Enzymatic defenses against oxygen toxicity in the hydrothermal vent animals Riftia pachyptila and Calyptogena magnifica

Two deep-sea hydrothermal vent organisms, the tube worm Riftia pachyptila and the clam Calyptogena magnifica, contain superoxide dismutase, dianisidine peroxidase, and glutathione peroxidase. The tube worm trophosome exhibits an iron-containing superoxide dismutase, ordinarily associated with prokaryotes and not previously seen in an animal tissue, in accord with the presence of symbiotic bacteria in this tissue. The enzymes which provide a defense against oxygen toxicity are thus present in these animals.     

The functional morphology of the organs of feeding and digestion of the hydrothermal vent bivalve Calyptogena magnifica (Vesicomyidae)

The discovery, around Galapagos Rift hydrothermal vents, of an unique community of animals dependent upon the chemoautotrophic oxidation of hydrogen sulphide by bacteria, has aroused wide interest. In the gutless pogonophoran Riftia pachyptila. trophosomal symbiotic bacteria are thought to be principally responsible for this unique form of nutrition. Similar symbiotic bacteria have been postulated for the ctenidia of the Rift clam Calyptogena magnifica. Such a mode of nutrition was deemed necessary since Calyptogena was thought not to possess ctenidial food grooves, thereby making normal filter-feeding impossible. This study reports upon the anatomy of a specimen of C. magnifica and demonstrates the presence of narrow ctenidial food grooves and the normal bivalve complement of feeding and digestive organs. Using a variety of general bacterial and DNA specific stains, no evidence of symbiotic intracellular bacteria has been found in the ctenidia (or any other tissues). It is concluded that C. magnifica is principally a filter feeder, albeit with modification for collecting and processing a diet of bacterial cells, with the possibility (as in all bivalves) of direct absorption. High chemoautotrophic activity levels in the ctenidia probably result from entrapment of vent water bacteria collected during filter feeding.     

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.     

Diversifying selection and host adaptation in two endosymbiont genomes

Background  

The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host:symbiont coevolution. Wolbachia 's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, w Mel and the nematode Brugia malayi, w Bm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts.     

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.     

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days.Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane.The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.     

A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

The uncultivated bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a central role in providing their host with fixed carbon. While this intimate association between host and symbiont indicates tight integration and coordination of function via cellular communication mechanisms, no such systems have been identified. To elucidate potential signal transduction pathways in symbionts that may mediate symbiont-host communication, we cloned and characterized a gene encoding a histidine protein kinase homolog isolated from a symbiont fosmid library. The gene, designated rssA (for Riftia symbiont signal kinase), resembles known sensor kinases and encodes a protein capable of phosphorylating response regulators in Escherichia coli. A second open reading frame, rssB (for Riftia symbiont signal regulator), encodes a protein similar to known response regulators. These results suggest that the symbionts utilize a phosphotransfer signal transduction mechanism to communicate external signals that may mediate recognition of or survival within the host. The specific signals eliciting a response by the signal transduction proteins of the symbiont remain to be elucidated.     

Identification and characterization of a flagellin gene from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment. To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont fliC gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus sigma28 RNA polymerase recognition site lay upstream of the proposed translational start site. The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy. A 30,000-Mr protein subunit was identified in whole-cell extracts by Western blot analysis. These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of R. pachyptila is acquired with each new host generation.     

Characterization of the endosymbiont of a deep-sea bivalve, Calyptogena soyoae.

We have purified DNA from gill tissue of a marine bivalve, Calyptogena soyoae, collected from the deep-sea cold seep communities in Sagami Bay, Japan. An rRNA gene was amplified, cloned, and sequenced. In situ hybridization revealed that the sequence is that of a bacterial endosymbiont within the gill of C. soyoae.     

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background  

Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps.     

The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi

The Filarial Genome Project (FGP) was initiated in 1994 under the auspices of the World Health Organisation. Brugia malayi was chosen as the model organism due to the availability of all life cycle stages for the construction of cDNA libraries. To date, over 20000 cDNA clones have been partially sequenced and submitted to the EST database (dbEST). These ESTs define approximately 7000 new Brugia genes. Analysis of the EST dataset provides useful information on the expression pattern of the most abundantly expressed Brugia genes. Some highly expressed genes have been identified that are expressed in all stages of the parasite's life cycle, while other highly expressed genes appear to be stage-specific. To elucidate the structure of the Brugia genome and to provide a basis for comparison to the Caenorhabditis elegans genome, the FGP is also constructing a physical map of the Brugia chromosomes and is sequencing genomic BAC clones. In addition to the nuclear genome, B. malayi possesses two other genomes: the mitochondrial genome and the genome of a bacterial endosymbiont. Eighty percent of the mitochondrial genome of B. malayi has been sequenced and is being compared to mitochondrial sequences of other nematodes. The bacterial endosymbiont genome found in B. malayi is closely related to the Wolbachia group of rickettsia-like bacteria that infects many insect species. A set of overlapping BAC clones is being assembled to cover the entire bacterial genome. Currently, half of the bacterial genome has been assembled into four contigs. A consortium has been established to sequence the entire genome of the Brugia endosymbiont. The sequence and mapping data provided by the FGP is being utilised by the nematode research community to develop a better understanding of the biology of filarial parasites and to identify new vaccine candidates and drug targets to aid the elimination of human filariasis.     

