A structural framework for unidirectional transport by a bacterial ABC exporter.

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6.

Lignostilbene-α,ß-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from ß-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

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.

Description of Erythrobacter mangrovi sp. nov., an aerobic bacterium from rhizosphere soil of mangrove plant (Kandelia candel).

A novel Gram-stain negative, aerobic, non-motile, rod-shaped bacterium, designated as strain EB310T, was isolated from rhizosphere soil of mangrove plant Kandelia candel in Fugong village, Zhangzhou, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain EB310T belonged to the genus Erythrobacter, clustering with Erythrobacter pelagi JCM 17468T, Erythrobacter lutimaris KCTC 42109T and Erythrobacter marisflavi KCTC 62896T, and showed the highest 16S rRNA gene sequence similarity of 97.5% to Erythrobacter pelagi JCM 17468T. The genomic average nucleotide identity and in silico DNA-DNA hybridization values between strain EB310T and the reference strains were 71.0-75.5% and 19.8-20.0%, respectively. Growth ranges of the isolate occurred at 10-45 °C (optimum 28-30 °C), pH 5.5-9.5 (optimum pH 7.5) and 0-9.0% NaCl concentrations (optimum 2.0%, w/v). The strain did not produce bacteriochlorophyll a and flexirubin, but produced carotenoids. The strain contained Q-10 as the predominant ubiquinone and summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω6c/C18:1 ω7c) as the major fatty acids. The major polar lipids were sphingoglycolipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylcholine. Differential phenotypic characteristics, together with chemotaxonomic, phylogenetic and genomic distinctiveness, indicated that strain EB310T is distinguishable from other members of the genus Erythrobacter. On the basis of the data exhibited, strain EB310T is considered to represent a novel species of the genus Erythrobacter, for which the name Erythrobacter mangrovi sp. nov., is proposed. The type strain is EB310T (= KCTC 72109T = MCCC 1K03690T). The genomic DNA G + C content is 62.9 mol%.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates.

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere.

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-ß-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the ß-aryl ether bond of lignin.

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the ß-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the ß-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-ß-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate ß-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the ß-aryl ether bond) and Escherichia coli (which cannot break the ß-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.

Sphingobium aromaticivastans sp. nov., a novel aniline- and benzene-degrading, and antimicrobial compound producing bacterium.

A strictly aerobic, orange-pigmented strain was isolated and designated as UCM-25T. This strain is capable of degrading aniline and benzene, while is also producing antimicrobial compounds which inhibit the growth of some common pathogenic microbes. A near full-length 16S rRNA gene sequence revealed similarity to Sphingobium chlorophenolicum NBRC 16172T (98.6%). The level of DNA-DNA hybridization between the new isolate and the related species suggests UCM-25T to be a new species belonging to the genus Sphingobium. The bacterial cells contained phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, three unidentified polar lipids, and an unidentified aminophospholipid. Ubiquinone Q-10 was the major quinone and spermidine was the major polyamine. The G+C content in the DNA of strain UCM-25T was 62.9 mol%. Cells contained summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0, and C14:0 2-OH as major fatty acids. Based on the comparison of phenotypic, genotypic, and chemotaxonomic characteristics, strain UCM-25T represents a new member of the genus Sphingobium, for which the name S. aromaticivastans sp. nov. is proposed. The type strain is UCM-25T (=KACC 19288T =DSM 105181T).

Sulfur-Containing Carotenoids from A Marine Coral Symbiont Erythrobacter flavus Strain KJ5.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral Acropora nasuta in the Karimunjawa Islands, Indonesia. The complete genome sequence of the bacterium has been reported recently. In this study, we examined the carotenoid composition of this bacterium using high-performance liquid chromatography coupled with ESI-MS/MS. We found that the bacterium produced sulfur-containing carotenoids, i.e., caloxanthin sulfate and nostoxanthin sulfate, as the most abundant carotenoids. A new carotenoid zeaxanthin sulfate was detected based on its ESI-MS/MS spectrum. The unique presence of sulfated carotenoids found among the currently known species of the Erythrobacter genus were discussed.

X-ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2.

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.

Involvement of phosphoesterases in tributyl phosphate degradation in Sphingobium sp. strain RSMS.

A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous. Sphingobium SS22 also did not form any of the intermediates or end products of TBP or DBP degradation, namely DBP, butanol or inorganic phosphate. Proteomic analysis revealed the absence of three prominent proteins in Sphingobium SS22 as compared to wild type. These proteins were identified by MALDI mass spectrometry, and they showed similarities to phosphohydrolase- and exopolyphosphatase-like proteins from other bacteria, which belong to the class of phosphoesterases. Cellular proteins of Sphingobium SS22 showed none or negligible phosphodiesterase (PDE) and phosphomonoesterase (PME) activities at pH 7 and displayed approximately five- and approximately twofold less DBP and monobutyl phosphate (MBP) degradation activity, respectively, in comparison to the wild type strain. In-gel zymographic analysis revealed two PDE and PME activity bands in the wild type strain, one of which was absent in the Sphingobium SS22 mutant. The corresponding proteins from the wild type strain could degrade DBP and MBP. The results demonstrate the involvement of phosphoesterase enzymes in the TBP degradation pathway elucidated earlier.

Ligand-induced folding of a two-component signaling receiver domain.

