Identification and characterization of VapBC toxin-antitoxin system in Bosea sp. PAMC 26642 isolated from Arctic lichens.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Microvirga terrestris sp. nov., and Microvirga arvi sp. nov., isolated from soil in South Korea.

Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290T and BT689T were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290T and BT689T were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290T and BT689T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rR NA gene sequence similarity between two strains was 97.9%. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290T and BT689T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT290T were C18:1 ω7c (58.2%) and C16:0 (17.7%), while those of strain BT689T were C18:1 ω7c (61.8%) and C16:0 (10.8%). On the bases of polyphasic analysis (phylogenetic, chemotaxonomic, and biochemical), strains BT290T and BT689T can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290T (= KCTC 72367T = NBRC 114844T) and the type strain of Microvirga arvi is BT689T (= KACC 22016T = NBRC 114858T), respectively.

Microvirga terricola sp. nov. and Microvirga solisilvae sp. nov, isolated from forest soil.

Two Gram-staining-negative, aerobic and rod-shaped strains, designated c23x22T and sex2T, were isolated from forest soil collected from Chebaling National Nature Reserve in Guangdong Province and Limu Mountain National Forest Park in Hainan Province, P. R. China, respectively. Phylogenetic analyses based on 16S rRNA gene sequences revealed that they belonged to the genus Microvirga, and strain c23 x22T was most closely related to 'Microvirga alba' KCTC 72385, while strain sex2T showed close relationship with Microvirga guangxiensis CGMCC 1.7666T. The average nucleotide identity and digital DNA-DNA hybridization values between strains c23 x22T and sex2T and their close relatives, 'M. alba' KCTC 72385 and M. guangxiensis CGMCC 1.7666T, were all below the threshold values for species delimitation. The predominant quinones of the two novel strains were ubiquinone 10, and the major fatty acids contained C19:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Their predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The phenotypic, genotypic and chemotaxonomic analyses clearly supported that strains c23 x 22T and sex2T represent two novel species of the genus Microvirga, for which the name Microvirga terricola sp. nov. (type strain c23 x 22T = GDMCC 1.1700T = KCTC 62432T) and Microvirga solisilvae sp. nov. (type strain sex2T = GDMCC 1.1651T = KACC 21311T) are proposed, respectively.

Microvirga puerhi sp. nov., isolated from Puerh tea garden soil.

Strain WGZ8T was isolated from a soil sample of Puerh tea garden in Pu'er city, Southwest China. The isolate was rod-shaped, Gram-stain negative, facultative anaerobic, non-motile. Growth occurred within 0-3.0% (w/v) NaCl (optimal concentration, 0-1.0%), pH 5.0-11.0 (optimal pH, 7.0) and 10-40 °C (optimal temperature, 28 °C). 16S rRNA gene sequence-based phylogenetic and phylogenomic analysis revealed that WGZ8T belonged to the genus Microvirga. Its major cellular fatty acids were C19:0 cyclo ω8c, C16:0, C18:1ω7c and/or C18:1ω6c. The profile of polar lipids included phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and phosphatidylglycerol. The only respiratory quinone was detected as ubiquinone 10 (Q-10). The genome size of strain WGZ8T was 5.17 MB, and the content of DNA G + C was 61 mol%. Based on the results of digital DNA-DNA hybridization and phenotypic results, strain WGZ8T could be concluded as a novel species of the genus Microvirga, for which the name Microvirga puerhi sp. nov. is proposed. The type strain is WGZ8T (= CGMCC 1.19171 T = JCM 35317 T).

Microvirga roseola sp. nov. and Microvirga lenta sp. nov., isolated from Taklamakan Desert soil.

Two Gram-negative, rod-shaped, non-spore-forming bacteria, designated SM9T and SM2T, were isolated from Taklamakan Desert soil samples. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strains SM9T and SM2T had the highest sequence similarity to the type strains Microvirga indica BCRC 80972T and Microvirga soli NBRC 112417T with similarity values of 98.2 and 97.7 %, respectively, and Microvirga was among the predominant genera in the desert soil. The draft genomes of these two strains were 4.56 Mbp (SM9T) and 5.08 Mbp (SM2T) long with 65.1 mol% (SM9T) and 63.5 mol% (SM2T) G+C content. To adapt to the desert environment, these two strains possessed pathways for the synthesis of stress metabolite trehalose. The major fatty acids (>5 %) included C18 : 1 ω9c in SM2T, but C16 : 0, C18 : 0 and C19 : 0 cyclo ω8c in SM9T, while the major menaquinone was ubiquinone 10 in both strains. The major polar lipids of SM9T and SM2T were phosphatidylglycerol, phosphatidylethanolamine and phospholipid. The average nucleotide identity and digital DNA-DNA hybridization results further indicated that strains SM9T and SM2T were distinguished from phylogenetically related species and represented two novel species within the genus Microvirga, for which the names Microvirga roseola sp. nov. (type strain SM2T=KCTC 72792T=CGMCC 1.17776T) and Microvirga lenta sp. nov. (type strain SM9T=KCTC 82729T=CCTCC AB 2021131T) are proposed.

