Devosia sediminis sp. nov., isolated from subterranean sediment.

A novel Gram-negative, cream-colored, rod-shaped, aerobic, non-motile bacterium, designated MSA67T, was isolated from a subterranean sediment sample of the Mohe Basin in Northeast China. Strain MSA67T was detected to grow at 4-40 °C (optimum 28-30 °C), pH 5.0-10.0 (optimum, pH 7.0) and in 0.0-8.0% (w/v) NaCl (optimum 2.0-3.0%). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MSA67T was a member of the genus Devosia, with the highest similarity with D. riboflavina IFO13584T (98.0%) and D. chinhatensis IPL18T (97.0%). The major cellular fatty acids are C16:0, C18:1ω7c 11-methyl and C18:1ω6c and/or C18:1ω7c. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, glycolipids and three unidentified phospholipids. The major respiratory quinone is ubiquinone 10 (Q-10). The genomic size of strain MSA67T is 4.1 MB and DNA G + C content is 63.6%. Based on genotypic, phenotypic and phylogenetic results, strain MSA67T is concluded to represent a novel species of the genus Devosia, for which the name Devosia sediminis sp. nov. is proposed. The type strain is MSA67T (= CGMCC 1.18467T = KCTC 82192T).

Hongsoonwoonella zoysiae gen. nov., sp. nov., a new member of the family Stappiaceae isolated from a tidal mudflat.

A Gram stain-negative bacterial strain, designated SY4-7T, was isolated from rhizosphere mudflat of a halophyte (Zoysia sinica) collected around Seonyu Island, Republic of Korea. Cells of the organism were strictly aerobic, non-sporulating, non-motile rods and grew at 20-42 °C, pH 6-8 and 1-6% (w/v) NaCl. The 16S rRNA gene-based phylogenetic analyses revealed that strain SY4-7T formed an independent cluster separated from the recognized genera of the family Stappiaceae, which was also supported by phylogenomic analysis-based 92-core gene sequences. The type stains of the phylogenetically closest relatives were Stappia indica (95.6% sequence similarity), Stappia stellulata (95.1%) and Roseibium hamelinense (95.1%). The isoprenoid quinone was Q-10. The polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminophospholipids, an unidentified phosphoglycolipid, an unidentified aminolipid, two unidentified phospholipids and an unidentified lipid. The major cellular fatty acids are C18:1ω7c and C19:1 cyclo ω8c. The G + C content of the genomic DNA is 60.7%. Discrimination of the organism from all the recognized genera of the family Stappiaceae was apparent by the chemotaxonomic and phylogenetic features. Based on the results presented here, strain SY4-7T (= KCTC 72226T = NBRC 113902T) represents a novel species of a new genus in the family Stappiaceae, for which the name Hongsoonwoonella zoysiae sp. nov. is proposed.

Lycopene-Family Carotenoids Confer Thermostability on Photocomplexes from a New Thermophilic Purple Bacterium.

Blastochloris tepida is a newly described thermophilic purple bacterium containing bacteriochlorophyll b. Using purified light-harvesting 1 reaction center (LH1-RC) core complexes from Blc. tepida, we compared the biochemical, spectroscopic, and thermal denaturation properties of these complexes with those of its mesophilic counterpart, Blc. viridis. Besides their growth temperature optima, a striking difference between the two species was seen in the carotenoid composition of their LH1-RC complexes. The more thermostable Blc. tepida complex contained more carotenoids with longer conjugation lengths (n > 9), such as lycopenes (n = 11), and had a total carotenoid content significantly higher than that of the Blc. viridis complex, irrespective of the light intensity used for growth. The thermostability of LH1-RCs from both Blc. tepida and Blc. viridis decreased significantly in cells grown in the presence of diphenylamine, a compound that inhibits the formation of highly conjugated carotenoids. In contrast to the thermophilic purple bacterium Thermochromatium tepidum, where Ca2+ is essential for LH1-RC thermostability, Ca2+ neither was present in nor had any effect on the thermostability of the Blc. tepida LH1-RC. These results point to a mechanism that carotenoids with elongated conjugations enhance hydrophobic interactions with proteins in the Blc. tepida LH1-RC, thereby allowing the complexes to withstand thermal denaturation. This conclusion is bolstered by a structural model of the Blc. tepida LH1-RC and is the first example of photocomplex thermostability being linked to a carotenoid-based mechanism.

