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Light was found to strongly influence the infection of a freshwater cyanobacterium (Synechococcus elongatus PCC 7942) by a contractile DNA phage named AS-1. Phage progeny production was correlated with the amount of light in the laboratory and occurred in a diel pattern under natural light. At least one effect of light on AS-1 infection is at the level of adsorption.
Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.
Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and are frequently observed in close proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here, we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independent of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.IMPORTANCEPolyphosphate (polyP) is a ubiquitous storage polymer found across all three domains of life. In bacteria, polyP is involved in virulence, energy metabolism, DNA structure, and stress. Here, we investigate the physical and functional relationships between polyP and the carboxysome-a carbon-fixing organelle that encapsulates the most abundant enzyme on Earth and is responsible for a significant amount of atmospheric CO2 sequestration. We discover that polyP is important for proper carboxysome assembly and organization in the cell and is also required for robust cell growth under ambient CO2 conditions. As carboxysomes are one class of the broader group of bacterial microcompartments, these findings have broader implications for polyP in the function and organization of the bacterial cytoplasm.
Recent advances in understanding circadian (daily) rhythms in the genera Neurospora, Gonyaulax, and Synechococcus are reviewed and new complexities in their circadian systems are described. The previous model, consisting of a unidirectional flow of information from input to oscillator to output, has now expanded to include multiple input pathways, multiple oscillators, multiple outputs; and feedback from oscillator to input and output to oscillator. New posttranscriptional features of the frq/white-collar oscillator (FWC) of Neurospora are described, including protein phosphorylation and degradation, dimerization, and complex formation. Experimental evidence is presented for frq-less oscillator(s) (FLO) downstream of the FWC. Mathematical models of the Neurospora system are also discussed.
The molecular mechanism of circadian rhythmicity is usually modeled by a transcription/translation feedback oscillator in which clock proteins negatively feed back on their own transcription to produce rhythmic levels of clock protein mRNAs, which in turn cause the production of rhythmic levels of clock proteins. This mechanism has been applied to all model organisms for which molecular data are available. This review summarizes the increasing number of anomalous observations that do not fit the standard molecular mechanism for the model organisms Acetabularia, Synechococcus, Drosophila, Neurospora, and mouse. The anomalies fall into 2 classes: observations of rhythmicity in the organism when transcription of clock genes is held constant, and rhythmicity in the organism when clock gene function is missing in knockout mutants. It is concluded that the weight of anomalies is now so large that the standard transcription/translation mechanism is no longer an adequate model for circadian oscillators. Rhythmic transcription may have other functions in the circadian system, such as participating in input and output pathways and providing robustness to the oscillations. It may be most useful to think in terms of a circadian system that uses a noncircadian oscillator consisting of metabolic feedback loops, which acquires its circadian properties from additional regulatory molecules such as the products of canonical clock genes.
The time measurement system of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is analogous to the circadian clock of eukaryotic cells. Circadian clock-related genes have been identified in this strain. The clock-related gene pex is thought to maintain the normal clock period because constitutive transcription or deficiency of this gene causes respectively longer (approximately 28 h) or shorter (approximately 24 h) circadian periods than that of the wild type (approximately 25 h). Here, the authors report other properties of pex in the circadian system. Levels of pex mRNA increased significantly in a 12-h exposure to darkness. Western blotting with a GST-Pex antibody revealed a 13.5-kDa protein band in wild-type cells that were incubated in the dark, while this protein was not detected in pex-deficient mutant cells. Therefore, the molecular weight of the Pex protein appears to be 13.5 kDa in vivo. The PadR domain, which is conserved among DNA-binding transcription factors in lactobacilli, was found in Pex. In the pex mutant, several 12-h light/12-h dark cycles reset the phase of the clock by 3 h earlier (phase advance) compared to wild-type cells. The degree of the advance in the pex mutant was proportional to the number of exposed light-dark cycles. In addition, ectopic induction of pex with an inducible Escherichia coli promoter, Ptrc, delayed the phase in the examined recombinant cells by 2.5 h (phase delay) compared to control cells. These results suggest that the dark-responsive gene expression of pex delays the circadian clock under daily light-dark cycles.
