Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Structural characterization and functional activity of an exopolysaccharide secreted by Rhodopseudomonas palustris GJ-22.

One water exopolysaccharide, designated G-EPS, was secreted by Rhodopseudomonas palustris GJ-22 culture media. The structure of G-EPS was characterized with HPGPC, GC-MS, methylation, 1D and 2D NMR, along with UV and FT-IR spectrum. The G-EPS molecular weight was 10.026 kilodalton, and is composed of D-mannose (92.8%) and d-glucose (7.2%). The purified G-EPS promoted plant growth and induced systemic resistance against TMV in Nicotiana benthamiana. These results suggested that G-EPS is an important active component of the bio-control capacity of GJ-22.

PRE-driven protein NMR structures: an alternative approach in highly paramagnetic systems.

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein.

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

Immobilization of Rhodopseudomonas palustris P1 on glass pumice to improve the removal of NH4 + -N and NO2 - -N from aquaculture pond water.

We conducted this research in order to investigate the potential of a new material called glass pumice for use as a microorganism immobilization carrier to improve aquaculture pond water quality. The pH adjustment capacity and the Rhodopseudomonas palustris P1 cell adsorption capacity of glass pumice were measured. The immobilized Rps. palustris P1 and the free sample were compared to determine which had an enhanced NH4 + -N and NO2 - -N removal efficiency. The results showed that glass pumice significantly affected the pH of the acid solution (P < 0.05); the pH increased from 3.0 ± 0.08 to 7.21 ± 0.13 in 12 H. Rps. palustris P1 adsorption to glass pumice was rapid and reached equilibrium within 60 Min. The Langmuir adsorption parameter data showed that glass pumice had a higher affinity for Rps. palustris P1 than SiO2 powder, with an adsorption capacity of 4.02 × 108  cells g-1 . The maximum NH4 + -N and NO2 - -N removal rates by immobilized Rps. palustris P1 were 134.82 ± 0.67% and 93.68 ± 0.14% higher than those of nonimmobilized P1, respectively. Based on the above results, we propose that glass pumice is potential as a microorganism carrier material in aquaculture water treatment.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

Preparation and biological activities of an extracellular polysaccharide from Rhodopseudomonas palustris.

The photosynthetic bacterium, Rhodopseudomonas palustris has been widely used as probiotics in aquaculture, while the molecular basis underlying the probiotic properties of this organism remains largely unknown. In this study, a novel extracellular polysaccharides (RPEPS-30) extracted from the fermentation of Rhodopseudomonas palustris was characterized. Results illustrated that RPEPS-30 was an α-mannan with a molecular weight of 46.82 kDa, which possessed a backbone consisted of 1, 2-linked and 1, 4-linked mannose residues, with side chains composed of 1 → 6 linked and 1 → 2,6 linked mannose residues and substitution at O-6. The in vitro immunomodulatory tests revealed that RPEPS-30 could enhance phagocytic capacity, NO release and mRNA expression of cytokines in macrophages. In addition, RPEPS-30 was shown to promote the growth of resident beneficial gut microbiotasuch as Lactobacillus reuteri, Bacteroides thetaiotaomicron and Akkermansia muciniphila. These findings might help us to partially understand the molecular mechanism concerning the probiotic properties of Rhodopseudomonas palustris, in which the extracellular polysaccharide RPEPS-30 stimulated host immune response and favored the growth of specific benificial micriobiota in the gut.

Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins.

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.

Membrane-Bound Bacteriophytochrome-Like Complex of Phototrophic Purple Non-Sulfur Bacterium Rhodopseudomonas palustris.

A pigment-protein complex of yellow color with absorption maxima at 682 and 776 nm, characteristic for bacteriophytochromes, was isolated from the photosynthetic membranes of the purple bacterium Rhodopseudomonas palustris. Zinc-induced fluorescence of the complex indicated the presence of the biliverdin chromophore covalently bound to the protein. The parameters of low-temperature fluorescence (λ excitation at 680 nm, λ emission at 695 nm) indicated the ability of the complex to undergo photoconversion. These data, as well as the kinetics of accumulation of the red (Pr)-form on far red light, allowed the complex to be classified as a bacteriophytochrome-like complex with its localization in the photosynthetic membranes of Rps. palustris.

