Relationships of Bradyrhizobium strains nodulating three Algerian Genista species.

The Mediterranean world is the cradle for the diversification of a large number of plant species, including legumes belonging to the Tribe Genisteae. Nodule bacteria from three species of Genista legumes indigenous to northwestern Africa (G. ferox, G. numidica, G. tricuspidata) were sampled across a 150km region of Algeria in order to investigate symbiotic relationships. Partial 23S rRNA sequences from 107 isolates indicated that Bradyrhizobium was the predominant symbiont genus (96% of isolates), with the remainder belonging to Rhizobium or Mesorhizobium. A multilocus sequence analysis on 46 Bradyrhizobium strains using seven housekeeping (HK) genes showed that strains were differentiated into multiple clades with affinities to seven species: B. canariense (17 isolates), B. japonicum (2), B. ottawaense (2), B. cytisi/B. rifense (9), 'B. valentinum' (5), and B. algeriense (11). Extensive discordance between the HK gene phylogeny and a tree for four loci in the symbiosis island (SI) region implied that horizontal transfer of SI loci has been common. Cases of close symbiont relationship across pairs of legumes hosts were evident, with 33% of isolates having as their closest relative a strain sampled from a different Genista species. Nevertheless, tree permutation tests also showed that there was substantial host-related phylogenetic clustering. Thus, each of the three Genista hosts utilized a measurably different array of bacterial lineages.

A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling.

Estrogen exerts extensive and diverse effects throughout the body of women. In addition to the classical nuclear estrogen receptors (ERα and ERß), the G protein-coupled estrogen receptor GPER is an important mediator of estrogen action. Existing ER-targeted therapeutic agents act as GPER agonists. Here, we report the identification of a small molecule, named AB-1, with the previously unidentified activity of high selectivity for binding classical ERs over GPER. AB-1 also possesses a unique functional activity profile as an agonist of transcriptional activity but an antagonist of rapid signaling through ERα. Our results define a class of small molecules that discriminate between the classical ERs and GPER, as well as between modes of signaling within the classical ERs. Such an activity profile, if developed into an ER antagonist, could represent an opportunity for the development of first-in-class nuclear hormone receptor-targeted therapeutics for breast cancer exhibiting reduced acquired and de novo resistance.

Author Correction: Parallels between experimental and natural evolution of legume symbionts.

Clémence Genthon and Céline Lopez-Roques, who performed sequencing, were inadvertently omitted from the author list. This has now been corrected in the PDF and HTML versions of the Article.

Parallels between experimental and natural evolution of legume symbionts.

The emergence of symbiotic interactions has been studied using population genomics in nature and experimental evolution in the laboratory, but the parallels between these processes remain unknown. Here we compare the emergence of rhizobia after the horizontal transfer of a symbiotic plasmid in natural populations of Cupriavidus taiwanensis, over 10 MY ago, with the experimental evolution of symbiotic Ralstonia solanacearum for a few hundred generations. In spite of major differences in terms of time span, environment, genetic background, and phenotypic achievement, both processes resulted in rapid genetic diversification dominated by purifying selection. We observe no adaptation in the plasmid carrying the genes responsible for the ecological transition. Instead, adaptation was associated with positive selection in a set of genes that led to the co-option of the same quorum-sensing system in both processes. Our results provide evidence for similarities in experimental and natural evolutionary transitions and highlight the potential of comparisons between both processes to understand symbiogenesis.

Diversifying selection by Desmodiinae legume species on Bradyrhizobium symbionts.

Desmodium and Hylodesmum (Papilionoideae Subtribe Desmodiinae) are among the most common herbaceous perennial legumes native to eastern North America. To analyze the population structure of their Bradyrhizobium sp. root-nodule bacteria, 159 isolates were sampled from ten host species across a 1000 km region. Phylogenetic analysis of four housekeeping loci (2164 bp) and two loci in the symbiosis island (SI) chromosomal region (1374 bp) indicated extensive overlap in symbiont utilization, with each common bacterial clade found on 2-7 species of these legume genera. However, host species differed considerably in the relative proportion of symbionts belonging to different Bradyrhizobium clades. High phylogenetic incongruence between trees for housekeeping loci and SI loci suggested that diversification of these Bradyrhizobium lineages involved substantial horizontal gene transfer. Plant inoculation with strains from six Bradyrhizobium clades revealed marked disparity in relative bacterial reproductive success across four Desmodium species. Estimated yield of Bradyrhizobium progeny cells per plant ranged from zero to >10(9), and strains with high fitness on one host sometimes reproduced poorly on other host species. Diversifying selection on bacteria, arising from differential success in habitats with different Desmodium and Hylodesmum taxa, is therefore likely to affect Bradyrhizobium diversity patterns at the landscape level.

