Coherent intradimer dynamics in reaction centers of photosynthetic green bacterium Chloroflexus aurantiacus.

Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140 cm-1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945 nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ΔA spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.

Amphiphilic block copolymers as flexible membrane materials generating structural and functional mimics of green bacterial antenna complexes.

We describe the ability of a short-chain amphiphilic block copolymer to self-assemble to form an artificial supramolecular light-harvesting system. Specifically, we demonstrate that the 2.5 kDa, poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD), exhibits sufficient morphological flexibility as a membrane material and enables generation of mimics of three-dimensional chlorosomes as well as supported membrane bilayers containing energy acceptors. This overall architecture replicates green bacterial light-harvesting function whereby these assemblies exhibit long-range order and three-dimensional morphology similar to native chlorosomes and are capable of energy transfer internally and to external acceptors, located in a supporting biomimetic polymer membrane. Unlike native green bacterial systems that use multiple lipids as a matrix to generate the appropriate environment for chlorosome assembly and function, the described system matrix is comprised entirely of a single polymer amphiphile. This work demonstrates the potential of short-chain amphiphilic block copolymers in generating self-assembled, bio-mimetic membrane architectures, and in doing so, generates scalable, spatial-energetic landscapes for photonic applications. Finally, the results presented provide evidence of minimal requirements to induce chlorosome-like organization and function.

Gliding motility driven by individual cell-surface movements in a multicellular filamentous bacterium Chloroflexus aggregans.

Chloroflexus aggregans is an unbranched multicellular filamentous bacterium having the ability of gliding motility. The filament moves straightforward at a constant rate, ∼3 µm sec(-1) on solid surface and occasionally reverses the moving direction. In this study, we successfully detected movements of glass beads on the cell-surface along long axis of the filament indicating that the cell-surface movement was the direct force for gliding. Microscopic analyses found that the cell-surface movements were confined to a cell of the filament, and each cell independently moved and reversed the direction. To understand how the cellular movements determine the moving direction of the filament, we proposed a discrete-time stochastic model; sum of the directions of the cellular movements determines the moving direction of the filament only when the filament pauses, and after moving, the filament keeps the same directional movement until all the cells pause and/or move in the opposite direction. Monte Carlo simulation of this model showed that reversal frequency of longer filaments was relatively fixed to be low, but the frequency of shorter filaments varied widely. This simulation result appropriately explained the experimental observations. This study proposed the relevant mechanism adequately describing the motility of the multicellular filament in C. aggregans.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus.

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.

Community structure and activity of a highly dynamic and nutrient-limited hypersaline microbial mat in Um Alhool Sabkha, Qatar.

The Um Alhool area in Qatar is a dynamic evaporative ecosystem that receives seawater from below as it is surrounded by sand dunes. We investigated the chemical composition, the microbial activity and biodiversity of the four main layers (L1-L4) in the photosynthetic mats. Chlorophyll a (Chl a) concentration and distribution (measured by HPLC and hyperspectral imaging, respectively), the phycocyanin distribution (scanned with hyperspectral imaging), oxygenic photosynthesis (determined by microsensor), and the abundance of photosynthetic microorganisms (from 16S and 18S rRNA sequencing) decreased with depth in the euphotic layer (L1). Incident irradiance exponentially attenuated in the same zone reaching 1% at 1.7-mm depth. Proteobacteria dominated all layers of the mat (24%-42% of the identified bacteria). Anoxygenic photosynthetic bacteria (dominated by Chloroflexus) were most abundant in the third red layer of the mat (L3), evidenced by the spectral signature of Bacteriochlorophyll as well as by sequencing. The deep, black layer (L4) was dominated by sulfate reducing bacteria belonging to the Deltaproteobacteria, which were responsible for high sulfate reduction rates (measured using 35S tracer). Members of Halobacteria were the dominant Archaea in all layers of the mat (92%-97%), whereas Nematodes were the main Eukaryotes (up to 87%). Primary productivity rates of Um Alhool mat were similar to those of other hypersaline microbial mats. However, sulfate reduction rates were relatively low, indicating that oxygenic respiration contributes more to organic material degradation than sulfate reduction, because of bioturbation. Although Um Alhool hypersaline mat is a nutrient-limited ecosystem, it is interestingly dynamic and phylogenetically highly diverse. All its components work in a highly efficient and synchronized way to compensate for the lack of nutrient supply provided during regular inundation periods.

A variety of glycolipids in green photosynthetic bacteria.

