Pseudopontixanthobacter vadosimaris gen. nov., sp. nov., isolated from shallow sea near Kueishan Island.

A Gram-stain-negative and aerobic bacterial strain, designated as JL3514T, was isolated from surface water of the hydrothermal system around Kueishan Island. The isolate formed red colonies and cells were non-flagellated, rod-shaped and contained methanol-soluble pigments. Growth was observed at 10-50 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and in the presence of 0-9 % (w/v) NaCl (optimum, 2 %). Strain JL3514T was positive for catalase and weakly positive for oxidase. Results of 16S rRNA gene sequence analyses showed highest similarities to species in the family Erythrobacteraceae, namely Croceibacterium atlanticum (96.1 %), Pelagerythrobacter marensis (96.0 %), Tsuneonella rigui (96.0 %) and Altericroceibacterium xinjiangense (96.0 %). Phylogenetic analysis based on core gene sequences revealed that the isolate formed a distinct branch with the related species and it had a lower average amino acid identity value than the suggested threshold for genera boundaries. The major fatty acids (>5 %) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, C17 : 1 ω6c, C14 : 0 2-OH and C12 : 0. The dominant polar lipids comprised diphosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, glycolipid, two unidentified lipids and one unidentified phospholipid. The main respiratory quinones were ubiquinone-10 (95.7 %) and ubiquinone-9 (4.3 %). The DNA G+C content from the genome was 63.0 mol%. Based on the presented data, we consider strain JL3514T to represent a novel genus of the family Erythrobacteraceae, with the name Pseudopontixanthobacter vadosimaris gen. nov., sp. nov. The type strain is JL3514T (=KCTC 62623T=MCCC 1K03561T).

From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

Protein corona formation and its influence on biomimetic magnetite nanoparticles.

Biomimetic magnetite nanoparticles (BMNPs) synthesized in the presence of MamC, a magnetosome-associated protein from Magnetoccus marinus MC-1, have gained interest for biomedical applications because of their unique magnetic properties. However, their behavior in biological systems, like their interaction with proteins, still has to be evaluated prior to their use in clinics. In this study, doxorubicin (DOXO) as a model drug was adsorbed onto BMNPs to form nanoassemblies. These were incubated with human plasma to trigger protein corona (PC) formation. Proteins from the human plasma stably attached to either BMNPs or DOXO-BMNP nanoassemblies. In particular, fibrinogen was detected as the main component in the PC of DOXO-BMNPs that potentially provides advantages, e.g. protecting the particles from phagocytosis, thus prolonging their circulation time. Adsorption of PC to the BMNPs did not alter their magnetic properties but improved their colloidal stability, thus reducing their toxicity in human macrophages. In addition, PC formation enhanced cellular internalization and did not interfere with DOXO activity. Overall, our data indicate that the adsorption of PC onto DOXO-BMNPs in biological environment even increases their efficiency as drug carrier systems.

Labrenzbactin from a coral-associated bacterium Labrenzia sp.

A new catecholate-containing siderophore, labrenzbactin (1), was isolated from the fermentation broth of a coral-associated bacterium Labrenzia sp. The structure and absolute configuration of 1 was determined by spectroscopic methods and Marfey's analysis. Overall, 1 showed antimicrobial activity against Ralstonia solanacearum SUPP1541 and Micrococcus luteus ATCC9341 with MIC values of 25 and 50 µg ml-1, respectively, and cytotoxicity against P388 murine leukemia cells with an IC50 of 13 µM.

Development of emulgels formulated with sweet fennel oil and rhamsan gum, a biological macromolecule produced by Sphingomonas.

In this work, a current application of rhamsan gum, a biological macromolecule which belongs to the sphingans group, as an efficient stabilizer of emulsions and emulgels is investigated. The main objective of this investigation was 1) to study the influence of glycerol and sweet fennel oil concentration on microfluidized emulsion properties; and 2) to develop stable emulgels stabilized with rhamsan gum. The emulsions and emulgels were characterized by droplet size, rheological properties, physical stability and microstructure. An analysis by surface response methodology of the results obtained revealed that essential oil concentration was the most determining factor affecting emulsion mean droplet sizes and rheological properties. An optimal emulsion with minimum d4,3 was obtained for the sample formulated with 10 wt% sweet fennel oil and 0 wt% glycerol. However, all of these emulsions suffered destabilization by creaming. The results of the rheological characterization of emulsions formulated with the biological macromolecule showed that the addition of 0.2 wt% of rhamsan gum allows an emulgel with enhanced physical stability to be obtained. Thus, we provide valuable information concerning the use of rhamsan gum as emulsion stabilizer and the development of stable emulsions and emulgels for use in the food industry.

pH-Dependent Adsorption Release of Doxorubicin on MamC-Biomimetic Magnetite Nanoparticles.