To survive and adapt to environmental changes, bacteria commonly use two-component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when they are considered within full-length proteins. Here, we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium, Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering, and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg(2+) and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.

Stereochemical features of glutathione-dependent enzymes in the Sphingobium sp. strain SYK-6 ß-aryl etherase pathway.

Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave ß-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of ß-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are ß-S-glutathionyl-α-keto-thioethers that are degraded by a ß-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (ß-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (ß-guaiacyl-α-veratrylethanone). However, when ß-etherase assays were conducted with a racemic model substrate, ß-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (ß-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the ß-position (i.e. its ß-epimer)) was produced only in the LigF-catalyzed reaction. Thus, ß-etherase catalysis causes stereochemical inversion of the chiral center, converting a ß(R)-substrate to a ß(S)-product (LigE and LigP), and a ß(S)-substrate to a ß(R)-product (LigF). Further, LigG catalyzed glutathione-dependent ß-S-thioether cleavage with ß-S-glutathionyl-α-veratrylethanone and with ß(R)-configured ß-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced ß-S-thioether-cleaving activity with the ß(S)-epimer, demonstrating that LigG is a stereospecific ß-thioetherase. We therefore propose that multiple Lig enzymes are needed in this ß-aryl etherase pathway in order to cleave the racemic ß-ether linkages that are present in the backbone of the lignin polymer.

Sphingorhabdus pacificus sp. nov., isolated from sandy sediments of the Sea of Japan seashore.

An aerobic, Gram-negative, yellow-pigmented, non-motile rod-shaped bacterium designated KMM 9574(T) was isolated from a sand sediment sample collected from the Sea of Japan seashore. Comparative 16S rRNA gene sequence analysis showed that strain KMM 9574(T) belonged to the genus Sphingorhabdus sharing a highest sequence similarity to Sphingorhabdus marina JCM 14161(T) 96.8 %. Strain KMM 9574(T) was characterized by the major ubiquinone Q-10, and by the predominance of C(18:1) ω7c, C(16:0) 2-OH, C(16:1) ω7c, C(17:1), followed by C(15:0) 2-OH and C(14:0) 2-OH in its fatty acid profile. Polar lipids consisted of phosphatidylcholine, sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid, and an unknown lipid. The DNA G+C content was 56.5 mol %. Based on phylogenetic analysis and distinctive phenotypic characteristics, strain 9574(T) is concluded to represent a novel species of the genus Sphingorhabdus, for which the name Sphingorhabdus pacificus sp. nov., is proposed. The type strain of the species is strain KMM 9574(T) (= NRIC 0922(T) = JCM 30177(T)).

Sphingosinicella cucumeris sp. nov., isolated from soil of a cucumber garden.

A novel bacterial strain, THG-sc1(T), was isolated from a soil sample of a cucumber garden and was characterised by using a polyphasic approach. Cells were observed to be Gram-stain negative, non-motile and rod-shaped. The strain was found to be aerobic, catalase and oxidase positive, esculin and starch negative, and to have an optimum growth temperature and pH of 28 °C and 7.5, respectively. On the basis of 16S rRNA gene sequence analysis, strain THG-sc1(T) was found to belong to the genus Sphingosinicella and to be closely related to Sphingosinicella vermicomposti KCTC 22446(T), followed by Sphingosinicella xenopeptidilytica DSM 17130(T) and Sphingosinicella microcystinivorans KCTC 12019(T). The DNA G+C content was determined to be 60.8 mol% and the predominant respiratory quinone was identified as ubiquinone-10. The major polyamine was found to be sym-homospermidine. The major polar lipids were identified as sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. The major fatty acids were identified as C(18:1)ω7c, C(16:0) and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH, as defined by MIDI). The results of the genotypic analysis, in combination with chemotaxonomic and physiological data, demonstrated that strain THG-sc1(T) represents a novel species within the genus Sphingosinicella, for which the name Sphingosinicella cucumeris is proposed. The type strain is THG-sc1(T) (=KACC 18279(T) = CCTCC AB 2015120(T)).

Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67-85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.

Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum.

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 µM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol.

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Differences in crystallization of two LinB variants from Sphingobium japonicum UT26.

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.

Erythrobacter westpacificensis sp. nov., a marine bacterium isolated from the Western Pacific.

A novel Gram-negative, orange-pigmented bacterial strain JLT2008(T) was isolated from the surface seawater of the Western Pacific and subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JLT2008(T) belonged to the genus Erythrobacter, sharing the highest similarity (96.6 %) with Erythrobacter gangjinensis K7-2(T) and the lowest similarity (94.9 %) with Erythrobacter litoralis DSM 8509(T). Strain JLT2008(T) did not contain bacteriochlorophyll a, and the predominant respiratory lipoquinone was ubiquinone-10. The major fatty acids were C(18:1) ω7c, C(16:0), C(16:1) ω7c/C(16:1) ω6c. The prominent polar lipids were sphingoglycolipid, phosphatidylethanolamine, and phosphatidylglycerol. The genomic G + C content was 60.1 mol %. Based on the polyphasic taxonomic data, a novel species within the genus Erythrobacter, and with the name Erythrobacter westpacificensis sp. nov., is proposed. The type strain is JLT2008(T) (=CGMCC 1.10993(T) = JCM 18014(T)).