Postnovo: Postprocessing Enables Accurate and FDR-Controlled de Novo Peptide Sequencing.

De novo sequencing offers an alternative to database search methods for peptide identification from mass spectra. Since it does not rely on a predetermined database of expected or potential sequences in the sample, de novo sequencing is particularly appropriate for samples lacking a well-defined or comprehensive reference database. However, the low accuracy of many de novo sequence predictions has prevented the widespread use of the variety of sequencing tools currently available. Here, we present a new open-source tool, Postnovo, that postprocesses de novo sequence predictions to find high-accuracy results. Postnovo uses a predictive model to rescore and rerank candidate sequences in a manner akin to database search postprocessing tools such as Percolator. Postnovo leverages the output from multiple de novo sequencing tools in its own analyses, producing many times the length of amino acid sequence information (including both full- and partial-length peptide sequences) at an equivalent false discovery rate (FDR) compared to any individual tool. We present a methodology to reliably screen the sequence predictions to a desired FDR given the Postnovo sequence score. We validate Postnovo with multiple data sets and demonstrate its ability to identify proteins that are missed by database search even in samples with paired reference databases.

Structural basis of transcriptional regulation by CouR, a repressor of coumarate catabolism, in Rhodopseudomonas palustris.

The MarR family transcriptional regulator CouR, from the soil bacterium Rhodopseudomonas palustris CGA009, has recently been shown to negatively regulate a p-coumarate catabolic operon. Unlike most characterized MarR repressors that respond to small metabolites at concentrations in the millimolar range, repression by CouR is alleviated by the 800-Da ligand p-coumaroyl-CoA with high affinity and specificity. Here we report the crystal structures of ligand-free CouR as well as the complex with p-coumaroyl-CoA, each to 2.1-Å resolution, and the 2.85-Å resolution cocrystal structure of CouR bound to an oligonucleotide bearing the cognate DNA operator sequence. In combination with binding experiments that uncover specific residues important for ligand and DNA recognition, these structures provide glimpses of a MarR family repressor in all possible states, providing an understanding of the molecular basis of DNA binding and the conformation alterations that accompany ligand-induced dissociation for activation of the operon.

Lipid-Controlled Stabilization of Charge-Separated States (P+QB-) and Photocurrent Generation Activity of a Light-Harvesting-Reaction Center Core Complex (LH1-RC) from Rhodopseudomonas palustris.

The photosynthetic light-harvesting-reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, P+QB-) of LH1-RC (from Rhodopseudomonas palustris) and enhances its activity in photocurrent generation. Steady-state fluorometric analysis demonstrated that PG enhances the formation of the P+QB- state at lower irradiances. The photocurrent generation activity was analyzed via Michaelis-Menten kinetics, revealing that 38% of LH1-RCs reconstituted into the PG membrane generated photocurrent at a turnover frequency of 46 s-1. PG molecules, which interact with LH1-RC in vivo, play the role of an active effector component for LH1-RC to enhance its function in the biohybrid system.

Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position.

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric 'ring' of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 'rings' are present in "low-light" adapted cells and that an unknown chaperon process creates multiple sub-types of 'rings' with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.

Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.

The NMR solution structure and function of RPA3313: a putative ribosomal transport protein from Rhodopseudomonas palustris.