A single sym plasmid type predominates across diverse chromosomal lineages of Cupriavidus nodule symbionts.

Cupriavidus nodule symbionts from Mimosa host legumes indigenous to five locations around the Caribbean region were analyzed by sequencing portions of five chromosomal housekeeping loci and five sym plasmid loci in 80 isolates. Nodule symbionts did not form a single clade separated from non-symbiotic reference strains of Cupriavidus and Ralstonia, implying that either convergent losses or independent gains of the trait of legume symbiosis have taken place. Chromosomal genes exhibited significantly higher nucleotide polymorphism and haplotype diversity than sym plasmid loci. A single derived sym plasmid haplotype (A1) was found to predominate in four of the populations, and was shared by multiple housekeeping gene clades. This suggests that one sym plasmid variant has recently spread geographically and has been acquired by diverse chromosomal lineages within the region. Inoculation of two Mimosa host species indicated that strains carrying the predominant A1 haplotype ranked either first or second among the five major sym plasmid haplotype groups with respect to plant growth enhancement. Symbiotic outcomes also varied greatly among chromosomally diverse strains that all shared the A1 haplotype. Thus, chromosomal as well as sym plasmid variants likely contribute to differential interactions with Mimosa host species.

The spread of Bradyrhizobium lineages across host legume clades: from Abarema to Zygia.

To analyze macroevolutionary patterns in host use by Bradyrhizobium root-nodule bacteria, 420 strains from 75 legume host genera (sampled in 25 countries) were characterized for portions of six housekeeping genes and the nifD locus in the symbiosis island chromosomal region. Most Bradyrhizobium clades utilized very divergent sets of legume hosts. This suggests that Bradyrhizobium spread across the major legume lineages early in its evolution, with only a few derived clades subsequently developing a narrower pattern of host use. Significant modularity existed in the network structure of recent host jumps (inferred from cases where closely related strain pairs were found on different legume taxa). This implies that recent host switching has occurred most often within particular subgroups of legumes. Nevertheless, the observed link structure would allow a bacterial lineage to reach almost any of the 75 legume host genera in a relatively small number of steps. However, permutation tests also showed that symbionts from certain host plant clades were significantly more similar than would be the case if bacteria were distributed at random on the trees. Related legumes thus harbored related sets of symbionts in some cases, indicating some degree of phylogenetic conservatism in partner selection.

Disparate origins of Bradyrhizobium symbionts for invasive populations of Cytisus scoparius (Leguminosae) in North America.

To identify the geographic origin of nodule bacteria associated with invasion of the European legume Cytisus scoparius in the United States, isolates from 15 sites in six states were compared to > 200 Bradyrhizobium strains from indigenous legumes in the U.S., Mexico, Europe (six countries), Morocco, and Australia. Portions of five housekeeping loci (2849 bp) were sequenced, along with the nifD locus in the symbiosis island (SI) portion of the Bradyrhizobium chromosome. Bayesian phylogenetic analysis showed that North American C. scoparius symbionts had highly heterogeneous ancestry. Some were grouped into three distinct clades of European C. scoparius symbionts. One isolate had both housekeeping and SI genes belonging to a Bradyrhizobium clade from native legumes in western North America. Two other clades had mosaic ancestry: sequences for nifD as well as two other SI genes (nifH, nodC) were highly similar or identical to a C. scoparius strain from Spain, while their housekeeping loci belonged to American Bradyrhizobium clades. Thus, it appears that bacteria ancestrally associated with other North American legumes have evolved to utilize C. scoparius, by acquiring SI-region genes from European C. scoparius symbionts. Inoculation assays indicated that North American isolates were as competent as European strains in promoting plant growth, consistent with the findings on symbiont ancestry.

Mosaic origins of Bradyrhizobium legume symbionts on the Caribbean island of Guadeloupe.