New biomimetic magnetite nanoparticles (hereafter BMNPs) with sizes larger than most common superparamagnetic nanoparticles were produced in the presence of the recombinant MamC protein from Magnetococcus marinus MC-1 and functionalized with doxorubicin (DOXO) intended as potential drug nanocarriers. Unlike inorganic magnetite nanoparticles, in BMNPs the MamC protein controls their size and morphology, providing them with magnetic properties consistent with a large magnetic moment per particle; moreover, it provides the nanoparticles with novel surface properties. BMNPs display the isoelectric point at pH 4.4, being strongly negatively charged at physiological pH (pH 7.4). This allows both (i) their functionalization with DOXO, which is positively charged at pH 7.4, and (ii) the stability of the DOXO-surface bond and DOXO release to be pH dependent and governed by electrostatic interactions. DOXO adsorption follows a Langmuir-Freundlich model, and the coupling of DOXO to BMNPs (binary biomimetic nanoparticles) is very stable at physiological pH (maximum release of 5% of the drug adsorbed). Conversely, when pH decreases, these electrostatic interactions weaken, and at pH 5, DOXO is released up to ∼35% of the amount initially adsorbed. The DOXO-BMNPs display cytotoxicity on the GTL-16 human gastric carcinoma cell line in a dose-dependent manner, reaching about ∼70% of mortality at the maximum amount tested, while the nonloaded BMNPs are fully cytocompatible. The present data suggest that BMNPs could be useful as potential drug nanocarriers with a drug adsorption-release governed by changes in local pH values.

Sulfated O-polysaccharide with anticancer activity from the marine bacterium Poseidonocella sedimentorum KMM 9023T.

The sulfated polysaccharides are of study interest due to their high structural diversity and broad spectrum of biological activity including antitumor properties. In this paper, we report on the structural analysis of sulfated O-specific polysaccharide (OPS) and in vitro anticancer activity of O-deacylated lipopolysaccharide (DPS) of the marine-derived bacterium Poseidonocella sedimentorum KMM 9023T achieved by a multidisciplinary approach (chemical analysis, NMR, MS, and bioassay). The OPS is shown to include two rare monosaccharide derivatives: 3-deoxy-9-O-methyl-d-glycero-d-galacto-non-2-ulosonic acid (Kdn9Me) and 3-O-acetyl-2-O-sulfate-d-glucuronic acid (D-GlcA2S3Ac). The structure of polysaccharide moiety of a previously unknown carbohydrate-containing biopolymer is established: →4)-α-Kdnp9Me-(2→4)-α-d-GlcpA2S3Ac-(1→. From a biological point of view, we demonstrate that DPS of the P. sedimentorum KMM 9023T has no cytotoxicity and inhibits colony formation of human HT-29, MCF-7 and SK-MEL-5 cells in a dose-dependent manner. The investigated polysaccharide is the second glycan isolated from the bacteria of the genus Poseidonocella: previously we studied the OPS of P. pacifica KMM 9010T (Kokoulin et al., 2017). Both polysaccharides are sulfated and contain rare residues of ulosonic acids. Thus, obtained findings provide a new knowledge about kinds and antitumor properties of sulfated polysaccharides and can be a starting point for further investigations of mechanisms of anticancer action of carbohydrate-containing biopolymers from marine Gram-negative bacteria.

Phenylobacterium terrae sp. nov., isolated from a soil sample of Khyber-Pakhtun-Khwa, Pakistan.

