Trace metal interaction with thermophilic phototrophic anaerobic bacterium Chloroflexus aurantiacus.

Towards improving the knowledge of possible paleo-microorganisms interaction with trace metals (micro-nutrients and toxicants), we studied adsorption of Mn, Zn, Sr, Cd, and Pb onto modern Chloroflexus aurantiacus, thermophilic anoxygenic phototrophic bacterium which could be highly abundant in the Precambiran aquatic environments. Acid-base surface titrations allowed quantifying the number of proton-active surface groups, whereas non-electrostatic linear programming method (LPM) was used to assess the surface site concentrations and adsorption reaction constants between divalent cations (Zn, Mb, Sr, Cd, Pb) and bacterial surface, based on results of pH-dependent adsorption edge and constant-pH 'langmuirian' adsorption experiments. The total proton/hydroxyl binding site number of Chl. aurantiacus surfaces was sizably lower than that of other phototrophic anaerobic bacteria studied previously using similar experimental and modeling approach. Divalent metals exhibited a decreasing order of adsorption affinity (Pb > Cd ≥ Zn ≥ Mn > Sr), which reflected the order of cation hydrolysis and was similar to adsorption order on other phototrophic bacteria. At the same time, adsorption of Zn increased with increasing of temperature, from 4 °C to 60 °C and was stronger under light compared to the darkness. This suggested some active metabolic control involved in this metal interaction with bacterial surfaces. Overall, Chl. aurantiacus exhibited trace metal adsorption parameters (site number and binding constants) which were lower compared to other anoxygenic phototrophic bacteria (Rhodopseudomonas palustris; Rhodobacter blasticus) and cyanobacteria. This may reflect different bioavailability of trace metals in the paleo-ocean, given that thermophilic Chl. aurantiacus are among the oldest phototrophs on the planet.

Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.

Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Cryo-EM structure of HQNO-bound alternative complex III from the anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) couples quinol oxidation and electron acceptor reduction with potential transmembrane proton translocation. It is compositionally and structurally different from the cytochrome bc1/b6f complexes but functionally replaces these enzymes in the photosynthetic and/or respiratory electron transport chains (ETCs) of many bacteria. However, the true compositions and architectures of ACIIIs remain unclear, as do their structural and functional relevance in mediating the ETCs. We here determined cryogenic electron microscopy structures of photosynthetic ACIII isolated from Chloroflexus aurantiacus (CaACIIIp), in apo-form and in complexed form bound to a menadiol analog 2-heptyl-4-hydroxyquinoline-N-oxide. Besides 6 canonical subunits (ActABCDEF), the structures revealed conformations of 2 previously unresolved subunits, ActG and I, which contributed to the complex stability. We also elucidated the structural basis of menaquinol oxidation and subsequent electron transfer along the [3Fe-4S]-6 hemes wire to its periplasmic electron acceptors, using electron paramagnetic resonance, spectroelectrochemistry, enzymatic analyses, and molecular dynamics simulations. A unique insertion loop in ActE was shown to function in determining the binding specificity of CaACIIIp for downstream electron acceptors. This study broadens our understanding of the structural diversity and molecular evolution of ACIIIs, enabling further investigation of the (mena)quinol oxidoreductases-evolved coupling mechanism in bacterial energy conservation.

Femtosecond Exciton Relaxation in Chlorosomes of the Photosynthetic Green Bacterium Chloroflexus aurantiacus.

Process of photosynthesis in the green bacteria Chloroflexus (Cfx.) aurantiacus starts from absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, the excited states are formed in BChl c, energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by non-radiative electronic transitions between the numerous exciton states, that is, exciton relaxation. In this work, we studied dynamics of the exciton relaxation in Cfx. aurantiacus chlorosomes using differential femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20-fs light pulses at wavelengths in the range from 660 to 750 nm, and differential (light-dark) absorption kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the obtained data revealed kinetic components with characteristic times of 140, 220, and 320 fs, which are responsible for exciton relaxation. As the excitation wavelength decreased, the number and relative contribution of these components increased. Theoretical modelling of the obtained data was carried out based of the cylindrical model of BChl c. Nonradiative transitions between the groups of exciton bands were described by a system of kinetic equations. The model that takes into account energy and structural disorder of chlorosomes turned out to be the most adequate.

Primary charge separation in native and plant pheophytin a-modified reaction centers of Chloroflexus aurantiacus: Ultrafast transient absorption measurements at low temperature.

Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the BA binding site were monitored as indicators of the formation and decay of the P+BA- state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P⁎ excited state, which decay by distinct means, forming the state P+HA- (HA is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P⁎ decays in ~2 ps with the formation of P+HA- via a short-lived P+BA- intermediate in a two-step ET process P⁎ â†’ P+BA-→ P+HA-. In another population (~35 %), P⁎ decays in ~20 ps to form P+HA- via a superexchange mechanism without producing measurable amounts of P+BA-. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the HA binding site also showed the presence of two P⁎ populations (~2 and ~20 ps), with P⁎ decaying through P+BA- only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P⁎ â†’ P+HA- superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.

Structural and Functional Elucidation of IF-3 Protein of Chloroflexus aurantiacus Involved in Protein Biosynthesis: An In Silico Approach.

Chloroflexus aurantiacus is a thermophilic bacterium that produces a multitude of proteins within its genome. Bioinformatics strategies can facilitate comprehending this organism through functional and structural interpretation assessments. This study is aimed at allocating the structure and function through an in silico approach required for bacterial protein biosynthesis. This in silico viewpoint provides copious properties, including the physicochemical properties, subcellular location, three-dimensional structure, protein-protein interactions, and functional elucidation of the protein (WP_012256288.1). The STRING program is utilized for the explication of protein-protein interactions. The in silico investigation documented the protein's hydrophilic nature with predominantly alpha (α) helices in its secondary structure. The tertiary-structure model of the protein has been shown to exhibit reasonably high consistency based on various quality assessment methods. The functional interpretation suggested that the protein can act as a translation initiation factor, a protein required for translation and protein biosynthesis. Protein-protein interactions also demonstrated high credence that the protein interconnected with 30S ribosomal subunit involved in protein synthesis. This study bioinformatically examined that the protein (WP_012256288.1) is affiliated in protein biosynthesis as a translation initiation factor IF-3 of C. aurantiacus.

Pigment-modified reaction centers of Chloroflexus aurantiacus: chemical exchange of bacteriopheophytins with plant-type pheophytins.

The pigment composition of isolated reaction centers (RCs) of the green filamentous bacterium Chloroflexus (Cfl.) aurantiacus was changed by chemical exchange of native bacteriopheophytin a (BPheo) molecules with externally added pheophytin a (Pheo) or [3-acetyl]-Pheo upon incubation of RC/pheophytin mixtures at room temperature and 45 °C. The modified RCs were characterized by Vis/NIR absorption spectroscopy, and the effect of pigment exchange on RC photochemical activity was assessed by measuring the photoaccumulation of the reduced pigment at the binding site HA. It is shown that both pheophytins can be exchanged into the HA site instead of BPheo by incubation at room temperature. While the newly introduced Pheo molecule is not active in electron transfer, the [3-acetyl]-Pheo molecule is able to replace functionally the photoreducible HA BPheo molecule with the formation of the [3-acetyl]-Pheo- radical anion instead of the BPheo-. After incubation at 45 °C, the majority (~ 90%) of HA BPheo molecules is replaced by both Pheo and [3-acetyl]-Pheo. Only a partial replacement of inactive BPheo molecules with pheophytins is observed even when the incubation temperature is raised to 50 °C. The results are discussed in terms of (i) differences in the accessibility of BPheo binding sites for extraneous pigments depending on structural constraints and incubation temperature and (ii) the effect of the reduction potential of pigments introduced into the HA site on the energetics of the charge separation process. The possible implication of Pheo-exchanged preparations for studying early electron-transfer events in Cfl. aurantiacus RCs is considered.

Femtosecond excited-state dynamics in chlorosomal carotenoids of the photosynthetic bacterium Chloroflexus aurantiacus revealed by near infrared pump-probe spectroscopy.

In photosynthetic green bacteria, chlorosomes provide light harvesting with high efficiency. Chlorosomal carotenoids (Cars) participate in light harvesting together with the main pigment, bacteriochlorophyll (BChl) c/d/e. In the present work, we studied the excited-state dynamics in Cars from Chloroflexus (Cfx.) aurantiacus chlorosomes by near infrared pump-probe spectroscopy with 25 fs temporal resolution at room temperature. The S2 state of Cars was excited at a wavelength of ∼520 nm, and the absorption changes were probed at 860-1000 nm where the excited state absorption (ESA) of the Cars S2 state occurred. Global analysis of the spectroscopy data revealed an ultrafast (∼15 fs) and large (>130 nm) red shift of the S2 ESA spectrum together with the well-known S2 → S1 IC (∼190 fs) and Cars → BChl c EET (∼120 fs). The S2 lifetime was found to be ∼74 fs. Our findings are in line with earlier results on the excited-state dynamics in Cars in vitro. To explain the extremely fast S2 dynamics, we have tentatively proposed two alternative schemes. The first scheme assumed the formation of a vibrational wavepacket in the S2 state, the motion of which caused a dynamical red shift of the S2 ESA spectrum. The second scheme assumed the presence of two potential minima in the S2 state and incoherent energy transfer between them.