To analyze geographic affinities of Bradyrhizobium sp. symbionts associated with the diverse legume flora on the Caribbean island of Guadeloupe, 39 isolates from 18 legume genera were compared to a reference set of 269 Bradyrhizobium strains from North America, Central America, Puerto Rico and the Philippines. A multilocus sequence analysis (4192 bp) showed that nucleotide diversity in Guadeloupe equaled or exceeded that found in all other regional Bradyrhizobium populations examined. Bayesian phylogenetic tree analysis grouped the Guadeloupe Bradyrhizobium strains into clades with at least 20 distinct sets of non-Guadeloupe relatives, implying that the island was colonized numerous times from multiple source regions. However, for 18% of the Guadeloupe isolates, inferred geographic affinities for the nifD locus, in the symbiosis island region of the Bradyrhizobium chromosome, conflicted with the source region deduced from a tree based on six concatenated housekeeping genes. Geographic mosaic ancestry was therefore evident among Guadeloupe bradyrhizobia. Horizontal gene transfer subsequent to island colonization appears to have generated strains that carry combinations of genes from disparate source regions.

Crystal structure of a benzimidazole hepatitis C virus inhibitor free and in complex with the viral RNA target.

The crystal structure of 8-((dimethylamino)methyl)-1-(3-(dimethylamino)propyl)-1,7,8,9-tetrahydrochromeno[5,6-d]imidazol-2-amine (1), an inhibitor of the hepatitis C virus internal ribosome entry site, is described and compared to the structure of the compound in complex with the viral RNA target. Compound 1 crystallized by pentane vapor diffusion into dichloroethane solution. It crystallized in the monoclinic system, P21/c space group with unit cell parameters a = 15.7950(5) Å, b = 14.0128(4) Å, c = 8.8147(3) Å, ß = 94.357(2)° and a cell volume of 1945.34(11) A-3. Packing interactions in the small molecule crystal lattice correspond to key interactions of the compound with the viral RNA target.

New route to the ergoline skeleton via cyclization of 4-unsubstituted indoles.

A new route to the ergoline skeleton has been developed that does not require prior functionalization of the indole 4-position. The indole nucleus is introduced late in the synthesis to allow for eventual efficient introduction of substituents in this region. Key steps include Negishi coupling of a three-carbon chain to a bromonicotinate ester, Fischer indole synthesis to facilitate incorporation of substituents via phenylhydrazines, and Pd-catalyzed cyclization to form the ergoline C ring.

Phylogenetic clustering of Bradyrhizobium symbionts on legumes indigenous to North America.

To analyse determinants of biogeographic structure in members of the genus Bradyrhizobium, isolates were obtained from 41 legume genera, originating from North American sites spanning 48.5 ° of latitude (Alaska to Panama). Sequencing of portions of six gene loci (3674 bp) in 203 isolates showed that there was only a weak trend towards higher nucleotide diversity in tropical regions. Phylogenetic relationships for nifD, in the symbiosis island region of the Bradyrhizobium chromosome, conflicted substantially with a tree inferred for five housekeeping gene loci. For both nifD and housekeeping gene trees, bacteria from each region were significantly more similar, on average, than would be expected if the source location was permuted at random on the tree. Within-region permutation tests also showed that bacteria clustered significantly on particular host plant clades at all levels in the phylogeny of legumes (from genus up to subfamily). Nevertheless, some bacterial groups were dispersed across multiple regions and were associated with diverse legume host lineages. These results indicate that migration, horizontal gene transfer and host interactions have all influenced the geographical divergence of Bradyrhizobium populations on a continental scale.

Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches.

The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution. Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.

American origin of Cupriavidus bacteria associated with invasive Mimosa legumes in the Philippines.

To identify the origins of Cupriavidus nodule symbionts associated with two invasive Mimosa species in the Philippines, 22 isolates were sequenced for portions of three chromosomal genes and two symbiotic plasmid loci. Eleven isolates were identical at all gene loci (2713 bp) to a lineage found in Central America. Four other Philippine isolates were identical to a second Cupriavidus lineage distributed both in Central America and in the Caribbean. None of the remaining Philippine strains had more than 0.6% sequence divergence from American Cupriavidus lineages. These results imply that the Philippine population was founded by multiple introductions from the native range of their Mimosa hosts.

Legumes select symbiosis island sequence variants in Bradyrhizobium.

