Phyllobacterium pellucidum sp. nov., isolated from soil.

A bacterial strain, BT25T, was isolated from soil in Korea. The bacterial cells were Gram-negative and rod-shaped. Phylogenetic analysis using 16S rRNA gene sequences showed that the BT25T strain was related to the genus Phyllobacterium. BT25T was 96.6 and 96.5% similar to Phyllobacterium brassicacearum STM 196T and Phyllobacterium myrsinacearum DSM 5892T, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between BT25T and the two closest phylogenetic neighbors were calculated to be 78.5 and 77.7, 21.1 and 21.2%, respectively. The major cellular fatty acids were summed feature 8 (C18:1 ω7c/C18:1 ω6c) (29.3%), cyclo-C19:0 ω8c (27.5%), and C16:0 (16.5%). The BT25T strain had menaquinone Q-10 as the predominant quinone, as well as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylcholine as the major polar lipids. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the BT25T strain was classified as a novel Phyllobacterium species. The name Phyllobacterium pellucidum sp. nov. was proposed. The type strain is BT25T (= KCTC 62765T = NBRC 114381T).

Chelativorans xinjiangense sp. nov., a novel bacterial species isolated from soil in Xinjiang, China.

A novel Gram-strain-negative, beige-pigmented, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain lm93T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alar city, located in Xinjiang province, China. Growth optimally occurred at 30 °C, pH 6.5-7.5, and 0-2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain lm93T belonged to the genus Chelativorans, with highest sequence similarity to Chelativorans multitrophicus DSM 9103T (96.9%). Genome sequencing revealed a genome size of 5 689 708 bp and a G + C content of 64.3 mol%. The ANI, POCP and the dDDH between strain lm93T and C. multitrophicus DSM 9103T were 76.4%, 54.8% and 0.8%, respectively. The prediction result of secondary metabolites based on genome showed that the strain lm93T contained one cluster of bacteriocin, one cluster of terpene production, two clusters of ectoine production, one cluster of non-ribosomal peptide synthetase, one cluster of type I polyketide synthases, three clusters of homoserine lactone production, one cluster of N-acetylglutaminylglutamine amide production and one cluster of phosphonate production. The major respiratory quinone was Q-10. The major fatty acids were C19:0 cyclo ω8c, iso-C17:0 and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, aminoglycolipid, three unknown lipids and diphosphatidylglycerol. On the basis of these data, strain lm93T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans xinjiangense sp. nov. is proposed. The type strain is lm93T (= KCTC 72857T = CCTCC AB2019376T).

Nitratireductor mangrovi sp. nov., a Nitrate-Reducing Bacterium Isolated from Mangrove Soil.

A Gram-stain-negative, non-motile and short-rod-shaped bacterium, designated as strain SY7T, was isolated from rhizosphere soil of the mangrove Kandelia obovata of Fugong village, in Zhangzhou, China. The isolate grew at 10-45 °C (optimum 30 °C), pH 6.0-10.0 (optimum pH 7.0) and 0-8% NaCl (optimum 3%, w/v). The 16S rRNA gene sequence and phylogenetic analysis revealed that strain SY7T located within the radiation of genus Nitratireductor and showed the highest sequence similarity of 97.23% to Nitratireductor pacificus MCCC 1A01024T. The DNA G+C content was 64.9%. In silico DNA-DNA hybridization and average nucleotide identity values between strain SY7T with reference strains of N. pacificus MCCC 1A01024T, N. basaltis KCTC 22119T and N. aquibiodomus DSM 15645T were 16.7%, 14.3%, 14.7% and 75.2%, 72.6%, 73.5%, respectively. The major isoprenoid quinone was Q-10. The dominant fatty acids were 11-methyl C18:1ω7c, iso-C17:0, C19:0ω8c cyclo and summed feature 8 (C18:1ω6c/C18:1ω7c), a profile that almost matched the other members of the genus Nitratireductor. The predominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol. On the basis of the phenotypic, phylogenetic and chemotaxonomic analysis, strain SY7T represents a novel species of the genus Nitratireductor, for which the name Nitratireductor mangrovi sp. nov., is proposed. The type strain is SY7T (= KCTC 72110T = MCCC 1K03723T).

Structure and molecular morphology of a novel moisturizing exopolysaccharide produced by Phyllobacterium sp. 921F.

Bacterial exopolysaccharides (EPSs) are widely applied in food, cosmetic, and medical industries. The EPS produced by Phyllobacterium sp. 921F was a novel polysaccharide, which exhibits attractive characteristics of high yield, favorable rheological properties, and excellent moisture retention ability. Considering the complexity of polysaccharide structures, specific enzymatic hydrolysis was employed here to resolve the structure of the EPS. End-products including tetra-, hexa- and octa-saccharides were isolated. According to their mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectra, the backbone of the EPS was found to be mainly comprising a → 4)-ß-d-Glcp-(1 → 3)-α-d-Galp(4,6-S-Pyr)-(1 → disaccharide repeating units. Based on atomic force microscopy results, EPS exhibited characteristics that were consistent with a stiff, elongated molecule with no branches. The length and height of the single molecular chain were approximately 600 and 0.7 nm, respectively. Our clarification of structure and molecular morphology of EPS from Phyllobacterium sp. 921F provide a foundation for the industrial application of this potential moisture-retaining material.

