, CDPP, MMS, HU, UV or IR. The double mutants Δrrp1 Δrhp51 and Δrrp2 Δrhp51 plus the triple Δrrp1 Δrrp2 Δrhp51 mutant did not display significant additional sensitivity. However, the double mutants Δrrp1 Δrhp57 and Δrrp2 Δrhp57 were significantly more sensitive to MMS, CPT, HU and IR than the Δrhp57 single mutant. The checkpoint response in these strains was functional. In S. pombe, Rhp55/57 acts in parallel with a second mediator complex, Swi5/Sfr1, to facilitate Rhp51-dependent DNA repair. Δrrp1 Δsfr1 and Δrrp2 Δsfr1 double mutants did not show significant additional sensitivity, suggesting a function for Rrp proteins in the Swi5/Sfr1 pathway of DSB repair. Consistent with this, Δrrp1 Δrhp57 and Δrrp2 Δrhp57 mutants, but not Δrrp1 Δsfr1 or Δrrp2 Δsfr1 double mutants, exhibited slow growth and aberrations in cell and nuclear morphology that are typical of Δrhp51.