All Kinds of Sunny Colors Synthesized from Methane: Genome-Encoded Carotenoid Production by Methylomonas Species.

Carotenoids are secondary metabolites that exhibit antioxidant properties and are characterized by a striking range of colorations from red to yellow. These natural pigments are synthesized by a wide range of eukaryotic and prokaryotic organisms. Among the latter, carotenoid-producing methanotrophic bacteria, which display fast growth on methane or natural gas, are of particular interest as potential producers of a feed protein enriched with carotenoids. Until recently, Methylomonas strain 16a and Methylomonas sp. ZR1 remained the only representatives of the genus for which detailed carotenoid profile was determined. In this study, we analyzed the genome sequences of five strains of Methylomonas species whose pigmentation varied from white and yellow to orange and red, and identified carotenoids produced by these bacteria. Carotenoids synthesized using four pigmented strains included C30 fraction, primarily composed of 4,4'-diaplycopene-4,4'-dioic acid and 4,4'-diaplycopenoic acid, as well as C40 fraction with the major compound represented by 1,1'-dihydroxy-3,4-didehydrolycopene. The genomes of studied Methylomonas strains varied in size between 4.59 and 5.45 Mb and contained 4201-4735 protein-coding genes. These genomes and 35 reference Methylomonas genomes available in the GenBank were examined for the presence of genes encoding carotenoid biosynthesis. Genomes of all pigmented Methylomonas strains harbored genes necessary for the synthesis of 4,4'-diaplycopene-4,4'-dioic acid. Non-pigmented "Methylomonas montana" MW1T lacked the crtN gene required for carotenoid production. Nearly all strains possessed phytoene desaturases, which explained their ability to naturally synthesize lycopene. Thus, members of the genus Methylomonas can potentially be considered as producers of C30 and C40 carotenoids from methane.

Gaussian Decomposition vs. Semiclassical Quantum Simulation: Obtaining the High-Order Derivatives of a Spectrum in the Case of Photosynthetic Pigment Optical Properties Studying.

In this paper, a procedure for obtaining undistorted high derivatives (up to the eighth order) of the optical absorption spectra of biomolecule pigments has been developed. To assess the effectiveness of the procedure, the theoretical spectra of bacteriochlorophyll a, chlorophyll a, spheroidene, and spheroidenone were simulated by fitting the experimental spectra using the differential evolution algorithm. The experimental spectra were also approximated using sets of Gaussians to calculate the model absorption spectra. Theoretical and model spectra can be differentiated without smoothing (high-frequency noise filtering) to obtain high derivatives. Superimposition of the noise track on the model spectra allows us to obtain test spectra similar to the experimental ones. Comparison of the high derivatives of the model spectra with those of the test spectra allows us to find the optimal parameters of the filter, the application of which leads to minimal differences between the high derivatives of the model and test spectra. For all four studied pigments, it was shown that smoothing the experimental spectra with optimal filters makes it possible to obtain the eighth derivatives of the experimental spectra, which were close to the eighth derivatives of their theoretical spectra.

Methylomonas rapida sp. nov., a novel species of fast-growing, carotenoid-producing obligate methanotrophs with high biotechnological potential.

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1T, which are phylogenetically distinct from the currently described Methylomonas species (94.0-97.3 % 16S rRNA gene sequence similarity). Strains 12 and MP1T were isolated from freshwater sediments collected in Moscow and Krasnodar regions, respectively. Cells of these strains are Gram-negative, red-pigmented, highly motile thick rods that contain a type I intracytoplasmic membrane system and possess a particulate methane monooxygenase (pMMO) enzyme. These bacteria grow between 8 and 45 °C (optimum 35 °C) in a relatively narrow pH range of 5.5-7.3 (optimum pH 6.6-7.2). Major carotenoids synthesized by these methanotrophs are 4,4'-diaplycopene-4,4'-dioic acid, 1,1'-dihydroxy-3,4-didehydrolycopene and 4,4'-diaplycopenoic acid. High biomass yield, of up to 3.26 g CDW/l, is obtained during continuous cultivation of MP1T on natural gas in a bioreactor at a dilution rate of 0.22 h-1. The complete genome sequence of strain MP1T is 4.59 Mb in size; the DNA G + C content is 52.8 mol%. The genome encodes four rRNA operons, one pMMO operon and 4,216 proteins. The genome sequence displays 82-85 % average nucleotide identity to those of earlier described Methylomonas species. We propose to classify these bacteria as representing a novel species of the genus Methylomonas, M. rapida sp. nov., with the type strain MP1T (=KCTC 92586T = VKM B-3663T).

ζ-Carotene: Generation and Quenching of Singlet Oxygen, Comparison with Phytofluene.