The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer.

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 104-105 bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S2 state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400-900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret â†’ Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100-270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S1 state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret â†’ Qy internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.

Rational Design of a Split Flavin-Based Fluorescent Reporter.

Protein-fragment complementation assays are used ubiquitously for probing protein-protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aß-Bß, Eα-Fα, and Hß-Iß, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein-protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.

Aerobic Production of Bacteriochlorophylls in the Filamentous Anoxygenic Photosynthetic Bacterium, Chloroflexus aurantiacus in the Light.

Filamentous anoxygenic photosynthetic bacteria grow by photosynthesis and aerobic respiration. The present study investigated the effects of light and O2 on bacteriochlorophyll contents and the transcription levels of photosynthesis-related genes in Chloroflexus aurantiacus J-10-fl T. Under aerobic conditions, C. aurantiacus produced marked amounts of bacteriochlorophylls in the presence of light, although their production was strongly suppressed in the dark. The transcription levels of genes related to the synthesis of bacteriochlorophylls, photosystems, and chlorosomes: bchM, bchU, pufL, pufBA, and csmM, were markedly increased by illumination. These results suggest that C. aurantiacus continuously synthesizes ATP by photophosphorylation even in the presence of O2.

Q-band hyperchromism and B-band hypochromism of bacteriochlorophyll c as a tool for investigation of the oligomeric structure of chlorosomes of the green photosynthetic bacterium Chloroflexus aurantiacus.

Chlorosomes of green photosynthetic bacteria are the most amazing example of long-range ordered natural light-harvesting antennae. Chlorosomes are the largest among all known photosynthetic light-harvesting structures (~ 104-105 pigments in the aggregated state). The chlorosomal bacteriochlorophyll (BChl) c/d/e molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. In the present work, we studied hyperchromism/hypochromism in the chlorosomal BChl c Q/B absorption bands of the green photosynthetic bacterium Chloroflexus (Cfx.) aurantiacus. The chlorosomes were isolated from cells grown under different light intensities and therefore, as we discovered earlier, they had different sizes of both BChl c antennae and their unit building blocks. We have shown experimentally that the Q-/B-band hyperchromism/hypochromism is proportional to the size of the chlorosomal antenna. We explained theoretically these findings in terms of excitonic intensity borrowing between the Q and B bands for the J-/H-aggregates of the BChls. The theory developed by Gülen (Photosynth Res 87:205-214, 2006) showed the dependence of the Q-/B-band hyperchromism/hypochromism on the structure of the aggregates. For the model of exciton-coupled BChl c linear chains within a unit building block, the theory predicted an increase in the hyperchromism/hypochromism with the increase in the number of molecules per chain and a decrease in it with the increase in the number of chains. It was previously shown that this model ensured a good fit with spectroscopy experiments and approximated the BChl c low packing density in vivo. The presented experimental and theoretical studies of the Q-/B-band hyperchromism/hypochromism permitted us to conclude that the unit building block of Cfx. aurantiacus chlorosomes comprises of several short BChl c chains.This conclusion is in accordance with previous linear and nonlinear spectroscopy studies on Cfx. aurantiacus chlorosomes.

On Excitation Energy Transfer within the Baseplate BChl a-CsmA Complex of Chloroflexus aurantiacus.

Recently, a hybrid approach combining solid-state NMR spectroscopy and cryo-electron microscopy showed that the baseplate in green sulfur bacterium Chlorobaculum tepidum is a 2D lattice of BChl a-CsmA dimers [Nielsen, J. T.; et al., Nat. Commun. 2016, 7, 12454-12465]. While the existence of the BChl a-CsmA subunit was previously known, the proposed orientations of the BChl a pigments had only been elucidated from spectral data up to this point. Regarding the electronic structure of the baseplate, two models have been proposed. 2D electronic spectroscopy data were interpreted as revealing that at least four excitonically coupled BChl a might be in close contact. Conversely, spectral hole burning data suggested that the lowest energy state was localized, yet additional states are sometimes observed because of the presence of the Fenna-Matthews-Olson (FMO) antenna protein. To solve this conundrum, this work studies the chlorosome-baseplate complex from Chloroflexus aurantiacus, which does not contain the FMO protein. The results confirm that in both C. tepidum and C. aurantiacus, excitation energy is transferred to a localized low-energy trap state near 818 nm with similar rates, most likely via exciton hopping.