基于宏基因组学重建来自深海热液喷口的Epsilonproteobacteria基因组及其代谢分析

【背景】非致病性Epsilonproteobacteria广泛存在于全球各种不同的自然环境中,特别是一些极端生境如深海热液喷口,并且经常在微生物群落中作为优势物种被发现。然而,由于现阶段培养技术的限制,仅有为数不多的深海热液Epsilonproteobacteria被分离培养,极大限制了对其生理特征、代谢方式以及生态功能的深入认识。【目的】研究深海热液未培养Epsilonproteobacteria的进化地位、代谢潜能及其在原位生态系统中可能发挥的作用。【方法】基于宏基因组学Binning的方法,从采集自东太平洋海隆深海热液烟囱体样本中构建4个高质量的Epsilonproteobacteria基因组Bin225、Bin51、Bin54和Bin189,并进行了系统发育和代谢途径的分析。【结果】Bin189在系统发育树上相对独立于其他所有已知的Epsilonproteobacteria类群,而其余3个重构基因组都与Nitratiruptor sp. SB155-2具有较近的亲缘关系。在代谢潜能方面,所有的基因组除了都含有sqr硫氧化和rTCA碳固定途径的基因以外,也同时具有脂多糖输出转运子和多种分泌系统。Bin189显示出与其它基因组显著不同的代谢特征,其中还检测到与有机物和氨基酸转运相关的功能基因。而其他的3个基因组均具有完整的反硝化途径的功能基因,其中2个还具有Sox系统、氢化酶和鞭毛移动系统。【结论】Bin189可能是一种新发现的深海热液兼性化能营养型Epsilonproteobacteria,推测其余的3个类群能够利用硫化物和氢气作为能源进行化能自养生长。考虑到它们多样的代谢潜能,这些Epsilonproteobacteria类群很可能在深海热液微生物群落的形成发展和地球化学元素循环中发挥重要作用。     

Pyrosequencing analysis of endosymbiont population structure: co-occurrence of divergent symbiont lineages in a single vesicomyid host clam

Bacteria–eukaryote endosymbioses are perhaps the most pervasive co-evolutionary associations in nature. Here, intracellular chemosynthetic symbionts of deep-sea clams ( Vesicomyidae ) were analysed by amplicon pyrosequencing to explore how symbiont transmission mode affects the genetic diversity of the within-host symbiont population. Vesicomyid symbionts ( Gammaproteobacteria ) are presumed to be obligately intracellular, to undergo nearly strict vertical transmission between host generations, and to be clonal within a host. However, recent data show that vesicomyid symbionts can be acquired laterally via horizontal transfer between hosts or uptake from the environment, potentially creating opportunities for multiple symbiont strains to occupy the same host. Here, genotype-specific PCR and direct sequencing of the bacterial internal transcribed spacer initially demonstrated the co-occurrence of two symbiont strains, symA and symB (93.5% nt identity), in 8 of 118 Vesicomya sp. clams from 3 of 7 hydrothermal vent sites on the Juan de Fuca Ridge. To confirm multiple strains within individual clams, amplicon pyrosequencing of two symbiont loci was used to obtain deep-coverage measurements (mean: ∼1500× coverage per locus per clam) of symbiont population structure. Pyrosequencing confirmed symA–symB co-occurrence for two individuals, showing the presence of both genotypes in amplicon pools. However, in the majority of clams, the endosymbiont population was remarkably homogenous, with > 99.5% of sequences collapsing into a single symbiont genotype in each clam. These results support the hypothesis that a predominantly vertical transmission strategy leads to the fixation of a single symbiont strain in most hosts. However, mixed symbiont populations do occur in vesicomyids, potentially facilitating the exchange of genetic material between divergent symbiont lineages.     