The compositions of glycolipids in the following seven strains of green photosynthetic bacteria were investigated at the molecular level using LC-MS coupled with an evaporative light scattering detector: Chlorobium (Chl.) limicola strains Larsen (30 °C as the optimal cultivation temperature) and DSM245 (30 °C), Chlorobaculum (Cba.) tepidum strain ATCC49652 (45 °C), Cba. parvum strain NCIB8327 (30 °C), Cba. limnaeum strain 1549 (30 °C), Chl. phaeovibrioides DSM269 (30 °C), and Chloroflexus (Cfl.) aurantiacus strain J-10-fl (55 °C). Dependence of the molecular structures of glycolipids including the chain-length of their acyl groups upon bacterial cultivation temperatures was clearly observed. The organisms with their optimal temperatures of 30, 45, and 55 °C dominantly accumulated glycolipids possessing the acyl chains in the range of C(15)-C(16), C(16)-C(17), and C(18)-C(20), respectively. Cba. tepidum with an optimal temperature of 45 °C preferred the insertion of a methylene group to produce finally a C(17)-cyclopropane chain. Cfl. aurantiacus cultured optimally at 55 °C caused a drastic increase in the chain-length. Notably, the length of such acyl groups corresponded to that of the esterifying chain in the 17-propionate residues of self-aggregative bacteriochlorophylls-c/d/e, indicating stabilization of their supramolecular structures through hydrophobic interactions among those hydrocarbon chains. Based on the detailed compositions of glycolipids, a survival strategy of green photosynthetic bacteria grown in the wide range of temperatures is discussed.

Colors of extreme exo-Earth environments.

The search for extrasolar planets has already detected rocky planets and several planetary candidates with minimum masses that are consistent with rocky planets in the habitable zone of their host stars. A low-resolution spectrum in the form of a color-color diagram of an exoplanet is likely to be one of the first post-detection quantities to be measured for the case of direct detection. In this paper, we explore potentially detectable surface features on rocky exoplanets and their connection to, and importance as, a habitat for extremophiles, as known on Earth. Extremophiles provide us with the minimum known envelope of environmental limits for life on our planet. The color of a planet reveals information on its properties, especially for surface features of rocky planets with clear atmospheres. We use filter photometry in the visible as a first step in the characterization of rocky exoplanets to prioritize targets for follow-up spectroscopy. Many surface environments on Earth have characteristic albedos and occupy a different color space in the visible waveband (0.4-0.9 µm) that can be distinguished remotely. These detectable surface features can be linked to the extreme niches that support extremophiles on Earth and provide a link between geomicrobiology and observational astronomy. This paper explores how filter photometry can serve as a first step in characterizing Earth-like exoplanets for an aerobic as well as an anaerobic atmosphere, thereby prioritizing targets to search for atmospheric biosignatures.

Comparing the temperature dependence of photosynthetic electron transfer in Chloroflexus aurantiacus and Rhodobactor sphaeroides reaction centers.

The process of electron transfer from the special pair, P, to the primary electron donor, H(A), in quinone-depleted reaction centers (RCs) of Chloroflexus (Cf.) aurantiacus has been investigated over the temperature range from 10 to 295 K using time-resolved pump-probe spectroscopic techniques. The kinetics of the electron transfer reaction, P* → P(+)H(A)(-), was found to be nonexponential, and the degree of nonexponentiality increased strongly as temperature decreased. The temperature-dependent behavior of electron transfer in Cf. aurantiacus RCs was compared with that of the purple bacterium Rhodobacter (Rb.) sphaeroides . Distinct transitions were found in the temperature-dependent kinetics of both Cf. aurantiacus and Rb. sphaeroides RCs, at around 220 and 160 K, respectively. Structural differences between these two RCs, which may be associated with those differences, are discussed. It is suggested that weaker protein-cofactor hydrogen bonding, stronger electrostatic interactions at the protein surface, and larger solvent interactions likely contribute to the higher transition temperature in Cf. aurantiacus RCs temperature-dependent kinetics compared with that of Rb. sphaeroides RCs. The reaction-diffusion model provides an accurate description for the room-temperature electron transfer kinetics in Cf. aurantiacus RCs with no free parameters, using coupling and reorganization energy values previously determined for Rb. sphaeroides , along with an experimental measure of protein conformational diffusion dynamics and an experimental literature value of the free energy gap between P* and P(+)H(A)(-).

In silico approaches to study mass and energy flows in microbial consortia: a syntrophic case study.