Structural Basis for the Catalytic Mechanism of Ethylenediamine- N, N'-disuccinic Acid Lyase, a Carbon-Nitrogen Bond-Forming Enzyme with a Broad Substrate Scope.

The natural aminocarboxylic acid product ethylenediamine- N, N'-disuccinic acid [( S, S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediaminetetraacetic acid (EDTA), which is currently used on a large scale in numerous applications. Previous studies have demonstrated that biodegradation of ( S, S)-EDDS may be initiated by an EDDS lyase, converting ( S, S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited because of the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA, and ( S, S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of ( S, S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids.

NADPH-Driven Organohalide Reduction by a Nonrespiratory Reductive Dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-dependent enzymes that mostly act as the terminal oxidoreductases in the bacterial organohalide respiration process. This process often leads to detoxification of recalcitrant organohalide pollutants. While low cell yields and oxygen sensitivity hamper the study of many reductive dehalogenases, this is not the case for the nonrespiratory reductive dehalogenase NpRdhA from Nitratireductor pacificus. We here report in vitro and in vivo reconstitution of an NADPH-dependent reducing system for NpRdhA. Surprisingly, NpRdhA mediated organohalide reduction could not be supported using N. pacificus ferredoxin-NAD(P)H oxidoreductase and associated ferredoxins. Instead, we found a nonphysiological system comprised of the Escherichia coli flavodoxin reductase (EcFldr) in combination with spinach ferredoxin (SpFd) was able to support NADPH-dependent organohalide reduction by NpRdhA. Using this system, organohalide reduction can be performed under both anaerobic and aerobic conditions, with 1.1 ± 0.1 and 3.5 ± 0.3 equiv of NADPH consumed per product produced, respectively. No significant enzyme inactivation under aerobic conditions was observed, suggesting a Co(I) species is unlikely to be present under steady state conditions. Furthermore, reduction of the Co(II) resting state was not observed in the absence of substrate. Only the coexpression of EcFldr, SpFd, and NpRdhA in Bacillus megaterium conferred the latter with the ability to reduce brominated NpRdhA substrates in vivo, in agreement with our in vitro observations. Our work provides new insights into biological reductive dehalogenase reduction and establishes a blueprint for the minimal functional organohalide reduction module required for bioremediation in situ.

Studies on lipid A isolated from Phyllobacterium trifolii PETP02T lipopolysaccharide.

The structure of lipid A from lipopolysaccharide of Phyllobacterium trifolii PETP02T, a nitrogen-fixing symbiotic bacterium, was studied. It was found that the lipid A backbone was composed of two 2,3-diamino-2,3-dideoxy-D-glucose (GlcpN3N) residues connected by a ß-(1 â†’ 6) glycosidic linkage, substituted by galacturonic acid (GalpA) at position C-1 and partly decorated by a phosphate residue at C-4' of the non-reducing GlcpN3N. Both diaminosugars were symmetrically substituted by 3-hydroxy fatty acids (14:0(3-OH) and 16:0(3-OH)). Ester-linked secondary acyl residues [i.e. 19:0cyc and 28:0(27-OH) or 28:0(27-4:0(3-OMe))] were located in the distal part of lipid A. A high similarity between the lipid A of P. trifolii and Mesorhizobium was observed and discussed from the perspective of the genetic context of both genomes.

Nitratireductor soli sp. nov., isolated from phenol-contaminated soil.

Strain ZZ-1(T), a Gram-negative, rod-shaped bacterium, motile by flagella, was isolated from phenol-contaminated soil. Strain ZZ-1(T) was found to grow at 15-37 °C (optimum 25-30 °C), at pH 6.0-10.0 (optimum pH 7.5) and with 0-8.0% (w/v) NaCl (optimum 0.5%). The isolate was found to be able to reduce nitrate to nitrite, but not to nitrogen. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZZ-1(T) is a member of the genus Nitratireductor, and shows high sequence similarities to Nitratireductor pacificus MCCC 1A01024(T) (98.5%) and lower (<97%) sequence similarities to all other Nitratireductor species. Chemotaxonomic analysis revealed that strain ZZ-1(T) possesses Q-10 as the predominant ubiquinone and Summed feature 8(C(18:1) ω6c and/or C(18:1) ω7c; 66.6%), C(19:0) ω8c cyclo (23.3%), C(18:0) (3.4%), iso-C(17:0) (2.3%) and C(17:0) (1.0%) as the major fatty acids. The polar lipids of strain ZZ-1(T) were determined to be diphosphatidylglycerol, phosphatidylcholine, phospholipids, aminolipids, a glycolipid and an aminophospholipid. The DNA G+C content was determined to be 64.1 mol%. Based on the draft genome sequence, the DNA-DNA hybridization estimate value between strain ZZ-1(T) and N. pacificus MCCC 1A01024(T) was 46.5 ± 3.0% and ANI was 75.9 %. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization supports the conclusion that strain ZZ-1(T) represents a novel species of the genus Nitratireductor, for which the name Nitratireductor soli sp. nov. is proposed. The type strain is ZZ-1(T) (=JCM 30640(T) = MCCC 1K00508(T)).

