Adaxial, abaxial phylloplane (leaf), and spermoplane (seed) are proximal yet contrasting habitats for a microbiota that needs to be adequately explored. Here, we proposed novel methods to decipher the adaxial/abaxial-phylloplane and spermoplane-microbiomes. Comparison of 22 meta barcoded-NGS datasets (size of total data set-1980.48 Mb) enabled us to fine-map the microbiome of the rice foliar niche, which encompasses the lower, middle, top leaf as well panicle. Here, the total- and the cultivable-microbiome profiling revealed 157 genera representing ten phyla and 87 genera from 4 bacterial phyla, respectively, with a predominance of Proteobacteria and Actinobacteria. Interestingly, more bacterial communities (124-genera) preferred the abaxial than the adaxial phylloplane (104-genera) and spermoplane (67-genera) for colonization. The microbiome profiles were nearly identical on the aromatic (125-genera) and non-aromatic rice (116-genera) with high representation of Pantoea, Methylobacterium, Curtobacterium, Sphingopyxis, and Microbacterium. The culturomics investigation confirmed the abundance of Pantoea, Chryseobacterium, Pseudomonas, Acinetobacter, Sphingobacterium, and Exiguobacterium. One hundred bacterial isolates characterized and identified by polyphasic-taxonomic tools revealed the dominance of Acinetobacter, Chryseobacterium, Enterobacter, Massilia, Pantoea, Pseudomonas, and Stenotrophomonas on adaxial/abaxial-phylloplane and spermoplane. The study culminated in identifying hitherto unexplored bacterial communities on the adaxial/abaxial phylloplane and spermoplane of rice that can be harnessed for microbiome-assisted rice cultivation in the future.

• Klíčová slova
• Abaxial, Adaxial, Metabarcoding, Microbiome, Phylloplane, Rice, Spermoplane,
• MeSH
• genotyp MeSH
• listy rostlin mikrobiologie   MeSH
• mikrobiota * MeSH
• rýže (rod) * MeSH
• Sphingomonadaceae * MeSH
• Publikační typ
• časopisecké články MeSH

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

• Klíčová slova
• ancestral sequence reconstruction, haloalkane dehalogenase, halogenated pollutants, structural analysis,
• MeSH
• hydrolasy chemie   metabolismus   MeSH
• hydrolýza MeSH
• katalytická doména MeSH
• krystalografie rentgenová metody   MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• Mycobacterium bovis enzymologie   MeSH
• proteinové inženýrství metody   MeSH
• sekvenční analýza proteinů metody   MeSH
• Sphingomonadaceae enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

• MeSH
• cystein chemie   MeSH
• flavinmononukleotid chemie   MeSH
• fotochemické procesy MeSH
• fototropiny chemie   MeSH
• glutamin chemie   MeSH
• isomerie MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• normální rozdělení MeSH
• oves chemie   MeSH
• sekvence aminokyselin MeSH
• spektrofotometrie infračervená MeSH
• Sphingomonadaceae chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• flavinmononukleotid MeSH
• fototropiny MeSH
• glutamin MeSH

Technologies based on synthetic biology to produce bacterial natural carotenoids depend on information regarding their biosynthesis. Although the biosynthetic pathway of common carotenoids is known, there are carotenoids whose pathways are not completely described. This work aimed to mine the genome of the deep-sea bacterium Erythrobacter citreus LAMA 915, an uncommon bacterium that forms yellow colonies under cultivation. This work further explores the potential application of the carotenoids found and low-cost substrates for bacterial growth. A combined approach of genome mining and untargeted metabolomics analysis was applied. The carotenoid erythroxanthin sulfate was detected in E. citreus LAMA 915 cell extract. A proposal for carotenoid biosynthesis by this bacterium is provided, involving the genes crtBIYZWG. These are responsible for the biosynthesis of carotenoids from the zeaxanthin pathway and their 2,2'-hydroxylated derivatives. E. citreus LAMA 915 extracts showed antioxidant and sun protection effects. Based on the high content of proteases and lipases, it was possible to rationally select substrates for bacterial growth, with residual oil from fish processing the best low-cost substrate selected. This work advances in the understanding of carotenoid biosynthesis and provides a genetic basis that can be further explored as a biotechnological route for carotenoid production.

• MeSH
• biosyntetické dráhy * genetika   MeSH
• karotenoidy * metabolismus   MeSH
• Sphingomonadaceae * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karotenoidy * MeSH

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer-fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants ( kon = 1822 s-1, koff = 60 s-1) corresponding well with those from the pre-steady-state kinetics ( k-1 = 1100 s-1, k1 = 20 s-1). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

• MeSH
• biokatalýza MeSH
• hydrolasy chemie   genetika   MeSH
• katalytická doména MeSH
• kinetika MeSH
• konformace proteinů MeSH
• mutace MeSH
• proteinové inženýrství MeSH
• simulace molekulární dynamiky MeSH
• Sphingomonadaceae enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Zymomonas mobilis is a Gram-negative bacterium studied primarily as a spoilage organism and ethanol producer. As with many bacteria, much remains to be learned about its ecology. It can serve as a model organism for examining microbial interactions, as well as interactions between plants and bacteria. Better understanding of its ecology can help with biotechnological applications, such as process improvement, new uses of the bacterium, and the search for new strains.