In the cyanobacterium, Synechococcus elongatus, most promoters are regulated by a circadian clock under continuous light (LL) conditions. Nevertheless, the basic circadian oscillation is primarily generated by alternating KaiC phosphorylation/dephosphorylation reactions at the posttranslational level. Indeed, the KaiC phosphorylation cycle was recently reconstituted in vitro by incubating KaiA, KaiB, and KaiC proteins with ATP. However, the molecular dynamics of this chemical oscillation and the mechanism that drives the circadian transcription/translation rhythms remain unknown. In this report, the KaiC phosphorylation cycle and the gene regulatory network in the cyanobacterial circadian system have been modeled. The model reproduces the robust KaiC phosphorylation cycle in the absence of de novo gene expression as is observed in vitro, as well as its coupling to transcriptional/translational feedback in LL conditions in vivo. Moreover, the model is consistent with most previous experiments, including various combinations of genetic knockout or overexpression of kai genes. It also predicts that multiple KaiC phosphorylation states and dynamic Kai protein interactions may be required for the cyanobacterial circadian system.
Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.
Nine typical phytoplankton species were chosen and cultivated under two temperatures (20 and 15 degrees C) and two illumi- nations (7000 and 1100 lux), and their absorption spectra at different growth period were measured. Firstly, singular value decomposition was used on the matrix, which is composed of the autoscaled spectra data. The S1/msigma(i=1)Si was used to compare the similarities of spectra. Twenty-five representative spectra were obtained for the nine species of phytoplankton. Among them, there are one spectrum for each of Isochrysis galbana and Chaetoceros Debilis, two for each of Platymonas helgolanidica and Chaetoceros curvisetus, three for Gymnodinium sp., and four for each of Alexandrium tamarense, Prorocentrum dentatum, Skeletonema costatuma and Synechococcus sp. Then, feature extraction was processed to obtain the characteristic spectra, including seven incontinuous wavelengths that have great ability to differentiate species. They are 340. 5-420. 5 nm, 423. 5-431 nm, 440.5-525.5 nm, 760.5, 763.5, 769.5 and 856.5 nm. Consequently, they form characteristic spectra. The discriminant result is 80%.
A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria. The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock. A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes. Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation. BioEssays 22:10-15, 2000.
Three strains of picoplankton designated as brown, green, and pink belonging to the Synechococcus genus in cyanobacteria (approximately 1 microm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria such as Proteobacteria and Bacillus by microscopy. In this study, we attempted to use the polymerase chain reaction (PCR) analysis of the 16S ribosomal DNA (rDNA) from picoplankton for the diagnosis of death by drowning. A segment of 16S rDNA was sequenced in order to investigate their phylogenetic relationships and to design the specific primers. The PCR products from three picoplanktons were compared with those from five other cyanobacteria, Melosira (diatom), Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The efficiency of PCR was increased more than hundredfold by isolating the picoplankton from human lung tissue. The specific PCR products of the picoplankton were obtained from a formalin-fixed drowning body (lung and liver) that was found in a downstream river and Lake Biwa. The PCR analysis of the picoplanktion 16S rDNA is considered useful for the diagnosis of death by drowning.
BACKGROUND: The Bay of Marseilles is under the influence of a large urban concentration and its maritime activities. All of them discharge compounds (hydrocarbons, excess nutrients, heavy metals, chemicals, etc.) that can alter the marine ecosystem. To investigate whether ultraphytoplankton (<10 microm) could be used as biosensors for their own ecosystem, a 2-year survey was conducted in the Bay of Marseilles. METHODS: Seven stations monitored water mass and potential anthropic effects in the bay. Seawater samples were collected monthly or bimonthly at three depths, prefiltered, fixed, and kept in liquid nitrogen until flow cytometric analysis. RESULTS: Five categories were created: Prochlorococcus, picoeukaryotes (<2 microm), nanoeukaryotes I (2--6 microm), nanoeukaryotes II (6--10 microm), and Synechococcus (<1.5 microm). Artificial neural network analysis (Kohonen self-organizing maps) produced the same number of clusters as cluster analysis with Winlist software (Verity Software House). CONCLUSIONS: In addition to the wide variabilities in abundance and biomass, there were a strong seasonal signal and sporadic events. Lessons are derived from this study for future monitoring of marine microorganisms.