It is known that C40 carotenoids with a short chain of conjugated double bonds (CDB) (5 and 7, respectively) are universal precursors in the biosynthesis of colored carotenoids in plant cells. Previously, using mainly stationary measurements of photosensitized phosphorescence of singlet oxygen (1O2), we discovered that phytofluene efficiently generates 1O2 in aerated solution and therefore, can serve as a source of the UV photodynamic stress in living cells [Ashikhmin et al., Biochemistry (Moscow), 2020, 85, 773]. In the present paper, by using novel pulsed light emitting diodes (LEDs), aerated hexafluorobenzene as a solvent and time-resolved measurements of 1O2 phosphorescence we confirmed that phytofluene efficiently photosensitizes 1O2 formation. The quantum yield of this process according to the novel experiments is about 0.4. An ability to generate 1O2 was also found in aerated solutions of ζ-carotene although the quantum yield of this process is 30-fold lower that in phytofluene solutions. Both carotenoids were found to quench 1O2 in the dark with the quenching rate constants equal to (3.6 ± 0.9)×107 and (2.1 ± 0.2)×108 M-1s-1, respectively. To our knowledge, the rate constant of 1O2 quenching by ζ-carotene has been reported in the present paper for the first time. It follows from the data obtained that the rate constants of 1O2 quenching by both carotenoids are much (by 2-3 orders of magnitude) smaller than the rate constant of the diffusion-limited biomolecular reactions. Hence, both carotenoids are weak protectors against 1O2 oxidative activity. It is more likely that they are potential promoters of photodynamic stress in living cells.

Reconstitution of the functional Carotenoid-Binding Protein from silkworm in E. coli.

Natural water-soluble carotenoproteins are promising antioxidant nanocarriers for biomedical applications. The Carotenoid-Binding Protein from silkworm Bombyx mori (BmCBP) is responsible for depositing carotenoids in cocoons. This determines the silk coloration, which is relevant for sericulture for four thousand years. While BmCBP function is well-characterized by molecular genetics, its structure and carotenoid-binding mechanism remain to be studied. To facilitate this, here we report on successful production of soluble BmCBP in Escherichia coli, its purification and characterization. According to CD spectroscopy and SEC-MALS, this protein folds into a ~ 27-kDa monomer capable of dose-dependent binding of lutein, a natural BmCBP ligand, in vitro. Binding leads to a >10 nm red-shift of the carotenoid absorbance and quenches tryptophan fluorescence of BmCBP. Using zeaxanthin, a close lutein isomer that can be stably produced in engineered E.coli strains, we successfully reconstitute the BmCBP holoform and characterize its properties. While BmCBP successfully matures into the holoform, BmCBP-zeaxanthin complexes are contaminated by the apoform. We demonstrate that the yield of the holoform can be increased by adding bovine serum albumin during cell lysis and that the remaining BmCBP apoform is efficiently removed using hydroxyapatite chromatography. Bacterial production of BmCBP paves the way for its structural studies and applications.

'Candidatus Chloroploca mongolica' sp. nov. a new mesophilic filamentous anoxygenic phototrophic bacterium.

A mesophilic filamentous anoxygenic phototrophic bacterium, designated M50-1, was isolated from a microbial mat of the Chukhyn Nur soda lake (northeastern Mongolia) with salinity of 5-14 g/L and pH 8.0-9.3. The organism is a strictly anaerobic phototrophic bacterium, which required sulfide for phototrophic growth. The cells formed short undulate trichomes surrounded by a thin sheath and containing gas vesicles. Motility of the trichomes was not observed. The cells contained chlorosomes. The antenna pigments were bacteriochlorophyll d and ß- and γ-carotenes. Analysis of the genome assembled from the metagenome of the enrichment culture revealed all the enzymes of the 3-hydroxypropionate bi-cycle for autotrophic CO2 assimilation. The genome also contained the genes encoding a type IV sulfide:quinone oxidoreductase (sqrX). The organism had no nifHDBK genes, encoding the proteins of the nitrogenase complex responsible for dinitrogen fixation. The DNA G + C content was 58.6%. The values for in silico DNA‒DNA hybridization and average nucleotide identity between M50-1 and a closely related bacterium 'Ca. Chloroploca asiatica' B7-9 containing bacteriochlorophyll c were 53.4% and 94.0%, respectively, which corresponds to interspecies differences. Classification of the filamentous anoxygenic phototrophic bacterium M50-1 as a new 'Ca. Chloroploca' species was proposed, with the species name 'Candidatus Chloroploca mongolica' sp. nov.

'Candidatus Oscillochloris kuznetsovii' a novel mesophilic filamentous anoxygenic phototrophic Chloroflexales bacterium from Arctic coastal environments.

