Reductive genome evolution in chemoautotrophic intracellular symbionts of deep-sea <Emphasis Type="Italic">Calyptogena</Emphasis> clams

To understand reductive genome evolution (RGE), we comparatively analyzed the recently reported small genomes of two chemoautotrophic, intracellular symbionts of deep-sea clams, Calyptogena okutanii and C. magnifica. Both genomes lack most genes for DNA recombination and repair such as recA and mutY. Their genome architectures were highly conserved except one inversion. Many deletions from small (<100 bp) to large (1–11 kbp) sizes were detected and the deletion numbers decreased exponentially with size. Densities of deletions and short-repeats, as well as A+T content were higher in non-coding regions than in coding regions. Because Calyptogena symbiont genomes lack recA, we propose that deletions and the single inversion occurred by RecA-independent recombination (RIR) at short-repeats with simultaneous consumption of repeats, and that short-repeats were regenerated by accelerated mutations with enhanced A+T bias due to the absence of mutY. We further propose that extant Calyptogena symbiont genomes are in an actively reducing stage of RGE consisting of small and large deletions, and the deletions are caused by short-repeat dependent RIR along with regeneration of short-repeats. In future, the RGE rate will slowdown when the gene repertoires approach the minimum gene set necessary for intracellular symbiotic life. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Kuwahara and Y. Takaki equally contributed to this work.     

Expression of genes for sulfur oxidation in the intracellular chemoautotrophic symbiont of the deep-sea bivalve Calyptogena okutanii

To understand sulfur oxidation in thioautotrophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox) (soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.     

Coupling of Bacterial Endosymbiont and Host Mitochondrial Genomes in the Hydrothermal Vent Clam Calyptogena magnifica

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) depends for its nutrition on sulfur-oxidizing symbiotic bacteria housed in its gill tissues. This symbiont is transmitted vertically between generations via the clam's eggs; however, it remains uncertain whether occasionally symbionts are horizontally transmitted or acquired from the environment. If symbionts are transmitted strictly vertically through the egg cytoplasm, inheritance of symbiont lineages should behave as if coupled to the host's maternally inherited mitochondrial DNA. This coupling would be obscured, however, with low rates of horizontal or environmental transfers, the equivalent of recombination between host lineages. Population genetic analyses of C. magnifica clams and associated symbionts from eastern Pacific hydrothermal vents clearly supported the hypothesis of strictly maternal cotransmission. Host mitochondrial and symbiont DNA sequences were coupled in a clam population that was polymorphic for both genetic markers. These markers were not similarly coupled with sequence variation at a nuclear gene locus, as expected for a randomly mating sexual population. Phylogenetic analysis of the two cytoplasmic genes also revealed no evidence for recombination. The tight association between vesicomyid clams and their vertically transmitted bacterial endosymbionts is phylogenetically very young (<50 million years) and may serve as a model for the origin and evolution of eukaryotic organelles.     

Chromosome Stability and Gene Loss in Cockroach Endosymbionts

Insect endosymbiont genomes reflect massive gene loss. Two Blattabacterium genomes display colinearity and similar gene contents, despite high orthologous gene divergence, reflecting over 140 million years of independent evolution in separate cockroach lineages. We speculate that distant homologs may replace the functions of some eliminated genes through broadened substrate specificity.Obligate symbionts of insects exhibit extreme patterns of genome evolution and include the smallest known bacterial genomes (10, 11, 14). Two recently published sequences of Blattabacterium, the obligate symbiont of cockroaches (7, 16), present the opportunity to analyze genome evolution in an additional symbiont lineage with extreme genome reduction.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Phylogenetic diversity of Mesorhizobium in chickpea

Crop domestication, in general, has reduced genetic diversity in cultivated gene pool of chickpea (Cicer arietinum) as compared with wild species (C. reticulatum, C. bijugum). To explore impact of domestication on symbiosis, 10 accessions of chickpeas, including 4 accessions of C. arietinum, and 3 accessions of each of C. reticulatum and C. bijugum species, were selected and DNAs were extracted from their nodules. To distinguish chickpea symbiont, preliminary sequences analysis was attempted with 9 genes (16S rRNA, atpD, dnaJ, glnA, gyrB, nifH, nifK, nodD and recA) of which 3 genes (gyrB, nifK and nodD) were selected based on sufficient sequence diversity for further phylogenetic analysis. Phylogenetic analysis and sequence diversity for 3 genes demonstrated that sequences from C. reticulatum were more diverse. Nodule occupancy by dominant symbiont also indicated that C. reticulatum (60%) could have more various symbionts than cultivated chickpea (80%). The study demonstrated that wild chickpeas (C. reticulatum) could be used for selecting more diverse symbionts in the field conditions and it implies that chickpea domestication affected symbiosis negatively in addition to reducing genetic diversity.     

