The sll0886 gene, controlling light-activated heterotrophic growth, is involved in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6893

The sll0886 gene, controlling light-activated heterotrophic growth (LAHG), was tested for the role in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6803. Insertional inactivation of the gene in the genome of a wildtype strain did not affect positive (toward light) or negative (away from high light) phototaxis. However, cells lost motility when sll0886 inactivation was combined with the prqRL17Q mutation, which determined negative phototaxis at low light. Immotile cells with the prqRL17Q mutation and the inactivated sll0886 gene did not display any defect in the formation of type IV pili, essential for phototaxis. Hence, the function, rather than biogenesis, of pili was affected. It was concluded that the sll0886 gene, coding for a TPR family protein, is involved in controlling negative phototaxis of cyanobacteria at the level of photoreception and signal transduction and that its role is mediated by the unidentified redundant gene whose function is suppressed by the prqRL17Q mutation.     

Cyanobacterial hybrid kinase Sll0043 regulates phototaxis by suppressing pilin and twitching motility protein

The unicellular cyanobacterium Synechocystis sp. PCC 6803 glides toward a light source through the interplay of positive phototaxis genes and proteins. In genetic analysis, the complete disruption of the hybrid sensory kinase sll0043 produced negative phototaxis. Furthermore, Sll0043 was found to be a hub protein by in silico prediction of protein-protein interaction, in which Sll0043 was predominantly linked to seven two-component proteins with high confidence. To understand the regulation and networking of positive phototaxis proteins, the proteomic profile of the sll0043 mutant was compared to that of wild-type. In the sll0043 mutant, 18 spots corresponding to 15 unique proteins were altered by 1.3 to 59 fold; the spots were identified by 2-DE/MALDI-MS analysis. Down-regulated proteins in the sll0043 null-mutant included chaperonins, superoxide dismutase, and phycocyanin beta-subunit. In contrast, nine proteins involved in photosynthesis, translation, regulatory function, and other functions were up-regulated. In particular, a twitching motility protein (PilT1) was induced over 2-fold in sll0043 mutant. Moreover, semi-quantitative and quantitative RT-PCR analysis revealed that pilin (pilA1), pili motor (pilT1), and pili switch gene (pilT2) were significantly increased in sll0043 mutant. These results suggest that the hybrid kinase Sll0043 regulates positive phototaxis by suppressing the expression of pili biosynthesis and regulatory genes and through the interplay with positive phototaxis/motility two-component proteins.     

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.     

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.     

Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.     

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.     

Negative Phototaxis from Blue Light and the Role of Third Rhodopsinlike Pigment in Halobacterium Cutirubrum

Wild-type cells of Halobacterium cutirubrum show phototaxis. In negative phototaxis the cells are repelled by blue-near ultraviolet light, and in positive phototaxis the cells are attracted to green-red light. The extent of the responses are measured by monitoring the changes in the reversal frequency of the swimming direction of cells using a computer-linked automated method as described previously (Takahashi, T., and Y. Kobatake, 1982, Cell. Struct. Funct., 7:183-192). When the intensity of the background light (illumination for the observation) was dramatically reduced, the sensitivity of the cells to the repellent light decreased markedly. This result has been previously explained by Bogomolni and Spudich (1982, Proc. Natl. Acad. Sci. USA, 79:6250-6254), who proposed that the photoreceptor for negative phototaxis is the long-lifetime intermediate in the photocycle of slow-rhodospin. The behavioral response in the negative phototaxis is dependent upon the intensity of the actinic light and the background light. This agrees quantitatively with our model based on the aforementioned hypothesis.     

Photoresponses of Chaoborus larvae

The diel vertical migration of Chaoborus larvae is a well known phenomenon. In order to quantify the ability of larvae to utilize underwater light cues in their migration, we measured photoresponses of fourth-instar Chaoborus punctipennis larvae in the laboratory. The action spectrum for these larvae was characterized by a maximum in sensitivity at 400 nm, a plateau at a lower sensitivity from 480 to 560 nm, and a region of much lower sensitivity at wavelengths longer than 620 nm. Dark-adapted larvae exhibited a positive phototaxis at low light intensity which shifted to a negative phototaxix as light intensity increased. At 540 nm the threshold intensity was 1.5 × 10^?9 W/m² for positive phototaxis and about 10^?6 W/m² for negative phototaxis. Light adaptation decreased sensitivity and altered the phototactic pattern. Larvae have a clear circadian rhythm in negative phototaxis, in which greatest responsiveness occurs early in the day. We suggest that the rhythm in photoresponsiveness primarily controls the timing of the downward migration at dawn.     