A Gram-stain negative, aerobic, motile, non-spore-forming and rod-shaped bacterial strain, designated YIM 730227T, was isolated from a soil sample, collected from Karak district, Khyber-Pakhtun-Khwa, Pakistan. The bacterium was characterized using a polyphasic taxonomic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strain YIM 730227T is closely related to Phenylobacterium lituiforme FaiI3T (97.5% sequence similarity), Phenylobacterium muchangponense A8T (97.4%), Phenylobacterium panacis DCY109T (97.1%), Phenylobacterium immobile ET (97.1%) and Phenylobacterium composti 4T-6T (97.0%), while also sharing 98.0% sequence similarity with Phenylobacterium hankyongense HKS-05T after NCBI blast, showing it represents a member of the family Caulobacteraceae. The major respiratory quinone was Q-10 and the major fatty acids were C16:0, summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c), C18:1ω7c 11-methyl and C17:0. The polar lipids were phosphatidylglycerol, unidentified glycolipids, phospholipid and unidentified lipid. The G + C content of the genomic DNA was 68.2 mol%. The DNA-DNA relatedness values of strain YIM 730227T with P. hankyongense HKS-05T, P. lituiforme FaiI3T, P. muchangponense A8T, P. panacis DCY109T, P. immobile ET and P. composti 4T-6T were 31.3 ± 0.6, 26.1 ± 0.2, 24.3 ± 0.1, 21.8 ± 0.9, 19.8 ± 0.6 and 18.2 ± 1.1%, respectively, values lower than 70%. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of 16S rRNA gene sequences and the biochemical characteristics indicated that the strain YIM 730227T represents a novel member of the genus Phenylobacterium, for which the name Phenylobacterium terrae sp. nov. (type strain YIM 730227T = KCTC62324T = CGMCC 1.16326T) is proposed.

Chemical analysis of the Alphaproteobacterium strain MOLA1416 associated with the marine lichen Lichina pygmaea.

Alphaproteobacterium strain MOLA1416, related to Mycoplana ramosa DSM 7292 and Chelativorans intermedius CC-MHSW-5 (93.6% 16S rRNA sequence identity) was isolated from the marine lichen, Lichina pygmaea and its chemical composition was characterized by a metabolomic network analysis using LC-MS/MS data. Twenty-five putative different compounds were revealed using a dereplication workflow based on MS/MS signatures available through GNPS (https://gnps.ucsd.edu/). In total, ten chemical families were highlighted including isocoumarins, macrolactones, erythrinan alkaloids, prodiginines, isoflavones, cyclohexane-diones, sterols, diketopiperazines, amino-acids and most likely glucocorticoids. Among those compounds, two known metabolites (13 and 26) were isolated and structurally identified and metabolite 26 showed a high cytotoxic activity against B16 melanoma cell lines with an IC50 0.6 ± 0.07 µg/mL.

Molecular basis of ion permeability in a voltage-gated sodium channel.

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) â‰« K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase.

Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.

Sub-micrometer-scale mapping of magnetite crystals and sulfur globules in magnetotactic bacteria using confocal Raman micro-spectrometry.

The ferrimagnetic mineral magnetite Fe3O4 is biomineralized by magnetotactic microorganisms and a diverse range of animals. Here we demonstrate that confocal Raman microscopy can be used to visualize chains of magnetite crystals in magnetotactic bacteria, even though magnetite is a poor Raman scatterer and in bacteria occurs in typical grain sizes of only 35-120 nm, well below the diffraction-limited optical resolution. When using long integration times together with low laser power (<0.25 mW) to prevent laser induced damage of magnetite, we can identify and map magnetite by its characteristic Raman spectrum (303, 535, 665 cm(-1)) against a large autofluorescence background in our natural magnetotactic bacteria samples. While greigite (cubic Fe3S4; Raman lines of 253 and 351 cm(-1)) is often found in the Deltaproteobacteria class, it is not present in our samples. In intracellular sulfur globules of Candidatus Magnetobacterium bavaricum (Nitrospirae), we identified the sole presence of cyclo-octasulfur (S8: 151, 219, 467 cm(-1)), using green (532 nm), red (638 nm) and near-infrared excitation (785 nm). The Raman-spectra of phosphorous-rich intracellular accumulations point to orthophosphate in magnetic vibrios and to polyphosphate in magnetic cocci. Under green excitation, the cell envelopes are dominated by the resonant Raman lines of the heme cofactor of the b or c-type cytochrome, which can be used as a strong marker for label-free live-cell imaging of bacterial cytoplasmic membranes, as well as an indicator for the redox state.

Magnetic properties of uncultivated magnetotactic bacteria and their contribution to a stratified estuary iron cycle.