Monte Carlo simulation and thermodynamic integration applied to protein charge transfer.

We introduce a combination of Monte Carlo simulation and thermodynamic integration methods to address a model problem in free energy computations, electron transfer in proteins. The feasibility of this approach is tested using the ferredoxin protein from Clostridium acidurici. The results are compared to numerical solutions of the Poisson-Boltzmann equation and data from recent molecular dynamics simulations on charge transfer in a protein complex, the NrfHA nitrite reductase of Desulfovibrio vulgaris. Despite the conceptual and computational simplicity of the Monte Carlo approach, the data agree well with those obtained by other methods. A link to experiments is established via the cytochrome subunit of the bacterial photosynthetic reaction center of Rhodopseudomonas viridis.

Blastochloris tepida, sp. nov., a thermophilic species of the bacteriochlorophyll b-containing genus Blastochloris.

A new taxon is created for the thermophilic purple nonsulfur bacterium previously designated as Rhodopseudomonas strain GI. Strain GI was isolated from a New Mexico (USA) hot spring microbial mat and grows optimally above 40 °C and to a maximum of 47 °C. Strain GI is a bacteriochlorophyll b-containing species of purple nonsulfur bacteria and displays a budding morphology, typical of species of the genus Blastochloris. Although resembling the species Blc. viridis in many respects, the absorption spectrum, carotenoid content, and lipid fatty acid profile of strain GI is distinct from that of Blc. viridis strain DSM133T and other recognized Blastochloris species. Strain GI forms its own subclade within the Blastochloris clade of purple nonsulfur bacteria based on comparative 16S rRNA gene sequences, and its genome is significantly larger than that of strain DSM133T; average nucleotide identity between the genomes of Blc. viridis and strain GI was below 85%. Moreover, concatenated sequence analyses of PufLM and DnaK clearly showed strain GI to be distinct from both Blc. viridis and Blc. sulfoviridis. Because of its unique assortment of properties, it is proposed to classify strain GI as a new species of the genus Blastochloris, as Blc. tepida, sp.n., with strain GIT designated as the type strain (= ATCC TSD-138 = DSM 106918).

Phospholipid distributions in purple phototrophic bacteria and LH1-RC core complexes.

In contrast to plants, algae and cyanobacteria that contain glycolipids as the major lipid components in their photosynthetic membranes, phospholipids are the dominant lipids in the membranes of anoxygenic purple phototrophic bacteria. Although the phospholipid compositions in whole cells or membranes are known for a limited number of the purple bacteria, little is known about the phospholipids associated with individual photosynthetic complexes. In this study, we investigated the phospholipid distributions in both membranes and the light-harvesting 1-reaction center (LH1-RC) complexes purified from several purple sulfur and nonsulfur bacteria. 31P NMR was used for determining the phospholipid compositions and inductively coupled plasma atomic emission spectroscopy was used for measuring the total phosphorous contents. Combining these two techniques, we could determine the numbers of specific phospholipids in the purified LH1-RC complexes. A total of approximate 20-30 phospholipids per LH1-RC were detected as the tightly bound lipids in all species. The results revealed that while cardiolipin (CL) exists as a minor component in the membranes, it became the most abundant phospholipid in the purified core complexes and the sum of CL and phosphatidylglycerol accounted for more than two thirds of the total phospholipids for most species. Preferential association of these anionic phospholipids with the LH1-RC is discussed in the context of the recent high-resolution structure of this complex from Thermochromatium (Tch.) tepidum. The detergent lauryldimethylamine N-oxide was demonstrated to selectively remove phosphatidylethanolamine from the membrane of Tch. tepidum.