Screening and Comparison of Lignin Degradation Microbial Consortia from Wooden Antiques.

Lignin, which is a component of wood, is difficult to degrade in nature. However, serious decay caused by microbial consortia can happen to wooden antiques during the preservation process. This study successfully screened four microbial consortia with lignin degradation capabilities (J-1, J-6, J-8 and J-15) from decayed wooden antiques. Their compositions were identified by genomic sequencing, while the degradation products were analyzed by GC-MS. The lignin degradation efficiency of J-6 reached 54% after 48 h with an initial lignin concentration of 0.5 g/L at pH 4 and rotation speed of 200 rpm. The fungal consortium of J-6 contained Saccharomycetales (98.92%) and Ascomycota (0.56%), which accounted for 31% of the total biomass. The main bacteria in J-6 were Shinella sp. (47.38%), Cupriavidus sp. (29.84%), and Bosea sp. (7.96%). The strongest degradation performance of J-6 corresponded to its composition, where Saccharomycetales likely adapted to the system and improved lignin degradation enzymes activities, and the abundant bacterial consortium accelerated lignin decomposition. Our work demonstrated the potential utilization of microbial consortia via the synergy of microbial consortia, which may overcome the shortcomings of traditional lignin biodegradation when using a single strain, and the potential use of J-6 for lignin degradation/removal applications.

Rhizobial Microsymbionts of Kamchatka Oxytropis Species Possess Genes of the Type III and VI Secretion Systems, Which Can Affect the Development of Symbiosis.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.).

Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.

Lipid A from Oligotropha carboxidovorans Lipopolysaccharide That Contains Two Galacturonic Acid Residues in the Backbone and Malic Acid A Tertiary Acyl Substituent.

The free-living Gram-negative bacterium Oligotropha carboxidovorans (formerly: Pseudomonas carboxydovorans), isolated from wastewater, is able to live in aerobic and, facultatively, in autotrophic conditions, utilizing carbon monoxide or hydrogen as a source of energy. The structure of O. carboxidovorans lipid A, a hydrophobic part of lipopolysaccharide, was studied using NMR spectroscopy and high-resolution mass spectrometry (MALDI-ToF MS) techniques. It was demonstrated that the lipid A backbone is composed of two d-GlcpN3N residues connected by a ß-(1→6) glycosidic linkage, substituted by galacturonic acids (d-GalpA) at C-1 and C-4' positions. Both diaminosugars are symmetrically substituted by 3-hydroxy fatty acids (12:0(3-OH) and 18:0(3-OH)). Ester-linked secondary acyl residues (i.e., 18:0, and 26:0(25-OH) and a small amount of 28:0(27-OH)) are located in the distal part of lipid A. These very long-chain hydroxylated fatty acids (VLCFAs) were found to be almost totally esterified at the (ω-1)-OH position with malic acid. Similarities between the lipid A of O. carboxidovorans and Mesorhizobium loti, Rhizobium leguminosarum, Caulobacter crescentus as well as Aquifex pyrophylus were observed and discussed from the perspective of the genomic context of these bacteria.

AidB, a Novel Thermostable N-Acylhomoserine Lactonase from the Bacterium Bosea sp.

Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. strain F3-2. Ultrahigh-performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone (HSL) ring. AidB was thermostable in the range 30 to 80°C and showed maximum activity after preincubation at 60°C for 30 min. The optimum temperature of AidB was 60°C, and the enzyme could be stably stored in double-distilled water (ddH2O) at 4°C or room temperature. AidB homologs were found only in Rhizobiales and Rhodospirillales of the Alphaproteobacteria AidB from Agrobacterium tumefaciens and AidB from Rhizobium multihospitium (with amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.IMPORTANCE A quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase, AidB, from a soil bacterial strain, Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum The homologs of AidB with AHL-degrading activities were found only in several genera belonging to the Alphaproteobacteria Remarkably, AidB is a thermostable enzyme that retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4°C and room temperature. These properties might make it more suitable for practical application.

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.

Microvirga soli sp. nov., an alphaproteobacterium isolated from soil.