Rhizobium laguerreae is the main nitrogen-fixing symbiont of cultivated lentil (Lens culinaris) in Morocco

Genetic diversity and population structure of 268 Lens culinaris symbiotic rhizobia collected from 40 cultivated fields in the main lentil production regions in Morocco were estimated. Three chromosomal housekeeping genes (recA, glnII and atpD) and one common symbiotic gene (nodC) were sequenced and analyzed in order to identify the local symbionts of lentil. The molecular phylogeny of the concatenated housekeeping genes clustered more than 95% of the isolates in one main clade together with Rhizobium laguerreae species. R. laguerreae represents the main symbiont of cultivated lentil in Morocco and, for the first time, a large sample of individuals is obtained for this species. There is a significant and high genetic differentiation of bacterial populations among the four regions for their symbiotic gene, and much lower for their housekeeping genes. The reasons why R. laguerreae is so frequently recovered in our study is discussed.     

Swapping symbionts in spittlebugs: evolutionary replacement of a reduced genome symbiont

Bacterial symbionts that undergo long-term maternal transmission experience elevated fixation of deleterious mutations, resulting in massive loss of genes and changes in gene sequences that appear to limit efficiency of gene products. Potentially, this dwindling of symbiont functionality impacts hosts that depend on these bacteria for nutrition. One evolutionary escape route is the acquisition of a novel symbiont with a robust genome and metabolic capabilities. Such an acquisition has occurred in an ancestor of Philaenus spumarius, the meadow spittlebug (Insecta: Cercopoidea), which has replaced its ancient association with the tiny genome symbiont Zinderia insecticola (Betaproteobacteria) with an association with a symbiont related to Sodalis glossinidius (Gammaproteobacteria). Spittlebugs feed exclusively on xylem sap, a diet that is low both in essential amino acids and in sugar or other substrates for energy production. The new symbiont genome has undergone proliferation of mobile elements resulting in many gene inactivations; nonetheless, it has selectively maintained genes replacing functions of its predecessor for amino-acid biosynthesis. Whereas ancient symbiont partners typically retain perfectly complementary sets of amino-acid biosynthetic pathways, the novel symbiont introduces some redundancy as it retains some pathways also present in the partner symbionts (Sulcia muelleri). Strikingly, the newly acquired Sodalis-like symbiont retains genes underlying efficient routes of energy production, including a complete TCA cycle, potentially relaxing the severe energy limitations of the xylem-feeding hosts. Although evolutionary replacements of ancient symbionts are infrequent, they potentially enable evolutionary and ecological novelty by conferring novel metabolic capabilities to host lineages.     

Loss of DNA recombinational repair enzymes in the initial stages of genome degeneration

Many obligate intracellular pathogens and symbionts undergo genome degeneration during long-term association with eukaryotic hosts; however, very little is known about genome changes that occur in the initial stages of such intracellular associations. By focusing on a clade of bacteria that have recently established symbiotic associations with insect hosts, we have identified events that may contribute to the reduction and degeneration of symbiont genomes. Unlike virtually all other bacteria, the obligate symbionts of maize and rice weevils each display substantial sequence divergence between multiple copies of their rDNA genes, resulting from a reduction in the efficacy of recombinational gene conversion, coincident with the inactivation of the recombinational repair gene recF in the common ancestor of both symbionts. The maize weevil endosymbiont also lacks a functional recA, resulting in further reduction in the efficacy of gene conversion between paralogous rDNAs and in a novel IS-mediated deletion in a 23S rDNA gene. Similar events may be pervasive during the evolution of symbiosis because symbiont genomes typically lack recombinational repair genes and have reduced numbers of ribosomal operons.     

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.     

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.     

Pantothenate mediates the coordination of whitefly and symbiont fitness

Intracellular symbionts in insects often have reduced genomes. Host acquisition of genes from bacteria is an important adaptation that supports symbionts. However, the function of horizontally transferred genes in insect symbiosis remains largely unclear. The primary symbiont Portiera housed in bacteriocytes lacks pantothenate synthesis genes: panB and panC, which is presumably complemented by a fused gene panB-panC (hereafter panBC) horizontally transferred from bacteria in Bemisia tabaci MEAM1. We found panBC in many laboratory cultures, and species of B. tabaci shares a common evolutionary origin. We demonstrated that complementation with whitefly panBC rescued E. coli pantothenate gene knockout mutants. Portiera elimination decreased the pantothenate level and PanBC abundance in bacteriocytes, and reduced whitefly survival and fecundity. Silencing PanBC decreased the Portiera titer, reduced the pantothenate level, and decreased whitefly survival and fecundity. Supplementation with pantothenate restored the symbiont titer, PanBC level, and fitness of RNAi whiteflies. These data suggest that pantothenate synthesis requires cooperation and coordination of whitefly PanBC expression and Portiera. This host–symbiont co-regulation was mediated by the pantothenate level. Our findings demonstrated that pantothenate production, by the cooperation of a horizontally acquired, fused bacteria gene and Portiera, facilitates the coordination of whitefly and symbiont fitness. Thus, this study extends our understanding on the basis of complex host–symbiont interactions.Subject terms: Applied microbiology, Functional genomics     

Diversification of MIF immune regulators in aphids: link with agonistic and antagonistic interactions

Background

The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions.

Results

In contrast to the limited antibacterial arsenal, we showed that the pea aphid Acyrthosiphon pisum expresses 5 members of Macrophage Migration Inhibitory Factors (ApMIF), known to be key regulators of the innate immune response. In silico searches for MIF members in insect genomes followed by phylogenetic reconstruction suggest that evolution of MIF genes in hemipteran species has been shaped both by differential losses and serial duplications, raising the question of the functional importance of these genes in aphid immune responses. Expression analyses of ApMIFs revealed reduced expression levels in the presence, or during the establishment of secondary symbionts. By contrast, ApMIFs expression levels significantly increased upon challenge with a parasitoid or a Gram-negative bacteria. This increased expression in the presence of a pathogen/parasitoid was reduced or missing, in the presence of facultative symbiotic bacteria.

Conclusions

This work provides evidence that while aphid’s antibacterial arsenal is reduced, other immune genes widely absent from insect genomes are present, diversified and differentially regulated during antagonistic or agonistic interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-762) contains supplementary material, which is available to authorized users.     

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.     

Bacterial Endosymbiosis in a Chordate Host: Long-Term Co-Evolution and Conservation of Secondary Metabolism

Intracellular symbiosis is known to be widespread in insects, but there are few described examples in other types of host. These symbionts carry out useful activities such as synthesizing nutrients and conferring resistance against adverse events such as parasitism. Such symbionts persist through host speciation events, being passed down through vertical transmission. Due to various evolutionary forces, symbionts go through a process of genome reduction, eventually resulting in tiny genomes where only those genes essential to immediate survival and those beneficial to the host remain. In the marine environment, invertebrates such as tunicates are known to harbor complex microbiomes implicated in the production of natural products that are toxic and probably serve a defensive function. Here, we show that the intracellular symbiont Candidatus Endolissoclinum faulkneri is a long-standing symbiont of the tunicate Lissoclinum patella, that has persisted through cryptic speciation of the host. In contrast to the known examples of insect symbionts, which tend to be either relatively recent or ancient relationships, the genome of Ca. E. faulkneri has a very low coding density but very few recognizable pseudogenes. The almost complete degradation of intergenic regions and stable gene inventory of extant strains of Ca. E. faulkneri show that further degradation and deletion is happening very slowly. This is a novel stage of genome reduction and provides insight into how tiny genomes are formed. The ptz pathway, which produces the defensive patellazoles, is shown to date to before the divergence of Ca. E. faulkneri strains, reinforcing its importance in this symbiotic relationship. Lastly, as in insects we show that stable symbionts can be lost, as we describe an L. patella animal where Ca. E. faulkneri is displaced by a likely intracellular pathogen. Our results suggest that intracellular symbionts may be an important source of ecologically significant natural products in animals.     

