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PubMed2005

In situ functional gene analysis: recognition of individual genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) using specific probes certainly is one of the most commonly applied molecular techniques in microbial ecology. Monitoring of community composition and dynamics can be combined with localization and identification of individual cells in situ. However, the resolution power of the method is limited by the need for high target numbers per cell. Apart from standard targets (ribosomal RNAs), mRNAs could be used successfully for in situ visualization in some cases. A new promising variant of in situ hybridization could be established that should provide access to any low copy number nucleic acid targets, such as chromosomal genes. The recognition of individual genes by FISH (RING-FISH) technology is based on target visualization mediated by polynucleotide probe network formation. The specificity of the approach is provided by intracellular probe-target hybridization. This initial hybridization apparently acts as a focal point for inter-probe hybridization within and mainly in the periphery of the cells. Probe-conferred fluorescence typically appears halo-like around the cells. RING-FISH can be used in combination with conventional oligonucleotide FISH. Thus, genetic potential can be assigned to in situ identified cells.

Escherichia coli↗
PubMed2005

Fourier transform infrared spectrum of the secondary quinone electron acceptor Q(B) in photosystem II.

Fourier transform infrared difference spectra upon single reduction of the secondary quinone electron acceptor Q(B) in photosystem II (PSII), without a contribution from the electron donor-side signals, were obtained for the first time using Mn-depleted PSII core complexes of the thermophilic cyanobacterium Thermosynechococcus elongatus. The Q(B)(-)/Q(B) difference spectrum exhibited a strong C...O stretching band of the semiquinone anion at 1480 cm(-)(1), the frequency higher by 2 cm(-)(1) than that of the corresponding band of Q(A)(-), in agreement with the previous S(2)Q(B)(-)/S(1)Q(B) spectrum of the PSII membranes of spinach [Zhang, H., Fischer, G., and Wydrzynski, T. (1998) Biochemistry 37, 5511-5517]. Also, several peaks originating from the Fermi resonance of coupled His modes with its strongly H-bonded NH vibration were observed in the 2900-2600 cm(-)(1) region, where the peak frequencies were higher by 7-24 cm(-)(1) compared with those of the Q(A)(-)/Q(A) spectrum. These frequency differences suggest that H-bond interactions of the CO groups, especially with a His side chain, are different between Q(B)(-) and Q(A)(-). Furthermore, a prominent positive peak was observed at 1745 cm(-)(1) in the C=O stretching region of COOH or ester groups in the Q(B)(-)/Q(B) spectrum. The peak frequency was unaffected by D(2)O substitution, indicating that this peak does not arise from a COOH group but probably from the 10a-ester C=O group of the pheophytin molecule adjacent to Q(B). The absence of protonation of carboxylic amino acids upon Q(B)(-) formation in contrast to the previous observation in the purple bacterium Rhodobacter sphaeroides suggests that the protonation mechanism of Q(B) in PSII is different from that of bacterial reaction centers.

Photosystem II Protein Complex↗
PubMed2006

The free energy of dissociation of oligomeric structure in phycocyanin is not linear with denaturant.

Using SEC HPLC and fluorescence anisotropy, absorption spectra were assigned to the specific oligomeric structures found with phycocyanin. The absorption spectra were used to quantify the population of each oligomeric form of the protein as a function of both urea concentration and temperature. Phycocyanin hexamers dissociate to trimers with equilibrium constants of 10(-6) to 10(-5). Phycocyanin trimers dissociate to monomers with equilibrium constants of 10(-15) to 10(-12). Both dissociation constants increase linearly with increasing urea concentration, and deltaG(o) values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, deltaG(urea)(o) = deltaG(water)(o) + A[denaturant], in which a linear relationship exists between the free energy of protein unfolding or loss of quaternary structure and the denaturant concentration. Our data examines a smaller range of denaturant concentration than generally used, which might partially explain the inconsistency.

Bacterial Proteins↗
PubMedDate not supplied

In situ monitoring of cell concentration in a photobioreactor using image analysis: comparison of uniform light distribution model and artificial neural networks.