Sulfide-dependent Photoautotrophy in the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aggregans.

Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated "ACA-12". A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats.

A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies.

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.

Ultrafast excited-state dynamics in chlorosomes isolated from the photosynthetic filamentous green bacterium Chloroflexus aurantiacus.

Bacteriochlorophyll (BChl) c pigments in the aggregated state are responsible for efficient light harvesting in chlorosomes of the filamentous anoxygenic photosynthetic bacterium, Chloroflexus (Cfx.) aurantiacus. Absorption of light creates excited states in the BChl c aggregates. After subpicosecond intrachlorosomal energy transfer, redistribution and relaxation, the excitation is transferred to the BChl a complexes and further to reaction centers on the picosecond time scale. In this work, the femtosecond excited state dynamics within BChl c oligomers of isolated Cfx. aurantiacus chlorosomes was studied by double difference pump-probe spectroscopy at room temperature. Difference (Alight - Adark ) spectra corresponding to excitation at 725 nm (blue side of the BChl c absorption band) were compared with those corresponding to excitation at 750 nm (red side of the BChl c absorption band). A very fast (time constant 70 ± 10 fs) rise kinetic component was found in the stimulated emission (SE) upon excitation at 725 nm. This component was absent at 750-nm excitation. These data were explained by the dynamical red shift of the SE due to excited state relaxation. The nature and mechanisms of the ultrafast excited state dynamics in chlorosomal BChl c aggregates are discussed.

Estimation of the bacteriochlorophyll c oligomerisation extent in Chloroflexus aurantiacus chlorosomes by very low-frequency vibrations of the pigment molecules: A new approach.

In green photosynthetic bacteria, the chlorosomal bacteriochlorophyll molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. For the first time, we demonstrated by coherent femtosecond spectroscopy at cryogenic temperature that the very low-frequency (~101 cm-1) vibrations of bacteriochlorophyll c pigments in isolated Chloroflexus aurantiacus chlorosomes are sensitive to their oligomerisation extent which depends on the light intensity during the growth of the cell cultures. We explained this sensitivity in terms of the coupling of delocalised vibration modes of BChl c molecules aggregated into chains within their antenna unit building blocks. These findings, together with previously obtained spectroscopy and microscopy data, confirmed that the unit building blocks functioning within Chloroflexus aurantiacus chlorosomal antenna are built up from the rather short (2-5 BChl c pigments) quasi-linear chains. The approach presented here seems to be perspective since it directly reveals structural and dynamical properties of the oligomeric systems.

Variability of aggregation extent of light-harvesting pigments in peripheral antenna of Chloroflexus aurantiacus.

The stationary ground state and femtosecond time-resolved absorption spectra as well as spectra of circular dichroism were measured at room temperature using freshly prepared samples of chlorosomes isolated from fresh cultures of the green bacterium Chloroflexus aurantiacus. Cultures were grown by using as inoculum the same seed culture but under different light conditions. All measured spectra clearly showed the red shift of BChl c Qy bands (up to 5 nm) for low-light chlorosomes as compared to high-light ones, together with concomitant narrowing of these bands and increasing of their amplitudes. The sizes of the unit BChl c aggregates of the high-light-chlorosomes and the low-light ones were estimated. The fit of all experimental spectra was obtained within the framework of our model proposed before (Fetisova et al., Biophys J 71:995-101, 1996). The model assumes that a unit building block of the BChl c antenna has a form of a tubular aggregate of L = 6 linear single or double exciton-coupled pigment chains within a rod element, with the pigment packing density, approximating that in vivo. The simultaneous fit of all experimental spectra gave the number of pigments in each individual linear pigment chain N = 4 and N = 6 for the high-light and the low-light BChl c unit building blocks, respectively. The size of a unit building block in the BChl c antenna was found to vary from L × N = 24 to L × N = 36 exciton-coupled BChl c molecules being governed by the growth-light intensity. All sets of findings for Chloroflexus aurantiacus chlorosomes demonstrated the biologically expedient light-controlled variability, predicted by us, of the extent of BChl c aggregation within a unit building block in this antenna.