Protein function elucidation often relies heavily on amino acid sequence analysis and other bioinformatics approaches. The reliance is extended to structure homology modeling for ligand docking and protein-protein interaction mapping. However, sequence analysis of RPA3313 exposes a large, unannotated class of hypothetical proteins mostly from the Rhizobiales order. In the absence of sequence and structure information, further functional elucidation of this class of proteins has been significantly hindered. A high quality NMR structure of RPA3313 reveals that the protein forms a novel split ßßαß fold with a conserved ligand binding pocket between the first ß-strand and the N-terminus of the α-helix. Conserved residue analysis and protein-protein interaction prediction analyses reveal multiple protein binding sites and conserved functional residues. Results of a mass spectrometry proteomic analysis strongly point toward interaction with the ribosome and its subunits. The combined structural and proteomic analyses suggest that RPA3313 by itself or in a larger complex may assist in the transportation of substrates to or from the ribosome for further processing. Proteins 2016; 85:93-102. © 2016 Wiley Periodicals, Inc.

The Lipid†A from Rhodopseudomonas palustris Strain BisA53 LPS Possesses a Unique Structure and Low Immunostimulant Properties.

The search for novel lipid A analogues from any biological source that can act as antagonists, displaying inhibitory activity towards the production of pro-inflammatory cytokines, or as immunomodulators in mammals, is a very topical issue. To this aim, the structure and immunological properties of the lipopolysaccharide lipid A from the purple nonsulfur bacterium Rhodopseudomonas palustris strain BisA53 have been determined. This lipid A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manp-α-(1→4)-GlcpN3N-ß-1→6-GlcpN3N-α-(1→1)-α-GalpA, and four primary amide-linked 14:0(3-OH) and, as secondary O-acyl substituents, a 16:0 and the very long-chain fatty acid 26:0(25-OAc), appended on the GlcpN3N units. This lipid A architecture is definitely rare, so far identified only in the genus Bradyrhizobium. Immunological tests on both murine bone-marrow-derived and human monocyte-derived macrophages revealed an extremely low immunostimulant capability of this LPS lipid A.

An Ab Initio Description of the Excitonic Properties of LH2 and Their Temperature Dependence.

The spectroscopic properties of light-harvesting (LH) antennae in photosyntehtic organisms represent a fingerprint that is unique for each specific pigment-protein complex. Because of that, spectroscopic observations are generally combined with structural data from X-ray crystallography to obtain an indirect representation of the excitonic properties of the system. Here, an alternative strategy is presented which goes beyond this empirical approach and introduces an ab initio computational description of both structural and electronic properties and their dependence on the temperature. The strategy is applied to the peripheral light-harvesting antenna complex (LH2) present in purple bacteria. By comparing this model with the one based on the crystal structure, a detailed, molecular level explanation of the absorption and circular dichroism (CD) spectra and their temperature dependence is achieved. The agreement obtained with the experiments at both low and room temperature lays the groundwork for an atomistic understanding of the excitation dynamics in the LH2 system.

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.

Ultrafast energy relaxation in single light-harvesting complexes.

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.

Hydrogen production under salt stress conditions by a freshwater Rhodopseudomonas palustris strain.

Hydrogen represents a possible alternative energy carrier to face the growing request for energy and the shortage of fossil fuels. Photofermentation for the production of H2 constitutes a promising way for integrating the production of energy with waste treatments. Many wastes are characterized by high salinity, and polluted seawater can as well be considered as a substrate. Moreover, the application of seawater for bacterial culturing is considered cost-effective. The aims of this study were to assess the capability of the metabolically versatile freshwater Rhodopseudomonas palustris 42OL of producing hydrogen on salt-containing substrates and to investigate its salt stress response strategy, never described before. R. palustris 42OL was able to produce hydrogen in media containing up to 3 % added salt concentration and to grow in media containing up to 4.5 % salinity without the addition of exogenous osmoprotectants. While the hydrogen production performances in absence of sea salts were higher than in their presence, there was no significant difference in performances between 1 and 2 % of added sea salts. Nitrogenase expression levels indicated that the enzyme was not directly inhibited during salt stress, but a regulation of its expression may have occurred in response to salt concentration increase. During cell growth and hydrogen production in the presence of salts, trehalose was accumulated as a compatible solute; it protected the enzymatic functionality against salt stress, thus allowing hydrogen production. The possibility of producing hydrogen on salt-containing substrates widens the range of wastes that can be efficiently used in production processes.