Bradyrhizobium strains sampled from 14 legume genera native to eastern North America showed substantial host-related phylogenetic clustering at three loci in the symbiotic island (SI) region (nodC, nifD, nifH), indicating selection of distinct suites of SI lineages by different legumes. Bacteria assorted consistently with particular legumes across two regions separated by 800 km, implying recurrent assembly of the same symbiotic combinations. High genetic polymorphism of all three SI loci relative to four nonsymbiotic loci supported the inference that a form of multiple-niche balancing selection has acted on the SI region, arising from differential symbiont utilization by different legume taxa. Extensive discordance between the tree for SI variants and a phylogenetic tree inferred for four housekeeping loci implied that lateral transfer of the symbiosis island region has been common (at least 26 transfer events among 85 Bradyrhizobium strains analysed). Patterns of linkage disequilibrium also supported the conclusion that recombination has impacted symbiotic and nonsymbiotic regions unequally. The high prevalence of lateral transfer suggests that acquisition of a novel SI variant may often confer a strong selective advantage for recipient cells.

Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with ≥ 96.1% sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFB(T), with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8% similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Microvirga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), WSM3557(T) and WSM3693(T) from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6(T) was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100% identical nodA sequences of WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), WSM3557(T) and WSM3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6(T) =LMG 26460(T) =HAMBI 3236(T)), Microvirga lotononidis sp. nov. (type strain WSM3557(T) =LMG 26455(T) =HAMBI 3237(T)) and Microvirga zambiensis sp. nov. (type strain WSM3693(T) =LMG 26454(T) =HAMBI 3238(T)).

An efficient new route to dihydropyranobenzimidazole inhibitors of HCV replication.

A class of dihydropyranobenzimidazole inhibitors was recently discovered that acts against the hepatitis C virus (HCV) in a new way, binding to the IRES-IIa subdomain of the highly conserved 5' untranslated region of the viral RNA and thus preventing the ribosome from initiating translation. However, the reported synthesis of these compounds is lengthy and low-yielding, the intermediates are troublesome to purify, and the route is poorly structured for the creation of libraries. We report a streamlined route to this class of inhibitors in which yields are far higher and most intermediates are crystalline. In addition, a key variable side chain is introduced late in the synthesis, allowing analogs to be easily synthesized for optimization of antiviral activity.

Halophilic Archaea cultured from ancient halite, Death Valley, California.

Halophilic Archaea cultured from ancient fluid inclusions in a 90-m-long (0- to 100,000-year-old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34,000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface-sterilized halite crystals exclusively in one section of the core (13.0-17.8 m; 22,000-34,000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1-86.7 m (10,000-100,000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra-laboratory reproducibility. Inter-laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.

The role of lipophilicity in determining binding affinity and functional activity for 5-HT2A receptor ligands.

The lipophilicity of a set of 5-HT(2A) ligands was determined using immobilized-artificial-membrane chromatography, a method that generates values well correlated with octanol-water partition coefficients. For agonists, a highly significant linear correlation was observed between binding affinity and lipophilicity. For ligands exhibiting partial agonist or antagonist properties, the lipophilicity was consistently higher than would be expected for an agonist of comparable affinity. The results suggest a possible method for distinguishing agonists from antagonists in high-throughput screening when a direct assay for functional activity is either unavailable or impractical.

Symbiotic relationships of legumes and nodule bacteria on Barro Colorado Island, Panama: a review.

Abstract New data on 129 bacterial isolates were analyzed together with prior samples to characterize community-level patterns of legume-rhizobial symbiosis on Barro Colorado Island (BCI), Panama. Nodules have been sampled from 24 BCI legume species in 18 genera, representing about one quarter of the legume species and one half of the genera on the island. Most BCI legumes associated exclusively with nodule symbionts in the genus Bradyrhizobium, which comprised 86.3% of all isolates (315 of 365). Most of the remaining isolates (44 of 365) belonged to the beta-proteobacterial genus Burkholderia; these were restricted to two genera in the legume subfamily Mimosoideae. Multilocus sequence analysis indicated that BCI Bradyrhizobium strains were differentiated into at least eight lineages with deoxyribonucleic acid divergence of the same magnitude as found among currently recognized species in this bacterial genus. Two of these lineages were widely distributed across BCI legumes. One lineage was utilized by 15 host species of diverse life form (herbs, lianas, and trees) in 12 genera spanning two legume subfamilies. A second common lineage closely related to the taxon B. elkanii was associated with at least five legume genera in four separate tribes. Thus, BCI legume species from diverse clades within the family frequently share interaction with a few common lineages of nodule symbionts. However, certain host species were associated with unique symbiont lineages that have not been found on other coexisting BCI legumes. More comprehensive sampling of host taxa will be needed to characterize the overall diversity of nodule bacteria and the patterns of symbiont sharing among legumes in this community.