Maintenance of Motility Bias during Cyanobacterial Phototaxis

Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.     

Characterization of a Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and Sll0933

The de novo assembly of photosystem II (PSII) depends on a variety of assisting factors. We have previously shown that two of them, namely, YCF48 and Sll0933, mutually interact and form a complex (Rengstl et al. in J Biol Chem 286:21944–21951, 2011). To gain further insights into the importance of the YCF48/Sll0933 interaction, an ycf48 ^? sll0933 ^? double mutant was constructed and its phenotype was compared with the single mutants’ phenotypes. Analysis of fluorescence spectra and oxygen evolution revealed high-light sensitivity not only for YCF48 deficient strains but also for sll0933 ^? , which, in addition, showed reduced synthesis and accumulation of newly synthesized CP43 and CP47 proteins in pulse-labeling experiments. In general, the phenotypic characteristics of ycf48 ^? sll0933 ^? were dominated by the effect of the ycf48 deletion and additional inactivation of the sll0933 gene showed only negligible additional impairments with regard to growth, absorption spectra and accumulation of PSII-related proteins and assembly complexes. In yeast split-ubiquitin analyses, the interaction between YCF48 and Sll0933 was confirmed and, furthermore, support for direct binding of Sll0933 to CP43 and CP47 was obtained. Our data provide important new information which further refines our knowledge about the PSII assembly process and role of accessory protein factors within it.     

Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae. Received: 12 February 1999 / Revision accepted: 24 May 1999     

The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20°C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44°C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.     

Chorismate Pyruvate-Lyase and 4-Hydroxy-3-solanesylbenzoate Decarboxylase Are Required for Plastoquinone Biosynthesis in the Cyanobacterium Synechocystis sp. PCC6803

Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.     

Formation of the function of the photosensing system in early development

The function of simple eyes in two planarian species, two-eyed Girardia tigrina and multi-eyed Polycelis tenuis, has been studied. When exposed to light, planarians display a light avoidance reaction known as negative phototaxis. This reaction has been investigated in intact animals and in head and tail fragments in the course of eye regeneration after their section. Specific features of the phototaxis reaction have been described in all groups of animals. The differences in light response recovery were shown between two planarian species and two regenerating fragments. No correlation has been found between phototactic reactions and restoration of eye structure, the number of eyes, maturation of the ganglion, growth of regenerative blastema, and motor system. The phototactic response occurred two days after the recovery of the morphology of eyes and their connection with the brain. The participation of conserved and novel genes in early development of the eye function is discussed.     

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ∼30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

ubiJ,a New Gene Required for Aerobic Growth and Proliferation in Macrophage,Is Involved in Coenzyme Q Biosynthesis in Escherichia coli and Salmonella enterica Serovar Typhimurium

Ubiquinone (coenzyme Q or Q₈) is a redox active lipid which functions in the respiratory electron transport chain and plays a crucial role in energy-generating processes. In both Escherichia coli and Salmonella enterica serovar Typhimurium, the yigP gene is located between ubiE and ubiB, all three being likely to constitute an operon. In this work, we showed that the uncharacterized yigP gene was involved in Q₈ biosynthesis in both strains, and we have renamed it ubiJ. Under aerobic conditions, an ubiJ mutant was found to be impaired for Q₈ biosynthesis and for growth in rich medium but did not present any defect anaerobically. Surprisingly, the C-terminal 50 amino acids, predicted to interact with lipids, were sufficient to restore Q₈ biosynthesis and growth of the ubiJ mutant. Salmonella ubiE and ubiB mutants were impaired in Q₈ biosynthesis and in respiration using different electron acceptors. Moreover, ubiE, ubiJ, and ubiB mutants were all impaired for Salmonella intracellular proliferation in macrophages. Taken together, our data establish an important role for UbiJ in Q₈ biosynthesis and reveal an unexpected link between Q₈ and virulence. They also emphasize that Salmonella organisms in an intracellular lifestyle rely on aerobic respiration to survive and proliferate within macrophages.