Light intensity is a very important factor that determines the growth of photosynthetic cells. In this study, the light distribution in a photobioreactor was analyzed by processing the images captured with a digital camera. The contour images obtained by filtering the original images clearly showed the effects of the cell concentration and external light intensity on the light distribution. Image-processing techniques were then applied to predict the cell density in the photobioreactor. To correlate the cell concentration with the light intensity in the photobioreactor, the captured images were processed using two different approaches. The first method involved the use of an average gray value after deriving a simplified model equation that could be related to the cell density. The second method involved the use of local points instead of a representative value. In this case, an artificial neural network model was adopted to infer the cell density from the information of the local points. By using these two methods, it was possible to relate the image data to the cell concentration. Finally, we compared these two methods with regard to their accuracy, easiness, and effectiveness.

Bioreactors↗
PubMed2005

Redox potentials of chlorophylls and beta-carotene in the antenna complexes of photosystem II.

Electron transfer (ET) processes in reaction centers (RC) of photosystem II (PSII) are prerequisites of oxygen generation. They are promoted by energy transfer from antenna to RC. Here, we calculated the redox potentials of chlorophylla/beta-carotene (Chla/Car) in PSII CP43/CP47 antenna complexes, solving the linearized Poisson-Boltzmann (LPB) equation based on the PSII crystal structure. The majority of antenna Chla redox potentials for reduction/oxidation were lower than those of RC Chla. Hence, ET events with excess electrons remain localized in the RC. Simultaneously antenna Chla can serve as an efficient cation sink to rereduce RC Chla if normal PSII function is inhibited. Especially three antenna Chla (Chl-47, Chl-18, and Chl-12) and two Car bridging the space between Chl(Z(D1)) and cytochrome (cyt) b559 have the same level of oxidation redox potential. Together with Chl(Z(D2)) they form an electron hole transfer pathway and temporary storage device guiding from the oxidized P680(+.) Chla to the cyt b559. This path may play a photoprotective role as efficient electron hole quencher.

Chlorophyll↗
PubMed2005

Correlational analysis of proteins and nonmetallic nanoparticles in a deep-nulling microscope.

We present a method for label-free microscopic analysis of nonmetallic nanoparticles such as biopolymers or technical polymers diffusing freely in an aqueous environment. We demonstrate the principal feasibility of the approach with first measurements of 20-200 nm sized polystyrene spheres and of the approximately 10 nm protein complex Photosystem I (PS I) of Thermosynechococcus elongatus. The approach is based on the combination of a microscope setup with a deep-nulling interferometer for measuring minute refractive index changes or absorptions in the focal area of the microscope. It is possible to obtain transient nulls in a microscope setup on the order of 10(-5), corresponding to optical pathway differences of less than 0.6 nm and to stabilize the nulls to about 5.10(-4). With this level of stabilization it is possible to perform a fast (1 s) correlational analysis of aqueous solutions containing the protein complex PS I or 20 nm spheres and to detect in real time single diffusional transits of larger nanospheres through the focal area of the microscope. A modulated heating of the samples in the microscope focus is not necessary for detection. The interferometer correlation data correspond well to conventional two-photon excited fluorescence correlation (FCS) data measured in the same setup, providing evidence that the detection volumes are of a similar size (approximately 200 nm). We also conducted first nulling experiments using coherent near-field sources of about 30 nm diameter. Theoretical considerations indicate that this combination with nanometric near-field sources will even allow label-free single-protein analysis.

Bacterial Proteins↗
PubMed2006

One- and two-color photon echo peak shift studies of photosystem I.

Wavelength-dependent one- and two-color photon echo peak shift spectroscopy was performed on the chlorophyll Qy band of trimeric photosystem I from Thermosynechococcus elongatus. Sub-100 fs energy transfer steps were observed in addition to longer time scales previously measured by others. In the main PSI absorption peak (675-700 nm), the peak shift decays more slowly with increasing wavelength, implying that energy transfer between pigments of similar excitation energy is slower for pigments with lower site energies. In the far-red region (715 nm), the decay of the peak shift is more rapid and is complete by 1 ps, a consequence of the strong electron-phonon coupling present in this spectral region. Two-color photon echo peak shift data show strong excitonic coupling between pigments absorbing at 675 nm and those absorbing at 700 nm. The one- and two-color peak shifts were simulated using the previously developed energy transfer model (J. Phys. Chem. B 2002, 106, 10251; Biophysical Journal 2003, 85, 140). The simulations agree well with the experimental data. Two-color photon echo peak shift is shown to be far more sensitive to variations in the molecular Hamiltonian than one-color photon echo peak shift spectroscopy.

Color↗
PubMed2006

Kinetics and mechanism of electron transfer in intact photosystem II and in the isolated reaction center: pheophytin is the primary electron acceptor.