A biosensor for the detection of triazine- and phenylurea-type herbicides was constructed using isolated Photosystem II (PS II) complexes as a biosensing element. PSII isolated from the thermophilic cyanobacterium Synechococcus elongatus was immobilized on the surface of a screen-printed sensor composed of a graphite working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate. The biosensor was mounted in a flow microcell with illumination. The principle of the detection was based on the fact that herbicides selectively block PSII electron transport activity in a concentration-dependent manner. Changes of the activity were registered amperometrically as the rate of photoreduction of an artificial electron acceptor. The setup resulted in a reusable herbicide biosensor with a good stability (half-life of 24 h) and limit of detection of approximately 10(-9) M for diuron, atrazine and simazine.
Analysis of light energy distribution in culture is important for maximizing the growth efficiency of photosynthetic cells and the productivity of a photobioreactor. To characterize the irradiance conditions in a photobioreactor, we developed a light distribution model for a single-radiator system and then extended the model to multiple radiators using the concept of parallel translation. Mathematical expressions for the local light intensity and the average light intensity were derived for a cylindrical photobioreactor with multiple internal radiators. The proposed model was used to predict the irradiance levels inside an internally radiating photobioreactor using Synechococcus sp. PCC 6301 as a model photosynthetic microorganism. The effects of cell density and radiator number were interpreted through photographic and model simulation studies. The predicted light intensity values were found to be very close to those obtained experimentally, which suggests that the proposed model is capable of accurately interpreting the local light energy profiles inside the photobioreactor system. Due to the simplicity and flexibility of the proposed model, it was also possible to predict the light conditions in other complex photobioreactors, including optical-fiber and pond-type photobioreactors.
Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-microm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated.
Light intensity is a crucial factor that determines the growth of photosynthetic cells. This study analyzed the light distribution in a photobioreactor by processing images, captured with a digital camera, of a rectangular photobioreactor containing Synechococcus sp. PCC6801 as a model microorganism. The gray-scale images obtained clearly demonstrate the variation of the light-distribution profiles upon changing cell concentrations and external light intensity. Image-processing techniques were also used to predict the cell density in the photobioreactor. By analyzing the digitized image data with a neural network model, we were able to predict the cell concentrations in the photobioreactor with a <5% error.
A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small-sized picoplankton (below 2 microns) such as Synechococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger-sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.
We present an electron microscopical analysis of the photosystem I reaction centre, the membrane complex involved in the second light-driven step of photosynthetic electron transfer in plants and cyanobacteria. To this end, ordered two-dimensional arrays were reconstituted from detergent solubilized photosystem I reaction centres and phospholipids, and studied by electron microscopy and digital image processing. Small (P1) and large (P3) hexagonal lattices obtained with reaction centres of the thermophilic cyanobacterium Phormidium laminosum had unit cell sizes of a = b = 8.8 nm and 15.8 nm, respectively. Reaction centres of a second thermophilic strain, Synechococcus sp. OD24, gave square lattices (a = b = 14.5 nm; P2(1)). Irrespective of the packing arrangement, projections of negatively stained photosystem I complexes showed elongated asymmetric shapes with a large domain at one end which was tilted with respect to a small domain forming the tip of the other end. Such features were also found in averaged projections of solubilized reaction centre trimers. Surface reliefs reconstructed from freeze-dried metal-shadowed P2(1) lattices revealed that reaction centres had a ridge of 2.5 nm height projecting from one side of the membrane while their other side was rather flat and exhibited a shallow, central indentation.