Evolutionarily stable infection by a male-killing endosymbiont in Drosophila innubila: molecular evidence from the host and parasite genomes

Maternally inherited microbes that spread via male-killing are common pathogens of insects, yet very little is known about the evolutionary duration of these associations. The few examples to date indicate very recent, and thus potentially transient, infections. A male-killing strain of Wolbachia has recently been discovered in natural populations of Drosophila innubila. The population-level effects of this infection are significant: approximately 35% of females are infected, infected females produce very strongly female-biased sex ratios, and the resulting population-level sex ratio is significantly female biased. Using data on infection prevalence and Wolbachia transmission rates, infected cytoplasmic lineages are estimated to experience a approximately 5% selective advantage relative to uninfected lineages. The evolutionary history of this infection was explored by surveying patterns of polymorphism in both the host and parasite genomes, comparing the Wolbachia wsp gene and the host mtDNA COI gene to five host nuclear genes. Molecular data suggest that this male-killing infection is evolutionarily old, a conclusion supported with a simple model of parasite and mtDNA transmission dynamics. Despite a large effective population size of the host species and strong selection to evolve resistance, the D. innubila-Wolbachia association is likely at a stable equilibrium that is maintained by imperfect maternal transmission of the bacteria rather than partial resistance in the host species.     

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.     

Evolution of mitochondrial energy metabolism genes associated with hydrothermal vent adaption of Alvinocaridid shrimps

Most of Alvinocaridid shrimps live in hydrothermal vents, where is a wicked environment with highly toxic chemistry, hypoxia, acidic pH, and sulfide deposits. In order to adapt to this environment, change in energy metabolism may be one of the primary factors. However, the genetic basis of energy metabolism underlying this environment remains unexplored. Here, we present the first systematic investigation of mitochondrial genes in Alvinocarididae. The analysis demonstrated that ATP6, ND4 and ND6 were subjected to strong positive selection leading to last common ancestors of Alvinocarididae, and ATP8, ND5, COX1 and COX2 were determined to have undergone positive selection in the interior lineages of Alvinocarididae. Considering that about 95% of ATP is supplied by mitochondria via oxidative phosphorylation, and body detoxification process associated with cytochrome c. Positive selection in these genes suggested that Alvinocaridid shrimps might have acquired an enhanced capacity for energy metabolism and detoxification in extreme hydrothermal vent field.     

Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi--evidence of two active domains

The cDNA and deduced amino acid sequences for arginine kinase (AK) from the deep-sea clam Calyptogena kaikoi have been determined revealing an unusual two-domain (2D) structure with molecular mass of 80 kDa, twice that of normal AK. The amino acid sequences of both domains contain most of the residues thought to be required for substrate binding found in the horseshoe crab Limulus polyphemus AK, a well studied system for which several X-ray crystal structures exist. However, two highly conserved residues, D62 and R193, that form a salt bridge thereby stabilizing the substrate-bound structure have been replaced by G and N in domain 1, and G and P in domain 2, respectively. The present effort probes whether both domains of Calyptogena AK are catalytically competent. Recombinant constructs of the wild-type enzyme of both single domains, and of selected mutants of the Calyptogena AK have been expressed as fusion proteins with the maltose-binding protein. The wild-type two-domain enzyme (2D[WT]) had high AK activity (k(cat)=23 s(- 1), average value of the two domains), and the single domain 2 (D2[WT]) showed 1.5-times higher activity (k(cat)=38 s(- 1)) than the wild-type 2D[WT]. Interestingly, the single domain 1 (D1[WT]) showed only a very low activity (k(cat) approximately 0.016 s(- 1)). Introduction of a Y68A mutation in both domains virtually abolished catalytic activity. On the other hand, significant residual activity was observed (k(cat)=2.8 s(- 1)), when the Y68A mutation was introduced only into domain 2 of the two-domain enzyme. A similar mutation in domain 1 of the two-domain enzyme reduced activity to a much lower extent (k(cat)=11.1 s(- 1)). Although the domains of this "contiguous" dimeric AK each have catalytic capabilities, the presence of domain 2 strongly influences the stability and activity of domain 1.     

The hemoglobin gene of the deep-sea clam Calyptogena soyoae has a novel intron in A-helix

The cDNAs encoding two dimeric hemoglobins, Hbs I and II, of the deep-sea clam Calyptogena soyoae were amplified by PCR and the complete nucleotide sequences determined. The cDNA-derived amino acid sequences agreed completely with those determined chemically. Many of the molluscan intracellular globin genes have a characteristic four-exon/three-intron structure, with the precoding and two conventional introns conserved widely in animal globin genes. In this work we have determined the exon/intron organization of two hemoglobin genes of the deep-sea clam C. soyoae. Surprisingly, this gene has no precoding intron but instead contains an additional intron in the A-helix (A3.1), together with the two conventional introns (B12.2 and G6.3). This observation suggests that the precoding intron has been lost and the insertion of intron in A-helix occurred in the genes of Calyptogena. Alternatively, the sliding of intron from precoding to A-helix might have occurred.