Chloroflexales bacteria are mostly known as filamentous anoxygenic phototrophs that thrive as members of the microbial communities of hot spring cyanobacterial mats. Recently, we described many new Chloroflexales species from non-thermal environments and showed that mesophilic Chloroflexales are more diverse than previously expected. Most of these species were isolated from aquatic environments of mid-latitudes. Here, we present the comprehensive characterization of a new filamentous multicellular anoxygenic phototrophic Chloroflexales bacterium from an Arctic coastal environment (Kandalaksha Gulf, the White Sea). Phylogenomic analysis and 16S rRNA phylogeny indicated that this bacterium belongs to the Oscillochloridaceae family as a new species. We propose that this species be named 'Candidatus Oscillochloris kuznetsovii'. The genomes of this species possessed genes encoding sulfide:quinone reductase, the nitrogenase complex and the Calvin cycle, which indicate potential for photoautotrophic metabolism. We observed only mesophilic anaerobic anoxygenic phototrophic growth of this novel bacterium. Electron microphotography showed the presence of chlorosomes, polyhydroxyalkanoate-like granules and polyphosphate-like granules in the cells. High-performance liquid chromatography also revealed the presence of bacteriochlorophylls a, c and d as well as carotenoids. In addition, we found that this bacterium is present in benthic microbial communities of various coastal environments of the Kandalaksha Gulf.

Lichenicoccus roseus gen. nov., sp. nov., the first bacteriochlorophyll a-containing, psychrophilic and acidophilic Acetobacteraceae bacteriobiont of lichen Cladonia species.

Gram-negative, aerobic, chemo-organotrophic and bacteriochlorophyll a-containing bacterial strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152, were isolated from the thalli of Cladonia arbuscula and Cladonia stellaris lichens. Cells from the strains were coccoid and reproduced by binary division. They were motile at the early stages of growth and utilized sugars and alcohols. All strains were psychrophilic and acidophilic, capable of growth between pH 3.5 and 7.5 (optimum, pH 5.5), and at 4-30 °C (optimum, 10-15 °C). The major fatty acids were C18 : 1 ω7c and C18 : 0; the lipids were phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylglycerol, glycolipids, diphosphatidylglycerol and polar lipids with an unknown structure. The quinone was Q-10. The DNA G+C content was 67.8 mol%. Comparative 16S rRNA gene analysis together with other data, supported that the strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 belonged to the same species. Whole genome analysis of the strain KEBCLARHB70RT and average amino acid identity values confirmed its distinctive phylogenetic position within the family Acetobacteraceae. Phenotypic, ecological and genomic characteristics distinguished strains KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 from all genera in the family Acetobacteraceae. Therefore, we propose a novel genus and a novel species, Lichenicoccus roseus gen. nov., sp. nov., for these novel Acetobacteraceae members. Strain KEBCLARHB70RT (=KCTC 72321T=VKM B-3305T) has been designated as the type strain.

The Main Structural and Functional Characteristics of Photosystem-II-Enriched Membranes Isolated from Wild Type and cia3 Mutant Chlamydomonas reinhardtii.

Photosystem II (PSII)-enriched membranes retain the original PSII architecture in contrast to PSII cores or PSII supercomplexes, which are usually isolated from Chlamydomonas reinhardtii. Here, we present data that fully characterize the structural and functional properties of PSII complexes in isolated PSII-enriched membranes from C. reinhardtii. The preparations were isolated from wild-type (WT) and CAH3-deficient mutant cia3 as the influence of CAH3 on the PSII function was previously proposed. Based on the equal chlorophyll content, the PSII-enriched membranes from WT and cia3 have the same amount of reaction centers (RCs), cytochrome b559, subunits of the water-oxidizing complex, Mn ions, and carotenes. They differ in the ratio of other carotenoids, the parts of low/intermediate redox forms of cytochrome b559, and the composition of outer light-harvesting complexes. The preparations had 40% more chlorophyll molecules per RC compared to higher plants. Functionally, PSII-enriched membranes from WT and cia3 show the same photosynthetic activity at optimal pH 6.5. However, the preparations from cia3 contained more closed RCs even at pH 6.5 and showed more pronounced suppression of PSII photosynthetic activity at shift pH up to 7.0, established in the lumen of dark-adapted cells. Nevertheless, the PSII photosynthetic capacities remained the same.

Structural peculiarities of keto-carotenoids in water-soluble proteins revealed by simulation of linear absorption.