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail.

Dimeric avidins are a newly discovered subgroup of the avidin family that bind biotin with high affinity. Their dimeric configuration is a quaternary substructure of the classical tetrameric avidins which lacks the requirement of the critical Trp that defines the tetramer and dictates the tenacious interaction with biotin. Hoefavidin, derived from the bacterium Hoeflea phototrophica DFL-43(T), is the third characterized member of the dimeric avidin subfamily. Like the other members of this group, hoefavidin is a thermostable protein that contains a disulfide bridge between Cys57 and Cys88, thereby connecting and stabilizing the L3,4 and L5,6 loops. This represents a distinctive characteristic of dimeric avidins that compensates for the lack of Trp and enables their dimeric configuration. The X-ray structure of the intact hoefavidin revealed unique crystal packing generated by an octameric cylindrical structure wherein the C-termini segments of each monomer is introduced into the entrance of the biotin-binding site of an adjacent non-canonical monomer. This anomaly in the protein structure served as a lead toward the design of specific binding peptides. We screened for specific hoefavidin binding peptides derived from the C-terminal region and two peptides were obtained that bind a truncated form of hoefavidin (lacking the last 10 amino acids) with dissociation constants of 10(-5)M. The crystal structure of short hoefavidin complexed with a C-terminal derived peptide revealed the mode of binding. These peptides may form the basis of novel and reversible binders for dimeric avidins.

The O-specific polysaccharides from Phyllobacterium trifolii PETP02(T) LPS contain 3-C-methyl-D-rhamnose.

The O-specific polysaccharides of Phyllobacterium trifolii PETP02(T), a microsymbiont of Trifolium pratense, were obtained by mild acid hydrolysis of the lipopolysaccharide and studied by chemical analyses, mass spectrometry, and (1)H and (13)C NMR spectroscopy, including homonuclear ((1)H,(1)H DQF-COSY, TOCSY, NOESY) and heteronuclear ((1)H,(13)C HSQC, HMQC, HMBC) experiments. It was revealed that α-D-rhamnose and ß-3-C-methyl-D-rhamnose (evalose) were the only components of two identified O-polysaccharides. The major O-polysaccharide was found to consist of linear hexasaccharide repeating units, while the other minor one, is composed of disaccharide repeats. The following structures of two O-polysaccharides were established: → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 → 3)-α-D-Rhap-(1 → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 → 2)-α-D-Rhap-(1 → and → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 →.

Arsenic transforming abilities of groundwater bacteria and the combined use of Aliihoeflea sp. strain 2WW and goethite in metalloid removal.

Several technologies have been developed for lowering arsenic in drinking waters below the World Health Organization limit of 10 µg/L. When in the presence of the reduced form of inorganic arsenic, i.e. arsenite, one options is pre-oxidation of arsenite to arsenate and adsorption on iron-based materials. Microbial oxidation of arsenite is considered a sustainable alternative to the chemical oxidants. In this contest, the present study investigates arsenic redox transformation abilities of bacterial strains in reductive groundwater from Lombardia (Italy), where arsenite was the main arsenic species. Twenty isolates were able to reduce 75 mg/L arsenate to arsenite, and they were affiliated to the genera Pseudomonas, Achromobacter and Rhodococcus and genes of the ars operon were detected. Three arsenite oxidizing strains were isolated: they belonged to Rhodococcus sp., Achromobacter sp. and Aliihoeflea sp., and aioA genes for arsenite oxidase were detected in Aliihoeflea sp. strain 2WW and in Achromobacter sp. strain 1L. Uninduced resting cells of strain 2WW were used in combination with goethite for arsenic removal in a model system, in order to test the feasibility of an arsenic removal process. In the presence of 200 µg/L arsenite, the combined 2WW-goethite system removed 95% of arsenic, thus lowering it to 8 µg/L. These results indicate that arsenite oxidation by strain 2WW combined to goethite adsorption is a promising approach for arsenic removal from contaminated groundwater.

The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase--rational design of mutants and the crystal structures of the A85H and W104Y variants.

Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected.