Distinctive Mesorhizobium populations associated with Cicer arietinum L. in alkaline soils of Xinjiang, China

Background and aims

Rhizobia associated with chickpea in the main chickpea production zone of Xinjiang, China have never been investigated. Here, we present the first systematic investigation of these rhizobia’s genetic diversity and symbiotic interactions with their host plant.

Methods

Ninety-five isolates obtained from chickpea nodules in eight alkaline-saline (pH?8.24–8.45) sites in Xinjiang were characterized by nodulation test, symbiotic gene analysis, PCR-based restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and 16S–23S rRNA intergenic spacer (IGS), BOX-PCR, phylogenies of 16S rRNA and housekeeping genes (atpD, recA and glnII), multilocus sequence analysis (MLSA) and DNA–DNA hybridization.

Results

All 95 isolates were identified within the genus of Mesorhizobium. Similarities less than 96.5% in MLSA and DNA–DNA hybridization values (<50%) between the new isolates and the defined Mesorhizobium species, and high similarities (>98%) of symbiotic genes (nodC and nifH) with those of the well studied chickpea microsymbioints Mesorhizobium ciceri and Mesorhizobium mediterraneum were found.

Conclusions

Chickpea rhizobia in alkaline-saline soils of Xinjiang, China, form a population distinct from the defined Mesorhizobium species. All these chickpea rhizobia in Xinjiang harbored symbiotic genes highly similar to the type strains of two well-studied chickpea rhizobia, M. ciceri and M. mediterraneum, evidencing the possible lateral transfer of symbiotic genes among these different rhizobial species. On the other hand, chickpea may strongly select rhizobia with a unique symbiotic gene background.     

Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal

Background and Aims

This study was conducted to reveal the genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in various agroecological regions in Nepal.

Method

A total of 63 strains were isolated from common bean grown in the soils collected from seven bean fields in Nepal and characterized based on the partial sequences of 16S–23S internal transcribed spacer (ITS) regions, 16S rDNA, nodC, and nifH. Symbiotic properties of some representative strains with host plants were examined to elucidate their characteristics in relation to genotype and their origin.

Results

The isolated strains belonged to Rhizobium leguminosarum, Rhizobium etli, Rhizobium phaseoli, and one unknown Rhizobium lineage, all belonging to a common symbiovar (sv.) phaseoli. Nine ITS genotypes were detected mainly corresponding to a single site, including a dominant group at three sites harboring highly diverse multiple ITS sequences. Three symbiotic genotypes corresponded to a geographical region, not to the ribosomal DNA group, suggesting horizontal transfer of symbiotic genes separately in each region. Great differences in nitrogenase activity and nodule forming ability among the strains irrespective of their species and origin were observed.

Conclusions

Nepalese Himalaya harbor phylogenetically highly diverse and site-specific strains of common bean rhizobia, some of which could have high potential of symbiotic nitrogen fixation.     

Plasmids impact on rhizobia-legumes symbiosis in diverse environments

Rhizobia are a well-known group of soil bacteria that establish symbiotic relationship with leguminous plants, fix atmospheric nitrogen, and improve soil fertility. To fulfill multiple duties in soil, rhizobia are elaborated with a large and complex multipartite genome composed of several replicons. The genetic material is divided among various replicons, in a way to cope with, and satisfy the diverse functions of rhizobia. In addition to the main chromosome, which is carrying the essential (core) genes required for sustaining cell life, the rhizobia genomes contain several extra-chromosomal plasmids, carrying the nonessential (accessory) genes. Occasionally, some mega-plasmids, denoted as secondary chromosomes or chromids, carry some essential (core) genes. Furthermore, specific accessory gene sequences (the symbiotic chromosomal islands) are incorporated in the main chromosome of some rhizobia species in Bradyrhizobium and Mesorhizobium genera. Plasmids in rhizobia are of variable sizes. All of the plasmids in a Rhizobium cell constitute about 30–50% of the genome. Rhizobia plasmids have specific characters such as miscellaneous genes, independent replication system, self-transmissibility, and instability. The plasmids regulate several cellular metabolic functions and enable the host rhizobia to survive in diverse habitats and even under stress conditions. Symbiotic plasmids in rhizobia are receiving increased attention because of their significance in the symbiotic nitrogen fixation process. They carry the symbiotic (nod, nif and fix) genes, and some non-symbiotic genes. Symbiotic plasmids are conjugally-transferred by the aid of the non-symbiotic, self-transmissible plasmids, and hence, brings about major changes in the symbiotic interactions and host specificity of rhizobia. Besides, the rhizobia cells harbor one or more accessory, non-symbiotic plasmids, carrying genes regulating various metabolic functions, rhizosphere colonization, and nodulation competitiveness. The entire rhizobia-plasmid pool interacting in harmony and provides rhizobia with substantial abilities to fulfill their complex symbiotic and non-symbiotic functions in variable environments. The above concepts are extensively reviewed and fairly discussed.     

The dog and rat olfactory receptor repertoires

Background

Dogs and rats have a highly developed capability to detect and identify odorant molecules, even at minute concentrations. Previous analyses have shown that the olfactory receptors (ORs) that specifically bind odorant molecules are encoded by the largest gene family sequenced in mammals so far.

Results

We identified five amino acid patterns characteristic of ORs in the recently sequenced boxer dog and brown Norway rat genomes. Using these patterns, we retrieved 1,094 dog genes and 1,493 rat genes from these shotgun sequences. The retrieved sequences constitute the olfactory receptor repertoires of these two animals. Subsets of 20.3% (for the dog) and 19.5% (for the rat) of these genes were annotated as pseudogenes as they had one or several mutations interrupting their open reading frames. We performed phylogenetic studies and organized these two repertoires into classes, families and subfamilies.

Conclusion

We have established a complete or almost complete list of OR genes in the dog and the rat and have compared the sequences of these genes within and between the two species. Our results provide insight into the evolutionary development of these genes and the local amplifications that have led to the specific amplification of many subfamilies. We have also compared the human and rat ORs with the human and mouse OR repertoires.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

Enhanced nodulation and symbiotic effectiveness of Medicago truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer (Sinorhizobium) medicae WSM419

Aims

Low numbers of rhizobia in soil or inoculants delay nodulation and decrease symbiotic legume productivity. This study investigated the effect of co-inoculation with a helper bacterium, Pseudomonas fluorescens WSM3457 on the Medicago truncatula - Ensifer (Sinorhizobium) medicae WSM419 symbiosis challenged by a low inoculum dose.

Methods

In a glasshouse experiment the effect of co-inoculation with WSM3457 on the kinetics of nodule initiation and development was assessed 5, 7, 10, 14, 17, 21, and 42 days after inoculation of M. truncatula cv. Caliph with 10³ cells/plant of E. medicae WSM419.

Results

Co-inoculated plants had enhanced rate of nodule initiation and development, greater numbers of larger crown nodules, and by day 42 accumulated more N than plants inoculated with E. medicae WSM419 alone. Nodule development was altered by co-inoculation. Approximately 25% of nodule initials on co-inoculated plants formed in closely associated pairs, young nodules were larger with multiple meristems and developed into cluster-like multi-lobed nodules compared to those on WSM419 inoculated plants. Molecular typing showed WSM3457 occupied a significant proportion of root nodules on co-inoculated plants.

Conclusion

Co-inoculation with P. fluorescens WSM3457 enhanced symbiotic effectiveness of M. truncatula when inoculated with a low inoculum dose of E. medicae WSM419.     

Evolutionary history of bacteriophages with double-stranded DNA genomes

Background

Reconstruction of evolutionary history of bacteriophages is a difficult problem because of fast sequence drift and lack of omnipresent genes in phage genomes. Moreover, losses and recombinational exchanges of genes are so pervasive in phages that the plausibility of phylogenetic inference in phage kingdom has been questioned.

Results

We compiled the profiles of presence and absence of 803 orthologous genes in 158 completely sequenced phages with double-stranded DNA genomes and used these gene content vectors to infer the evolutionary history of phages. There were 18 well-supported clades, mostly corresponding to accepted genera, but in some cases appearing to define new taxonomic groups. Conflicts between this phylogeny and trees constructed from sequence alignments of phage proteins were exploited to infer 294 specific acts of intergenome gene transfer.

Conclusion

A notoriously reticulate evolutionary history of fast-evolving phages can be reconstructed in considerable detail by quantitative comparative genomics.

Open peer review

This article was reviewed by Eugene Koonin, Nicholas Galtier and Martijn Huynen.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.     

Establishment and survival of the South African legume Lessertia spp. and rhizobia in Western Australian agricultural systems

Background and aims

The South African herbaceous legume species Lessertia capitata, L. diffusa, L. excisa L. incana and L. herbacea were introduced to Australia to assess plant establishment and survival, as well as the saprophytic ability of their root nodule bacteria (RNB).

Methods

Five Lessertia spp., were inoculated with selected RNB strains and were sown in five different agroclimatic areas of the Western Australian wheat-belt during 2007 and 2008. Plant population and summer survival were evaluated in situ. Soil samples and nodules from host plants were also taken from each site. The re-isolated rhizobia were RPO1-PCR fingerprinted and their partial dnaK and nodA genes were sequenced to confirm their identity.

Results

Plants achieved only poor establishment followed by weak summer survival. More than 83 % of the rhizobia re-isolated from Lessertia did not correlate with the original inoculants’ fingerprints, and were identified as Rhizobium leguminosarum. The nodA sequences of the naturalised strains were also clustered with R. leguminosarum sequences, thus eliminating the likelihood of lateral gene transference from Mesorhizobium and suggesting a competition problem with indigenous rhizobia.

Conclusion

The stressful soil conditions and high numbers of resident R. leguminosarum strains in Western Australian soils, and their ability to rapidly nodulate Lessertia spp. but not fix nitrogen are likely to preclude the adoption of Lessertia as an agricultural legume in this region.     

Evolution of root nodule bacteria: Reconstruction of the speciation processes resulting from genomic rearrangements in a symbiotic system

The processes of speciation and macroevolution of root nodule bacteria (rhizobia), based on deep rearrangements of their genomes and occurring in the N₂-fixing symbiotic system, are reconstructed. At the first stage of rhizobial evolution, transformation of free-living diazotrophs (related to Rhodopseudomonas) to symbiotic N₂-fixers (Bradyrhizobium) occurred due to the acquisition of the fix gene system, which is responsible for providing nitrogenase with electrons and redox potentials, as well as for oxygen-dependent regulation of nitrogenase synthesis in planta, and then of the nod genes responsible for the synthesis of the lipo-chitooligosaccharide Nod factors, which induce root nodule development. The subsequent rearrangements of bacterial genomes included (1) increased volume of hereditary information supported by species, genera (pangenome), and individual strains; (2) transition from the unitary genome to a multicomponent one; and (3) enhanced levels of bacterial genetic plasticity and horizontal gene transfer, resulting in formation of new genera—of which Mesorhizobium, Rhizobium, and Sinorhizobium are the largest—and of over 100 species. Rhizobial evolution caused by development and diversification of the Nod factor-synthesizing systems may result in either relaxed host specificity range (transition of Bradyrhizobium from autotrophic to symbiotrophic carbon metabolism in interaction with a broad spectrum of legumes) or narrowed host specificity range (transition of Rhizobium and Sinorhizobium to “altruistic” interaction with legumes of the galegoid clade). Reconstruction of the evolutionary pathway from symbiotic N₂-fixers to their free-living ancestors makes it possible to initiate the studies based on up-to-date genome screening technologies and aimed at the issues of genetic integration of organisms into supraspecies complexes, ratios of the macro- and microevolutionary mechanisms, and development of cooperative adaptations based on altruistic interaction between the symbiotic partners.     

Detection of evolutionarily stable fragments of cellular pathways by hierarchical clustering of phyletic patterns

Background

Phyletic patterns denote the presence and absence of orthologous genes in completely sequenced genomes and are used to infer functional links between genes, on the assumption that genes involved in the same pathway or functional system are co-inherited by the same set of genomes. However, this basic premise has not been quantitatively tested, and the limits of applicability of the phyletic-pattern method remain unknown.

Results

We characterized a hierarchy of 3,688 phyletic patterns encompassing more than 5,000 known protein-coding genes from 66 complete microbial genomes, using different distances, clustering algorithms, and measures of cluster quality. The most sensitive set of parameters recovered 223 clusters, each consisting of genes that belong to the same metabolic pathway or functional system. Fifty-six clusters included unexpected genes with plausible functional links to the rest of the cluster. Only a small percentage of known pathways and multiprotein complexes are co-inherited as one cluster; most are split into many clusters, indicating that gene loss and displacement has occurred in the evolution of most pathways.