Addicted? Reduced host resistance in populations with defensive symbionts

Heritable symbionts that protect their hosts from pathogens have been described in a wide range of insect species. By reducing the incidence or severity of infection, these symbionts have the potential to reduce the strength of selection on genes in the insect genome that increase resistance. Therefore, the presence of such symbionts may slow down the evolution of resistance. Here we investigated this idea by exposing Drosophila melanogaster populations to infection with the pathogenic Drosophila C virus (DCV) in the presence or absence of Wolbachia, a heritable symbiont of arthropods that confers protection against viruses. After nine generations of selection, we found that resistance to DCV had increased in all populations. However, in the presence of Wolbachia the resistant allele of pastrel—a gene that has a major effect on resistance to DCV—was at a lower frequency than in the symbiont-free populations. This finding suggests that defensive symbionts have the potential to hamper the evolution of insect resistance genes, potentially leading to a state of evolutionary addiction where the genetically susceptible insect host mostly relies on its symbiont to fight pathogens.     

A Lover and a Fighter: The Genome Sequence of an Entomopathogenic Nematode Heterorhabditis bacteriophora

Heterorhabditis bacteriophora are entomopathogenic nematodes that have evolved a mutualism with Photorhabdus luminescens bacteria to function as highly virulent insect pathogens. The nematode provides a safe harbor for intestinal symbionts in soil and delivers the symbiotic bacteria into the insect blood. The symbiont provides virulence and toxins, metabolites essential for nematode reproduction, and antibiotic preservation of the insect cadaver. Approximately half of the 21,250 putative protein coding genes identified in the 77 Mbp high quality draft H. bacteriophora genome sequence were novel proteins of unknown function lacking homologs in Caenorhabditis elegans or any other sequenced organisms. Similarly, 317 of the 603 predicted secreted proteins are novel with unknown function in addition to 19 putative peptidases, 9 peptidase inhibitors and 7 C-type lectins that may function in interactions with insect hosts or bacterial symbionts. The 134 proteins contained mariner transposase domains, of which there are none in C. elegans, suggesting an invasion and expansion of mariner transposons in H. bacteriophora. Fewer Kyoto Encyclopedia of Genes and Genomes Orthologies in almost all metabolic categories were detected in the genome compared with 9 other sequenced nematode genomes, which may reflect dependence on the symbiont or insect host for these functions. The H. bacteriophora genome sequence will greatly facilitate genetics, genomics and evolutionary studies to gain fundamental knowledge of nematode parasitism and mutualism. It also elevates the utility of H. bacteriophora as a bridge species between vertebrate parasitic nematodes and the C. elegans model.     

Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of Paraburkholderia

The Chapada Diamantina in NE of Brazil is a biodiversity hotspot and a center of radiation for many Neotropical legume genera, such as Calliandra and Mimosa. The present study aimed to evaluate nodulation in Calliandra species endemic to various environments, and to characterize the diversity of their symbiotic rhizobia using housekeeping (16S rRNA, recA) and plasmid-borne, symbiosis-related (nifH and nodC) genes. The nodulation ability of selected isolates was assessed. All of the 126 bacterial isolates from 18 Calliandra species collected in six different vegetation types were identified as Paraburkholderia according to their housekeeping and symbiosis gene phylogenies. They were grouped in seven clades in relation to the dominant vegetation type in their native environments. The majority, particularly those from highland “campo rupestre” vegetation, were similar to Paraburkholderia nodosa, but had nodC genes identical to the Mimosa symbiont Paraburkholderia tuberum sv. mimosae. The other smaller groups were related to Paraburkholderia diazotrophica and Paraburkholderia sabiae, and some single strains were not close to any known species. The symbionts of Calliandra spp. in NE Brazil are Paraburkholderia strains closely-related to Mimosa symbionts from the same region. NE Brazil is a reservoir of symbiotic Paraburkholderia that have an affinity for genera in the Mimosoid clade.