The mechanism and kinetics of electron transfer in isolated D1/D2-cyt(b559) photosystem (PS) II reaction centers (RCs) and in intact PSII cores have been studied by femtosecond transient absorption and kinetic compartment modeling. For intact PSII, a component of approximately 1.5 ps reflects the dominant energy-trapping kinetics from the antenna by the RC. A 5.5-ps component reflects the apparent lifetime of primary charge separation, which is faster by a factor of 8-12 than assumed so far. The 35-ps component represents the apparent lifetime of formation of a secondary radical pair, and the approximately 200-ps component represents the electron transfer to the Q(A) acceptor. In isolated RCs, the apparent lifetimes of primary and secondary charge separation are approximately 3 and 11 ps, respectively. It is shown (i) that pheophytin is reduced in the first step, and (ii) that the rate constants of electron transfer in the RC are identical for PSII cores and for isolated RCs. We interpret the first electron transfer step as electron donation from the primary electron donor Chl(acc D1). Thus, this mechanism, suggested earlier for isolated RCs at cryogenic temperatures, is also operative in intact PSII cores and in isolated RCs at ambient temperature. The effective rate constant of primary electron transfer from the equilibrated RC* excited state is 170-180 ns(-1), and the rate constant of secondary electron transfer is 120-130 ns(-1).

Electron Transport↗
PubMed2006

Intersubunit signaling in glutamate-1-semialdehyde-aminomutase.

Enzymes are highly dynamic and tightly controlled systems. However, allosteric communication linked to catalytic turnover is poorly understood. We have performed an integrated approach to trap several catalytic intermediates in the alpha2-dimeric key enzyme of chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase. Our data reveal an active-site "gating loop," which undergoes a dramatic conformational change during catalysis, that is simultaneously open in one subunit and closed in the other. This loop movement requires a beta-sheet-to-alpha-helix transition to assume the closed conformation, thus facilitating transport of substrate toward, and concomitantly forming, an integral part of the active site. The accompanying intersubunit cross-talk, which controls negative cooperativity between the allosteric pair, was explored at the atomic level. The central elements of the communication triad are the cofactor bound to different catalytic intermediates, the interface helix, and the gating loop. Together, they form a molecular switch in which the cofactor acts as a central signal transmitter linking the subunit interface with the gating loop.

Binding Sites↗
PubMed2004

Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane.

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.

Cell Membrane↗
PubMed2005

In vivo subcellular ultrastructures recognized with Hilbert differential contrast transmission electron microscopy.

We describe the first application of a novel electron microscopic technique to visualize subcellular structures in a near-living state. Rapidly frozen ice-embedded cells provide the most realistic images, as they are free from artefacts induced by sample preparation methods, such as chemical fixation, dehydration, staining and sectioning. The application of the conventional transmission electron microscope to ice-embedded cell imaging, however, has been limited by the low image contrast. The recently developed Hilbert differential contrast transmission electron microscope, which exhibits an unexpectedly high contrast, akin to the differential interference contrast in visible light microscopy, enabled us to clearly discern detailed subcellular structures in ice-embedded cyanobacterial cells.

Cryoelectron Microscopy↗
PubMed2005

Exploitation of genomic sequences in a systematic analysis to access how cyanobacteria sense environmental stress.

The perception and subsequent transduction of environmental signals are primary events in the acclimation of living organisms to changes in their environment. Many of the molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Therefore, the genomic information has been exploited in a systematic approach to this problem, performing systematic mutagenesis of potential sensors and transducers, namely, histidine kinases and response regulators, respectively, in combination with DNA microarray analysis, to examine the genome-wide expression of genes in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Using targeted mutagenesis, 44 out of the 47 histidine kinases and 42 out of the 45 response regulators of this organism have successfully been inactivated. The resultant mutant libraries were screened by genome-wide DNA microarray analysis and by slot-blot hybridization analysis under various stress and non-stress conditions. Histidine kinases have been identified that perceive and transduce signals of low-temperature, hyperosmotic, and salt stress, as well as manganese deficiency.

Adaptation, Physiological↗
PubMed2004

Structure and molecular phylogeny of sasA genes in cyanobacteria: insights into evolution of the prokaryotic circadian system.