• MeSH
• biotechnologie metody   MeSH
• ethanol metabolismus   MeSH
• potravinářská mikrobiologie metody   MeSH
• Zymomonas metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• ethanol MeSH

The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product.

• Klíčová slova
• LinB, biocatalysis, enantioselectivity, haloalkane dehalogenase, hydrolysis, regioselectivity,
• MeSH
• butylenglykoly chemická syntéza   MeSH
• halogenace MeSH
• hydrolasy metabolismus   MeSH
• hydrolýza MeSH
• Sphingomonadaceae enzymologie   MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• butylenglykoly MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Light-harvesting complexes ensure necessary flow of excitation energy into photosynthetic reaction centers. In the present work, transient absorption measurements were performed on LH1-RC complexes isolated from two aerobic anoxygenic phototrophs (AAPs), Roseobacter sp. COL2P containing the carotenoid spheroidenone, and Erythrobacter sp. NAP1 which contains the carotenoids zeaxanthin and bacteriorubixanthinal. We show that the spectroscopic data from the LH1-RC complex of Roseobacter sp. COL2P are very similar to those previously reported for Rhodobacter sphaeroides, including the transient absorption spectrum originating from the intramolecular charge-transfer (ICT) state of spheroidenone. Although the ICT state is also populated in LH1-RC complexes of Erythrobacter sp. NAP1, its appearance is probably related to the polarity of the bacteriorubixanthinal environment rather than to the specific configuration of the carotenoid, which we hypothesize is responsible for populating the ICT state of spheroidenone in LH1-RC of Roseobacter sp. COL2P. The population of the ICT state enables efficient S1/ICT-to-bacteriochlorophyll (BChl) energy transfer which would otherwise be largely inhibited for spheroidenone and bacteriorubixanthinal due to their low energy S1 states. In addition, the triplet states of these carotenoids appear well-tuned for efficient quenching of singlet oxygen or BChl-a triplets, which is of vital importance for oxygen-dependent organisms such as AAPs.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• bakteriochlorofyly chemie   MeSH
• karotenoidy chemie   MeSH
• kinetika MeSH
• přenos energie MeSH
• Rhodobacter sphaeroides metabolismus   MeSH
• Roseobacter metabolismus   MeSH
• Sphingomonadaceae metabolismus   MeSH
• světlosběrné proteinové komplexy chemie   metabolismus   MeSH
• xanthofyly chemie   MeSH
• zeaxanthiny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriochlorofyly MeSH
• karotenoidy MeSH
• spheroidenone MeSH Prohlížeč
• světlosběrné proteinové komplexy MeSH
• xanthofyly MeSH
• zeaxanthiny MeSH

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

• Klíčová slova
• LinB, Sphingobium japonicum, haloalkane dehalogenase, macroseeding,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• biodegradace MeSH
• bromované uhlovodíky chemie   metabolismus   MeSH
• Escherichia coli chemie   genetika   MeSH
• ethylendibromid chemie   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• izoenzymy chemie   genetika   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Sphingomonadaceae chemie   enzymologie   genetika   MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bromované uhlovodíky MeSH
• butyl bromide MeSH Prohlížeč
• ethylendibromid MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• izoenzymy MeSH
• rekombinantní proteiny MeSH

A yellow-pigmented bacterial strain, designated LL01(T), was isolated from hexachlorocyclohexane (HCH)-contaminated soil at Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL01(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing highest similarity to Sphingobium rhizovicinum CC-FH12-1(T) (98.5 %). The DNA G+C content of strain LL01(T) was 66.1 mol%. The predominant respiratory pigment was ubiquinone Q-10. The polar lipid profile of strain LL01(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipids), supporting its identification as a member of the genus Sphingobium. Spermidine was the major polyamine observed. The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished strain LL01(T) from closely related species of the genus Sphingobium. Therefore, strain LL01(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium czechense sp. nov. is proposed (type strain LL01(T) = CCM 7979(T) = DSM 25410(T)).

• MeSH
• DNA bakterií genetika   MeSH
• fylogeneze * MeSH
• hexachlorcyklohexan * MeSH
• hybridizace nukleových kyselin MeSH
• látky znečišťující půdu * MeSH
• mastné kyseliny analýza   MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• spermidin analýza   MeSH
• Sphingomonadaceae klasifikace   genetika   izolace a purifikace   MeSH
• techniky typizace bakterií MeSH
• ubichinon analýza   MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA bakterií MeSH
• hexachlorcyklohexan * MeSH
• látky znečišťující půdu * MeSH
• mastné kyseliny MeSH
• RNA ribozomální 16S MeSH
• spermidin MeSH
• ubichinon MeSH
• Ubiquinone Q2 MeSH Prohlížeč