Cryo-EM structure of the Blastochloris viridis LH1-RC complex at 2.9 Å.

The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 ß-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between ß-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc 1 complex.

The enzymatic detoxification of the mycotoxin deoxynivalenol: identification of DepA from the DON epimerization pathway.

The biological detoxification of mycotoxins, including deoxynivalenol (DON), represents a very promising approach to address the challenging problem of cereal grain contamination. The recent discovery of Devosia mutans 17-2-E-8 (Devosia spp. 17-2-E-8), a bacterial isolate capable of transforming DON to the non-toxic stereoisomer 3-epi-deoxynivalenol, along with earlier reports of bacterial species capable of oxidizing DON to 3-keto-DON, has generated interest in the possible mechanism and enzyme(s) involved. An understanding of these details could pave the way for novel strategies to manage this widely present toxin. It was previously shown that DON epimerization proceeds through a two-step biocatalysis. Significantly, this report describes the identification of the first enzymatic step in this pathway. The enzyme, a dehydrogenase responsible for the selective oxidation of DON at the C3 position, was shown to readily convert DON to 3-keto-DON, a less toxic intermediate in the DON epimerization pathway. Furthermore, this study provides insights into the PQQ dependence of the enzyme. This enzyme may be part of a feasible strategy for DON mitigation within the near future.

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

Devosia nitraria sp. nov., a novel species isolated from the roots of Nitraria sibirica in China.

An aerobic, Gram-stain negative, short rod-shaped and motile strain, 36-5-1T, was isolated from the roots of Nitraria sibirica in Zhangye city, Gansu province, north-west of China. Phylogenetic analysis based on the 16S rRNA gene sequence and two housekeeping genes (glnA and atpD) indicated that the strain represents a novel species closely related to the Devosia, Rhizobium and Devosia genera with 98.3, 96.2 and 91.1% similarities, respectively. The strain 36-5-1T contained Q-10 as the predominant ubiquinone and 16:0 (36.8%) as the major fatty acid; a large amount of unidentified glycolipid, diphosphatidylglycerol, phosphatidylglycerol and a small amount of unidentified polar lipids were present as polar lipids. In addition, the G+C content of the genomic DNA was 61.7 mol% and the DNA-DNA hybridization with type strains Devosia geojensis BD-c194T and Devosia pacifica NH131T 44.1 ± 1.1 and 40.2 ± 1.7, respectively. Based on chemotaxonomic data and molecular properties, strain 36-5-1T represents a novel species within the genus Devosia, for which the name Devosia nitraria sp. nov. is proposed. The type strain is 36-5-1T (=CGMCC1.15704T=NBRC112416T).

An epimer of deoxynivalenol: purification and structure identification of 3-epi-deoxynivalenol.

In an investigation of deoxynivalenol (DON)-transformation products by Devosia mutans 17-2-E-8, the major product was identified as 3-epi-DON. This DON-transformation product was analysed by liquid chromatography and identified by congruent retention time and UV/Vis spectrum, as well as mass spectrometric data. Nuclear magnetic resonance (NMR) experiments including correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and nuclear overhauser effect (NOE) were conducted for structural characterisation of 3-epi-DON. High-speed counter-current chromatography (HSCCC) was applied to scale up the separation of 3-epi-DON from DON in a D. mutans 17-2-E-8 culture. From the culture where 100 mg DON was applied, 56 mg of 3-epi-DON (purity of 96.8%) was obtained from the HSCCC. The purified 3-epi-DON will be used for toxicological characterisation studies of this chemical.

Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

Carotenoids in Rhodoplanes species: variation of compositions and substrate specificity of predicted carotenogenesis enzymes.

Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: high-field EPR study.

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D+ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.

Light-induced structural changes in a photosynthetic reaction center caught by Laue diffraction.

Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

Fragment molecular orbital study of the electronic excitations in the photosynthetic reaction center of Blastochloris viridis.

All electron calculations were performed on the photosynthetic reaction center of Blastochloris viridis, using the fragment molecular orbital (FMO) method. The protein complex of 20,581 atoms and 77,754 electrons was divided into 1398 fragments, and the two-body expansion of FMO/6-31G* was applied to calculate the ground state. The excited electronic states of the embedded electron transfer system were separately calculated by the configuration interaction singles approach with the multilayer FMO method. Despite the structural symmetry of the system, asymmetric excitation energies were observed, especially on the bacteriopheophytin molecules. The asymmetry was attributed to electrostatic interaction with the surrounding proteins, in which the cytoplasmic side plays a major role.

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center.

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 x 100 x 100 microm, belonged to space group P4(3)2(1)2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 A resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C(380), revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.

Proton-transfer pathways in photosynthetic reaction centers analyzed by profile hidden markov models and network calculations.

In the bacterial reaction center (bRC) of Rhodobacter sphaeroides, the key residues of proton transfer to the secondary quinone (Q(B)) are known. Also, several possible proton entry points and proton-transfer pathways have been proposed. However, the mechanism of the proton transfer to Q(B) remains unclear. The proton transfer to Q(B) in the bRC of Blastochloris viridis is less explored. To analyze whether the bRCs of different species use the same key residues for proton transfer to Q(B), we determined the conservation of these residues. We performed a multiple-sequence alignment based on profile hidden Markov models. Residues involved in proton transfer but not located at the protein surface are conserved or are only exchanged to functionally similar amino acids, whereas potential proton entry points are not conserved to the same extent. The analysis of the hydrogen-bond network of the bRC from R. sphaeroides and that from B. viridis showed that a large network connects Q(B) with the cytoplasmic region in both bRCs. For both species, all non-surface key residues are part of the network. However, not all proton entry points proposed for the bRC of R. sphaeroides are included in the network in the bRC of B. viridis. From our analysis, we could identify possible proton entry points. These proton entry points differ between the two bRCs. Together, the results of the conservation analysis and the hydrogen-bond network analysis make it likely that the proton transfer to Q(B) is not mediated by distinct pathways but by a large hydrogen-bond network.

Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device.

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

Cucumibacter marinus gen. nov., sp. nov., a marine bacterium in the family Hyphomicrobiaceae.

A Gram-negative, rod-shaped, strictly aerobic micro-organism, designated strain CL-GR60(T), was isolated from coastal seawater from the East Sea, Korea. A 16S rRNA gene sequence analysis revealed a clear affiliation with the family Hyphomicrobiaceae. Phylogenetic analyses showed that strain CL-GR60(T) formed a robust cluster with the species of the genus Devosia at sequence similarity levels of 91.1-93.1 %; no other species in the family Hyphomicrobiaceae shared more than 90 % sequence similarity with strain CL-GR60(T). The strain grew optimally in the presence of 3-4 % sea salts at 30-35 degrees C and pH 7. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and an unidentified lipid. The strain contained C(18 : 1)omega7c (54.7 %), 11-methyl C(18 : 1)omega7c (19.3 %), C(18 : 0) (14.4 %) as the predominant fatty acids. Ubiquinone-10 was the major quinone. The DNA G+C content was 62.9 mol%. On the basis of physiological data, fatty acid composition and results of phylogenetic analysis of the 16S rRNA gene sequence, strain CL-GR60(T) represents a novel genus and species within the family Hyphomicrobiaceae, for which the name Cucumibacter marinus gen. nov., sp. nov. is proposed. The type strain of Cucumibacter marinus is CL-GR60(T) (=KCCM 90027(T) =DSM 18995(T)).