Cyanobacteria are the simplest organisms known to have a circadian system. In addition to the three well-studied kai genes, kaiA, kaiB, and kaiC, an important element of this system is a two-component sensory transduction histidine kinase sasA. Using publicly available data of complete prokaryotic genomes, we performed structural and phylogenetic analyses of the sasA genes. Results show that this gene has a triple-domain structure, and the domains are under different selective constraints. The sasA gene originated in cyanobacteria probably through the fusion of the ancestral kaiB gene with a double-domain, two-component sensory transduction histidine kinase. The results of the phylogenetic analyses suggest that sasA emerged before the kaiA gene, about 3,000-2,500 MYA, and has evolved in parallel with the evolution of the kaiBC cluster. The observed concordant patterns of the sasA and kaiBC evolution suggest that these genes might compose an ancient KaiBC-SasA-based circadian system, without the kaiA gene, and that such a system still exists in some unicellular cyanobacteria.

Amino Acid Sequence↗
PubMed2006

Subfamilies of cpmA, a gene involved in circadian output, have different evolutionary histories in cyanobacteria.

The cpmA gene mediates an output signal in the cyanobacterial circadian system. This gene and its homologues are evolutionarily old, and occur in some non-photosynthetic bacteria and archaea as well as in cyanobacteria. The gene has two functional domains that differ drastically in their level of polymorphism: the N-terminal domain is much more variable than the PurE homologous C-terminal domain. The phylogenetic tree of the cpmA homologues features four main clades (C1-C4), two of which (C1 and C3) belong to cyanobacteria. These cyanobacterial clades match respective ones in the previously reported phylogenetic trees of the other genes involved in the circadian system. The phylogenetic analysis suggested that the C3 subfamily, which comprises the genes from the cyanobacteria with the kaiBC-based circadian system, experienced a lateral transfer, probably from evolutionarily old proteobacteria about 1,000 million years ago. The genes of this subfamily have a significantly higher nonsynonymous substitution rate than those of C1 (2.13 x 10(-10) and 1.53 x 10(-10) substitutions per nonsynonymous site per year, respectively). It appears that the functional and selective constraints of the kaiABC-based system have slowed down the rate of sequence evolution compared to the cpmA homologues of the kaiBC-based system. On the other hand, the differences in the mutation rates between the two cyanobacterial clades point to the different functional constraints of the systems with or without kaiA.

Bacterial Proteins↗
PubMed2006

Phylogenetic analyses of cyanobacterial genomes: quantification of horizontal gene transfer events.

Using 1128 protein-coding gene families from 11 completely sequenced cyanobacterial genomes, we attempt to quantify horizontal gene transfer events within cyanobacteria, as well as between cyanobacteria and other phyla. A novel method of detecting and enumerating potential horizontal gene transfer events within a group of organisms based on analyses of "embedded quartets" allows us to identify phylogenetic signal consistent with a plurality of gene families, as well as to delineate cases of conflict to the plurality signal, which include horizontally transferred genes. To infer horizontal gene transfer events between cyanobacteria and other phyla, we added homologs from 168 available genomes. We screened phylogenetic trees reconstructed for each of these extended gene families for highly supported monophyly of cyanobacteria (or lack of it). Cyanobacterial genomes reveal a complex evolutionary history, which cannot be represented by a single strictly bifurcating tree for all genes or even most genes, although a single completely resolved phylogeny was recovered from the quartets' plurality signals. We find more conflicts within cyanobacteria than between cyanobacteria and other phyla. We also find that genes from all functional categories are subject to transfer. However, in interphylum as compared to intraphylum transfers, the proportion of metabolic (operational) gene transfers increases, while the proportion of informational gene transfers decreases.

Computational Biology↗
PubMed2006

Hourglass model for a protein-based circadian oscillator.

Many organisms possess internal biochemical clocks, known as circadian oscillators, which allow them to regulate their biological activity with a 24-hour period. It was recently discovered that the circadian oscillator of photosynthetic cyanobacteria is able to function in a test tube with only three proteins, KaiA, KaiB, and KaiC, and ATP. Biochemical events are intrinsically stochastic, and this tends to desynchronize oscillating protein populations. We propose that stability of the Kai-protein oscillator relies on active synchronization by (i) monomer exchange between KaiC hexamers during the day, and (ii) formation of clusters of KaiC hexamers at night. Our results highlight the importance of collective assembly or disassembly of proteins in biochemical networks, and may help guide design of novel protein-based oscillators.

Bacterial Proteins↗