Conclusions

Phyletic patterns of functionally linked genes are perturbed by differential gains, losses and displacements of orthologous genes in different species, reflecting the high plasticity of microbial genomes. Groups of genes that are co-inherited can, however, be recovered by hierarchical clustering, and may represent elementary functional modules of cellular metabolism. The phyletic patterns approach alone can confidently predict the functional linkages for about 24% of the entire data set.     

Towards precise classification of cancers based on robust gene functional expression profiles

Background

Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method.

Results

We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end.

Conclusions

De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.     

Genomic diversity of the human intestinal parasite Entamoeba histolytica

Background

Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome.

Results

The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually.

Conclusions

E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.     

Signatures of selection in natural populations adapted to chronic pollution

Background

Ectocarpus siliculosus virus-1 (EsV-1) is a lysogenic dsDNA virus belonging to the super family of nucleocytoplasmic large DNA viruses (NCLDV) that infect Ectocarpus siliculosus, a marine filamentous brown alga. Previous studies indicated that the viral genome is integrated into the host DNA. In order to find the integration sites of the viral genome, a genomic library from EsV-1-infected algae was screened using labelled EsV-1 DNA. Several fragments were isolated and some of them were sequenced and analyzed in detail.

Results

Analysis revealed that the algal genome is split by a copy of viral sequences that have a high identity to EsV-1 DNA sequences. These fragments are interspersed with DNA repeats, pseudogenes and genes coding for products involved in DNA replication, integration and transposition. Some of these gene products are not encoded by EsV-1 but are present in the genome of other members of the NCLDV family. Further analysis suggests that the Ectocarpus algal genome contains traces of the integration of a large dsDNA viral genome; this genome could be the ancestor of the extant NCLDV genomes. Furthermore, several lines of evidence indicate that the EsV-1 genome might have originated in these viral DNA pieces, implying the existence of a complex integration and recombination system. A protein similar to a new class of tyrosine recombinases might be a key enzyme of this system.

Conclusion

Our results support the hypothesis that some dsDNA viruses are monophyletic and evolved principally through genome reduction. Moreover, we hypothesize that phaeoviruses have probably developed an original replication system.     

The role of laterally transferred genes in adaptive evolution

Background

Bacterial genomes develop new mechanisms to tide them over the imposing conditions they encounter during the course of their evolution. Acquisition of new genes by lateral gene transfer may be one of the dominant ways of adaptation in bacterial genome evolution. Lateral gene transfer provides the bacterial genome with a new set of genes that help it to explore and adapt to new ecological niches.

Methods

A maximum likelihood analysis was done on the five sequenced corynebacterial genomes to model the rates of gene insertions/deletions at various depths of the phylogeny.

Results

The study shows that most of the laterally acquired genes are transient and the inferred rates of gene movement are higher on the external branches of the phylogeny and decrease as the phylogenetic depth increases. The newly acquired genes are under relaxed selection and evolve faster than their older counterparts. Analysis of some of the functionally characterised LGTs in each species has indicated that they may have a possible adaptive role.

Conclusion

The five Corynebacterial genomes sequenced to date have evolved by acquiring between 8 – 14% of their genomes by LGT and some of these genes may have a role in adaptation.

     

Genome-wide analysis of long,exact DNA repeats in rhizobia

The rhizobia are a group of bacteria widely studied for their capacity to form intimate symbiotic relationships with leguminous plants. However, they are also interesting for containing a remarkable abundance of repetitive genetic elements, such as long DNA repeats. In this study we deeply analyzed long, exact DNA repeats in five representative rhizobial genomes; Rhizobium etli, Rhizobium leguminosarum, Bradyrhizobium japonicum, Sinorhizobium meliloti and Mesorhizobium loti. The results suggest that a huge proportion of repeats can be located in either plasmid or chromosome replicons, except in B. japonicum, which lacks plasmids, but contains the largest number, and longest repeat elements of the genomes analyzed here. Interestingly, we detected a slight correlation between the density of repeats (either number or length) and genome size. As expected, the highest percentage of DNA repeats code for mobile genetic elements, including insertion sequences, recombinases, and transposases. Some repeats corresponded to non-coding or intergenic regions, while in genomes like that of R. etli, a significant percentage of large repeats, mainly located in plasmids, were strongly associated with symbiotic and nitrogen fixation activities. In conclusion, our analysis shows that rhizobial genomes contain a high density of long DNA repeats, which might facilitate recombination events and genome rearrangements, functioning in adaption and persistence during saprophytic or symbiotic life.