Of the two nanocrystal (magnetosome) compositions biosynthesized by magnetotactic bacteria (MTB), the magnetic properties of magnetite magnetosomes have been extensively studied using widely available cultures, while those of greigite magnetosomes remain poorly known. Here we have collected uncultivated magnetite- and greigite-producing MTB to determine their magnetic coercivity distribution and ferromagnetic resonance (FMR) spectra and to assess the MTB-associated iron flux. We find that compared with magnetite-producing MTB cultures, FMR spectra of uncultivated MTB are characterized by a wider empirical parameter range, thus complicating the use of FMR for fossilized magnetosome (magnetofossil) detection. Furthermore, in stark contrast to putative Neogene greigite magnetofossil records, the coercivity distributions for greigite-producing MTB are fundamentally left-skewed with a lower median. Lastly, a comparison between the MTB-associated iron flux in the investigated estuary and the pyritic-Fe flux in the Black Sea suggests MTB play an important, but heretofore overlooked role in euxinic marine system iron cycle.

NMR structures of α-proteobacterial ATPase-regulating ζ-subunits.

NMR structures of ζ-subunits, which are recently discovered α-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ζ-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ζ-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in α-proteobacteria.

The structural diversity of carbohydrate antigens of selected gram-negative marine bacteria.

Marine microorganisms have evolved for millions of years to survive in the environments characterized by one or more extreme physical or chemical parameters, e.g., high pressure, low temperature or high salinity. Marine bacteria have the ability to produce a range of biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents, and as a result, they have been a topic of research interest for many years. Among these biologically active molecules, the carbohydrate antigens, lipopolysaccharides (LPSs, O-antigens) found in cell walls of gram-negative marine bacteria, show great potential as candidates in the development of drugs to prevent septic shock due to their low virulence. The structural diversity of LPSs is thought to be a reflection of the ability for these bacteria to adapt to an array of habitats, protecting the cell from being compromised by exposure to harsh environmental stress factors. Over the last few years, the variety of structures of core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been discovered. In this review, we discuss the most recently encountered structures that have been identified from bacteria belonging to the genera Aeromonas, Alteromonas, Idiomarina, Microbulbifer, Pseudoalteromonas, Plesiomonas and Shewanella of the Gammaproteobacteria phylum; Sulfitobacter and Loktanella of the Alphaproteobactera phylum and to the genera Arenibacter, Cellulophaga, Chryseobacterium, Flavobacterium, Flexibacter of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention is paid to the particular chemical features of the LPSs, such as the monosaccharide type, non-sugar substituents and phosphate groups, together with some of the typifying traits of LPSs obtained from marine bacteria. A possible correlation is then made between such features and the environmental adaptations undertaken by marine bacteria.

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria.

Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to SAM-I aptamers. In this article, we describe a fifth class of SAM binding aptamer, which we have termed SAM-V. SAM-V was discovered by analyzing GC-rich intergenic regions preceding metabolic genes in the marine alpha-proteobacterium "Candidatus Pelagibacter ubique." Although the motif is nearly unrepresented in cultured bacteria whose genomes have been completely sequenced, SAM-V is prevalent in marine metagenomic sequences. The consensus sequence and structure of SAM-V show some similarities to that of the SAM-II riboswitch, and it is likely that the two aptamers form similar ligand binding pockets. In addition, we identified numerous examples of a tandem SAM-II/SAM-V aptamer architecture. In this arrangement, the SAM-II aptamer is always positioned 5' of the SAM-V aptamer and the SAM-II aptamer is followed by a predicted intrinsic transcription terminator stem. The SAM-V aptamer, however, appears to use a ribosome binding site occlusion mechanism for genetic regulation. This tandem riboswitch arrangement exhibits an architecture that can potentially control both the transcriptional and translational stages of gene expression.

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide-sensitive K(+) channel from Mesorhizobium loti. The protein consists of a wide anti-parallel beta-roll topped by a helical bundle comprising five alpha-helices and a short 3(10)-helix. In contrast to the dimeric arrangement ('dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains.

The recent characterization of the prokaryotic Cys(2)His(2) zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified approximately 300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys(2)His(2) zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros(56-142)C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys(2)His(2) coordination, in Ros homologues can either exploit a CysAspHis(2) coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.

Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov., isolated from wild legumes growing in desert soils of Xinjiang, China.