To prevent irreversible damage caused by an excess of incident light, the photosynthetic machinery of many cyanobacteria uniquely utilizes the water-soluble orange carotenoid protein (OCP) containing a single keto-carotenoid molecule. This molecule is non-covalently embedded into the two OCP domains which are interconnected by a flexible linker. The phenomenon of OCP photoactivation, causing significant changes in carotenoid absorption in the orange and red form of OCP, is currently being thoroughly studied. Numerous additional spectral forms of natural and synthetic OCP-like proteins have been unearthed. The optical properties of carotenoids are strongly determined by the interaction of their electronic states with vibrational modes, the surrounding protein matrix, and the solvent. In this work, the effects of the pigment-protein interaction and vibrational relaxation in OCP were studied by computational simulation of linear absorption. Taking into account Raman spectroscopy data and applying the multimode Brownian oscillator model as well as the cumulant expansion technique, we have calculated a set of characteristic microparameters sufficient to demarcate different carotenoid states in OCP forms, using the model carotenoids spheroidene and spheroidenone in methanol/acetone solution as benchmarks. The most crucial microparameters, which determine the effect of solvent and protein environment, are the Huang-Rhys factors and the frequencies of C[double bond, length as m-dash]C and C-C stretching modes, the low-frequency mode and the FWHM due to inhomogeneous line broadening. Considering the difference of linear absorption between spheroidene and spheroidenone, which remarkably resembles the photoinduced changes of OCP absorption, and applying quantum chemical calculations, we discuss structural and functional determinants of carotenoid binding proteins.

'Candidatus Oscillochloris fontis': a novel mesophilic phototrophic Chloroflexota bacterium belonging to the ubiquitous Oscillochloris genus.

We present the results of a study of mesophilic anoxygenic phototrophic Chloroflexota bacteria from Mechigmen hot spring (the Chukotka Peninsula) and Siberia. According to 16S rRNA phylogenetic analysis, these bacteria belong to Oscillochloris trichoides. However, sequencing the draft genome of the bacterium from the Chukotka and analysis of the average nucleotide identity, as well as in silico DNA-DNA hybridization, reveal that this bacterium belongs to a novel species within the Oscillochloris genus. We, therefore, propose 'Candidatus Oscillochloris fontis' as a novel taxon to represent this mesophilic alkaliphilic anaerobic anoxygenic phototrophic bacterium. Spectrophotometry and high-performance liquid chromatography analysis show that the bacterium possesses bacteriochlorophylls c and a, as well as lycopene, ß-carotene and γ-carotene. In addition, transmission electron microscopy shows the presence of chlorosomes, polyhydroxyalkanoate- and polyphosphate-like granules. The genome of 'Ca. Oscillochloris fontis' and the Siberian strains of Oscillochloris sp. possess the key genes for nitrogenase complex (nifH) and ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), as previously described for O. trichoides DG-6. The results presented here, and previously published data, show that Oscillochloris bacteria from different aquatic environments have the potential for CO2 and N2 fixation. Additionally, we describe a new primer system for the detection of RuBisCo form I.

'Candidatus Viridilinea mediisalina', a novel phototrophic Chloroflexi bacterium from a Siberian soda lake.

In this article, we present the description of a novel mesophilic phototrophic Chloroflexi bacterium, 'Candidatus Viridilinea mediisalina' Kir15-3F. We have isolated an anaerobic, highly enriched culture of this bacterium from the Kiran soda lake (Siberia) and optimized its cultivation. Metagenomic sequencing revealed that 'Ca. Viridilinea mediisalina' Kir15-3F is a bacteriochlorophyll-containing Chloroflexi bacterium in the enrichment culture. Fluorescent in situ hybridisation demonstrated a link between the phenotype described here and the 'Ca. Viridilinea mediisalina' Kir15-3F genome. Spectrophotometry and high-performance liquid chromatography analyses showed the presence of bacteriochlorophylls d, c and a, as well as lycopene, γ-carotene and ß-carotene. Transmission electron microscopy showed chlorosomes, gas vesicles, polyhydroxyalkanoate-like and polyphosphate-like granules. Our results illustrated that 'Ca. Viridilinea mediisalina' Kir15-3F is an alkaliphilic, salt-tolerant, obligately mesophilic, anaerobic, phototrophic bacterium. The genome sequences lack genes of the Calvin cycle and a sulphide:quinone reductase gene for sulphide oxidation. Owing to the lack of an axenic culture and based on the genomic and phenotypic data, we have presented the description of the bacterium in the Candidatus category.

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation.

As a basis of photosynthesis, photoinduced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from E. haloalkaliphila was first resolved by X-ray diffraction to 3.7 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed-bleaching and blue-shifting of the B850 exciton band and correlated emergence of a higher-energy C700 exciton band-are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls, with no noticeable damage to the pigment-binding protein scaffold. A prospective noninvasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.