An aerobic, Gram-stain-negative, catalase-positive and oxidase-negative, non-motile, non-spore-forming, rod-shaped, pink-pigmented bacterium designated strain R491T was isolated from soil. Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain R491T formed a lineage within the family Methylobacteriaceae of the phylum Proteobacteria that was distinct from various species of the genus Microvirga, including Microvirgaaerilata 5420S-16T (97.83 % 16S rRNA gene sequence similarity), Microvirga zambiensis WSM3693T (97.76 %), Microvirga flocculans ATCC BAA-817T (97.41 %), and Microvirga lupini Lut6T, Microvirga subterranea DSM 14364T, Microvirga vignae BR3299T, and Microvirga guangxiensis 25BT (96.99 %). The major polar lipids of strain R491T were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 71.2 %), C16 : 0 (12.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 4.7 %), and C18 : 0 (4.2 %). The DNA G+C content of strain R491T was 61.8 mol%. DNA-DNA hybridization values between strain R491T and other members of the genus Microvirga ranged from 27 to 57 %. On the basis of phenotypic, genotypic and phylogenetic analyses, strain R491T represents a novel species of the genus Microvirga, for which the name Microvirga soli sp. nov. is proposed. The type strain is R491T (=KEMB 9005-408T=KACC 18969T=NBRC 112417T).

Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria-rich microbiome.

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of Elaphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the Elaphomyces lineage is most closely related to Talaromyces and Trichocomaceae s.s. The genome of E. granulatus is reduced in carbohydrate-active enzymes, despite a large expansion in genome size, both of which are consistent with what is seen in Tuber melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E. granulatus genome, especially Gypsy-like long terminal repeats, and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue, this study has provided insights into Elaphomyces phylogenetics, genomics, metagenomics and the evolution of the ectomycorrhizal association.

Extra-slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Bosea vaviloviae sp. nov., a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia formosa (Stev.) Fed.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant.

The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L(-1) Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L(-1); and biomass dose, 2.0 g·L(-1). Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes.

Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus), average-sized genomes (Bacillus, Enterobacteriaceae), and large genomes (Pseudomonas, Bradyrhizobiaceae) to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

Phylogeny and photoheterotrophy in the acidophilic phototrophic purple bacterium Rhodoblastus acidophilus.

Norbert Pfennig isolated the first acidophilic purple bacterium over 40 years ago and named the organism Rhodopseudomonas acidophila (now Rhodoblastusacidophilus). Since the original work of Pfennig, no systematic study has been conducted on the phylogeny and carbon nutrition of a collection of strains of Rbl. acidophilus. We have isolated six new strains of Rbl. acidophilus from a Canadian peat bog. These strains, three of the original Pfennig strains and two additional putative R. acidophilus strains isolated several years ago in this laboratory,were characterized as to their pigments, phylogeny, and carbon sources supporting photoheterotrophic growth. Phototrophic cultures were either purple or orange in color,and the color of a particular strain was linked to phylogeny. As for the Pfennig strains of Rbl. acidophilus, all new strains grew photoheterotrophically at pH 5 on a variety of organic and fatty acids. However, in addition to methanol and ethanol, the new strains as well as the Pfennig strains grew on several other primary alcohols, results not reported in the original species description. Our work shows that some phylogenetic and physiological diversity exists within the species Rbl. acidophilus and supports the observation that few species of acidophilic purple bacteria appear to exist in nature.

Multilocus sequence analysis of Bosea species and description of Bosea lupini sp. nov., Bosea lathyri sp. nov. and Bosea robiniae sp. nov., isolated from legumes.

Gram-negative, rod-shaped bacteria were isolated from root nodules of Lupinus polyphyllus, Lathyrus latifolius and Robinia pseudoacacia. Based on the 16S rRNA gene phylogeny, they were closely related to Bosea species (100-97 % similarity), belonging to the class Alphaproteobacteria, family Bradyrhizobiaceae. The closest relatives of LMG 26383(T), LMG 26379(T) and LMG 26381(T) were respectively the type strains of Bosea thiooxidans (99.6 %), B. eneae (98.3 %) and B. minatitlanensis (99.0 %). Chemotaxonomic data, including major fatty acid profiles, supported the assignment of our strains to the genus Bosea. Analysis of the concatenated sequences of five housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) and the results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from each other and from the five Bosea species with validly published names. No nodA or nodC genes could be amplified, while nifH PCR gave non-specific products. On the basis of genotypic and phenotypic data, three novel species, Bosea lupini sp. nov. (type strain LMG 26383(T)  = CCUG 61248(T)  = R-45681(T)), Bosea lathyri sp. nov. (type strain LMG 26379(T)  = CCUG 61247(T)  = R-46060(T)) and Bosea robiniae sp. nov. (type strain LMG 26381(T)  = CCUG 61249(T)  = R-46070(T)), are proposed.