BACKGROUND: Three methods were developed for the application of stoichiometry-based network analysis approaches including elementary mode analysis to the study of mass and energy flows in microbial communities. Each has distinct advantages and disadvantages suitable for analyzing systems with different degrees of complexity and a priori knowledge. These approaches were tested and compared using data from the thermophilic, phototrophic mat communities from Octopus and Mushroom Springs in Yellowstone National Park (USA). The models were based on three distinct microbial guilds: oxygenic phototrophs, filamentous anoxygenic phototrophs, and sulfate-reducing bacteria. Two phases, day and night, were modeled to account for differences in the sources of mass and energy and the routes available for their exchange. RESULTS: The in silico models were used to explore fundamental questions in ecology including the prediction of and explanation for measured relative abundances of primary producers in the mat, theoretical tradeoffs between overall productivity and the generation of toxic by-products, and the relative robustness of various guild interactions. CONCLUSION: The three modeling approaches represent a flexible toolbox for creating cellular metabolic networks to study microbial communities on scales ranging from cells to ecosystems. A comparison of the three methods highlights considerations for selecting the one most appropriate for a given microbial system. For instance, communities represented only by metagenomic data can be modeled using the pooled method which analyzes a community's total metabolic potential without attempting to partition enzymes to different organisms. Systems with extensive a priori information on microbial guilds can be represented using the compartmentalized technique, employing distinct control volumes to separate guild-appropriate enzymes and metabolites. If the complexity of a compartmentalized network creates an unacceptable computational burden, the nested analysis approach permits greater scalability at the cost of more user intervention through multiple rounds of pathway analysis.

Optoelectronic energy transfer at novel biohybrid interfaces using light harvesting complexes from Chloroflexus aurantiacus.

In nature, nanoscale supramolecular light harvesting complexes initiate the photosynthetic energy collection process at high quantum efficiencies. In this study, the distinctive antenna structure from Chloroflexus aurantiacusthe chlorosomeis assessed for potential exploitation in novel biohybrid optoelectronic devices. Electrochemical characterization of bacterial fragments containing intact chlorosomes with the photosynthetic apparatus show an increase in the charge storage density near the working electrode upon light stimulation and suggest that chlorosomes contribute approximately one-third of the overall photocurrent. Further, isolated chlorosomes (without additional photosynthetic components, e.g., reaction centers, biochemical mediators) produce a photocurrent (approximately 8-10 nA) under light saturation conditions. Correlative experiments indicate that the main chlorosome pigment, bacteriochlorophyll-c, contributes to the photocurrent via an oxidative mechanism. The results reported herein are the first to demonstrate that isolated chlorosomes (lipid-enclosed sacs of pigments) directly transduce light energy in an electrochemical manner, laying an alternative, biomimetic approach for designing photosensitized interfaces in biofuel cells and biomedical devices, such as bioenhanced retinal prosthetics.

Direct counting of submicrometer-sized photosynthetic apparatus dispersed in medium at cryogenic temperature by confocal laser fluorescence microscopy: estimation of the number of bacteriochlorophyll c in single light-harvesting antenna complexes chlorosomes of green photosynthetic bacteria.

The number of pigments in single light-harvesting complexes (chlorosomes) were calculated by imaging single chlorosomes in a frozen buffer at cryogenic temperature with a confocal laser fluorescence microscope and pigment extraction. Chlorosomes were isolated from two types of green photosynthetic bacteria Chlorobium (Chl.) tepidum and Chloroflexus (Cfl.) aurantiacus and were individually imaged in the frozen medium. Each fluorescence spot observed mainly came from a single chlorosome and was ascribable to self-aggregates of bacteriochlorophyll (BChl) c molecules as core parts of chlorosomes. A three-dimensional distribution of fluorescence of single chlorosomes was analyzed, and the number of chlorosomes in a volume of 54,000 microm(3) was counted directly. On the basis of the results, averaged numbers of the BChl c molecules contained in a single chlorosome of Chl. tepidum and Cfl. aurantiacus were determined to be 1.4 x 10(5) and 9.6 x 10(4), respectively. The present numbers are almost comparable to those estimated by other methods (Martinez-Planells et al., Photosynth. Res. 2002, 71, 83 and Montaño et al., Biophys. J. 2003, 85, 2560).

Diversity and function of Chloroflexus-like bacteria in a hypersaline microbial mat: phylogenetic characterization and impact on aerobic respiration.

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and gamma-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.

Microbial diversity in natural environments: focusing on fundamental questions.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.