Twenty-four Mesorhizobium strains were isolated from desert soils in the Xinjiang region of China and were characterized by a polyphasic approach. These strains grouped into three clusters in IGS-RFLP, SDS-PAGE analysis of whole-cell proteins and BOX-PCR analysis, corresponding to genomic species V, VI and VII as found in a previous study. The results were supported by sequencing analyses of rrs, IGS, atpD and recA genes. Genospecies VII was most related to Mesorhizobium septentrionale, while genospecies V and VI were both most closely related to Mesorhizobium tianshanense, but were distinct from each other and from M. tianshanense. The DNA-DNA hybridization value between the representative strain CCBAU 83284 (genospecies VII) and the type strain of M. septentrionale was 90.1 %. Genospecies VII was thus defined as M. septentrionale. The DNA-DNA relatedness value for representative strains of genospecies V or VI with the related reference strains of recognized species were always lower than 60 %. Low values of DNA-DNA hybridization (32.79 %) between representative strains of genospecies V (CCBAU 83330(T)) and of VI (CCBAU 83306(T)) were also observed. Based upon these results, two novel species are proposed: Mesorhizobium gobiense sp. nov. represented by genospecies V (type strain, CCBAU 83330(T)=LMG 23949(T)=HAMBI 2974(T)) and Mesorhizobium tarimense sp. nov. represented by genospecies VI (type strain, CCBAU 83306(T)=LMG 24338(T)=HAMBI 2973(T)). Strain CCBAU 83278 grouped as the most peripheral member with genospecies VI in SDS-PAGE of whole-cell proteins and BOX-PCR analysis and in the phylogenetic tree of 16S-23S rRNA intergenic spacer (IGS) sequences. The results of analyses of rrs, atpD and recA gene sequences, as well as those of DNA-DNA hybridization studies, strongly supported the suggestion that this strain belonged to a species quite different from genospecies V and VI and from any other recognized species of the genus Mesorhizobium. As only one strain has been isolated to date, strain CCBAU 83278 was not proposed as a novel species in this study. Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov. could be differentiated from each other as well as from recognized species of the genus Mesorhizobium on the basis of phenotypic characteristics. The symbiotic loci (nodC and nifH) of the two novel species formed two phylogenetic branches related to Mesorhizobium loti and M. tianshanense. The type strains of the two novel species were able to nodulate Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra and Robinia pseudoacacia but not Astragalus membranaceus, Leucaena leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.

Labrys okinawensis sp. nov. and Labrys miyagiensis sp. nov., budding bacteria isolated from rhizosphere habitats in Japan, and emended descriptions of the genus Labrys and Labrys monachus.

Three strains, MAFF 210191(T), G24103(T) and G24116, assumed to be members of two novel species, were isolated from several rhizosphere habitats in different parts of Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates formed a distinct monophyletic group together with the two known species of the genus Labrys, suggesting that the isolates have a close affiliation with this genus. In this study, a polyphasic approach was used to characterize and compare the three isolates with the two species of the genus Labrys, Labrys monachus and Labrys methylaminiphilus. All three isolates were aerobic, Gram-negative, motile and non-sporulating and they ranged in shape from spherical to short rods. The cells multiplied by budding and utilized a wide variety of monosaccharides, disaccharides and sugar alcohols as sole carbon and energy sources, but they did not utilize C(1) compounds, salicin or d-melezitose. The strains were inhibited by dl-alpha-alanine and glycine (both at 10 mM). The major cellular fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 0) and C(18 : 1)omega7c. The three isolates shared <12 % and <11 % DNA-DNA relatedness with L. monachus DSM 5896(T) and L. methylaminiphilus DSM 16812(T), respectively. The G+C content of the isolates (61-62 mol%) was also significantly lower than those of the two previously characterized species. In spite of many morphological, physiological and chemotaxonomic similarities among the three isolates, strain MAFF 210191(T) could be differentiated from strains G24103(T) and G24116 on the basis of 16S rRNA gene sequence divergence, DNA-DNA relatedness (<46 %) and gelatin hydrolysis. Two novel species are therefore proposed, namely Labrys okinawensis sp. nov., with the type strain MAFF 210191(T) (=DSM 18385(T)), and Labrys miyagiensis sp. nov., with the type strain G24103(T) (=NBRC 101365(T)=NCIMB 14143(T)) and also including strain G24116 (=NBRC 101366=NCIMB 14144). Emended descriptions of the genus Labrys and Labrys monachus are also presented.