Identification in situ and phylogeny of uncultured bacterial endosymbionts.

The use of Koch's technique to isolate bacteria in pure cultures has enabled thousands of bacterial species to be characterized. But for the many microorganisms that have never been cultivated, DNA amplification in vitro using the polymerase chain reaction is now making their genes accessible. Here we use this technique to study bacteria of the genus Holospora, which live in ciliates and whose phylogenetic relationship has remained unknown because they are impossible to cultivate. Species of Holospora are highly infectious and live in the nuclei of their specific host cells: H. elegans and H. undulata infect micronuclei of Paramecium caudatum, whereas H. obtusa infects the macronucleus in other strains of the same host species; Holospora species have a common developmental cycle. We have amplified, cloned and sequenced gene fragments encoding ribosomal RNA of H. obtusa. The phylogenetic position of H. obtusa in the alpha group of Proteobacteria was determined by 16S rRNA sequence analysis. The sequences were then used to design species- as well as genus-specific rRNA hybridization probes, which enabled us to detect and differentiate individual cells of the endosymbionts in situ. The large amount of rRNA in the cells indicates a high physiological activity of the endosymbionts in the host nuclei.     

Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm

Stable associations of more than one species of symbiont within a single host cell or tissue are assumed to be rare in metazoans because competition for space and resources between symbionts can be detrimental to the host. In animals with multiple endosymbionts, such as mussels from deep-sea hydrothermal vents and reef-building corals, the costs of competition between the symbionts are outweighed by the ecological and physiological flexibility gained by the hosts. A further option for the coexistence of multiple symbionts within a host is if these benefit directly from one another, but such symbioses have not been previously described. Here we show that in the gutless marine oligochaete Olavius algarvensis, endosymbiotic sulphate-reducing bacteria produce sulphide that can serve as an energy source for sulphide-oxidizing symbionts of the host. Thus, these symbionts do not compete for resources but rather share a mutalistic relationship with each other in an endosymbiotic sulphur cycle, in addition to their symbiotic relationship with the oligochaete host.     

Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS

Almost all aphid species (Homoptera, Insecta) have 60-80 huge cells called bacteriocytes, within which are round-shaped bacteria that are designated Buchnera. These bacteria are maternally transmitted to eggs and embryos through host generations, and the mutualism between the host and the bacteria is so obligate that neither can reproduce independently. Buchnera is a close relative of Escherichia coli, but it contains more than 100 genomic copies per cell, and its genome size is only a seventh of that of E. coli. Here we report the complete genome sequence of Buchnera sp. strain APS, which is composed of one 640,681-base-pair chromosome and two small plasmids. There are genes for the biosyntheses of amino acids essential for the hosts in the genome, but those for non-essential amino acids are missing, indicating complementarity and syntrophy between the host and the symbiont. In addition, Buchnera lacks genes for the biosynthesis of cell-surface components, including lipopolysaccharides and phospholipids, regulator genes and genes involved in defence of the cell. These results indicate that Buchnera is completely symbiotic and viable only in its limited niche, the bacteriocyte.     

Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves

In a symbiotic association between an invertebrate host and chemoautotrophic bacteria, each partner has different metabolic requirements, and the host typically supplies the bacteria with necessary reduced chemicals (sulphide or methane). Some combination of anatomical, physiological and behavioural adaptations in the host often facilitates uptake and transport of reduced chemicals to the symbionts. We have studied five species of bivalve molluscs of the family Thyasiridae (that is, thyasirids) three of which harbour chemoautotrophic bacteria. Here we show that the symbiotic bivalves extend their feet to form elongated and ramifying burrows in the sediment, most probably to gain access to reduced sulphur. Closely related bivalves (including some thyasirid species) without bacterial symbionts show no comparable foot extension behaviour. The length and number of burrows formed by chemosymbiotic thyasirids are related to the concentration of hydrogen sulphide in the sediment. The burrows are formed by the foot of each bivalve, which can extend up to 30 times the length of the shell, and may be the most extreme case of animal structure elongation documented to date.     

Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.     

Phagosome acidification blocked by intracellular Toxoplasma gondii

Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.     

The evolution of alternative parasitic life histories in large blue butterflies

Large blue (Maculinea) butterflies are highly endangered throughout the Palaearctic region, and have been the focus of intense conservation research. In addition, their extraordinary parasitic lifestyles make them ideal for studies of life history evolution. Early instars consume flower buds of specific host plants, but later instars live in ant nests where they either devour the brood (predators), or are fed mouth-to-mouth by the adult ants (cuckoos). Here we present the phylogeny for the group, which shows that it is a monophyletic clade nested within Phengaris, a rare Oriental genus whose species have similar life histories. Cuckoo species are likely to have evolved from predatory ancestors. As early as five million years ago, two Maculinea clades diverged, leading to the different parasitic strategies seen in the genus today. Contrary to current belief, the two recognized cuckoo species show little genetic divergence and are probably a single ecologically differentiated species. On the other hand, some of the predatory morphospecies exhibit considerable genetic divergence and may contain cryptic species. These findings have important implications for conservation and reintroduction efforts.     

An ecological and evolutionary perspective on human-microbe mutualism and disease

The microbial communities of humans are characteristic and complex mixtures of microorganisms that have co-evolved with their human hosts. The species that make up these communities vary between hosts as a result of restricted migration of microorganisms between hosts and strong ecological interactions within hosts, as well as host variability in terms of diet, genotype and colonization history. The shared evolutionary fate of humans and their symbiotic bacteria has selected for mutualistic interactions that are essential for human health, and ecological or genetic changes that uncouple this shared fate can result in disease. In this way, looking to ecological and evolutionary principles might provide new strategies for restoring and maintaining human health.     

Coral bleaching: thermal adaptation in reef coral symbionts

Many corals bleach as a result of increased seawater temperature, which causes them to lose their vital symbiotic algae (Symbiodinium spp.) - unless these symbioses are able to adapt to global warming, bleaching threatens coral reefs worldwide. Here I show that some corals have adapted to higher temperatures, at least in part, by hosting specifically adapted Symbiodinium. If other coral species can host these or similar Symbiodinium taxa, they might adapt to warmer habitats relatively easily.     

Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells

Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.     

Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles

Listeria monocytogenes is an intracellular bacterial pathogen that replicates rapidly in the cytosol of host cells during acute infection. Surprisingly, these bacteria were found to occupy vacuoles in liver granuloma macrophages during persistent infection of severe combined immunodeficient (SCID) mice. Here we show that L. monocytogenes can replicate in vacuoles within macrophages. In livers of SCID mice infected for 21 days, we observed bacteria in large LAMP1(+) compartments that we termed spacious Listeria-containing phagosomes (SLAPs). SLAPs were also observed in vitro, and were found to be non-acidic and non-degradative compartments that are generated in an autophagy-dependent manner. The replication rate of bacteria in SLAPs was found to be reduced compared to the rate of those in the cytosol. Listeriolysin O (LLO, encoded by hly), a pore-forming toxin essential for L. monocytogenes virulence, was necessary and sufficient for SLAP formation. A L. monocytogenes mutant with low LLO expression was impaired for phagosome escape but replicated slowly in SLAPs over a 72 h period. Therefore, our studies reveal a role for LLO in promoting L. monocytogenes replication in vacuoles and suggest a mechanism by which this pathogen can establish persistent infection in host macrophages.     

A candidate NAD+ transporter in an intracellular bacterial symbiont related to Chlamydiae

Bacteria living within eukaryotic cells can be essential for the survival or reproduction of the host but in other cases are among the most successful pathogens. Environmental Chlamydiae, including strain UWE25, thrive as obligate intracellular symbionts within protozoa; are recently discovered relatives of major bacterial pathogens of humans; and also infect human cells. Genome analysis of UWE25 predicted that this symbiont is unable to synthesize the universal electron carrier nicotinamide adenine dinucleotide (NAD+). Compensation of limited biosynthetic capacity in intracellular bacteria is usually achieved by import of primary metabolites. Here, we report the identification of a candidate transporter protein from UWE25 that is highly specific for import of NAD+ when synthesized heterologously in Escherichia coli. The discovery of this candidate NAD+/ADP exchanger demonstrates that intact NAD+ molecules can be transported through cytoplasmic membranes. This protein acts together with a newly discovered nucleotide transporter and an ATP/ADP translocase, and allows UWE25 to exploit its host cell by means of a sophisticated metabolic parasitism.     

Nitrogen fixation. Endocrine disrupters and flavonoid signalling

Nitrogen fixation is a symbiotic process initiated by chemical signals from legumes that are recognized by soil bacteria. Here we show that some endocrine-disrupting chemicals (EDCs), so called because of their effect on hormone-signalling pathways in animal cells, also interfere with the symbiotic signalling that leads to nitrogen fixation. Our results raise the possibility that these phytochemically activated pathways may have features in common with hormonal signalling in vertebrates, thereby extending the biological and ecological impact of EDCs.     

Somatic stem cell niche tropism in Wolbachia

Wolbachia are intracellular bacteria found in the reproductive tissue of all major groups of arthropods. They are transmitted vertically from the female hosts to their offspring, in a pattern analogous to mitochondria inheritance. But Wolbachia phylogeny does not parallel that of the host, indicating that horizontal infectious transmission must also occur. Insect parasitoids are considered the most likely vectors, but the mechanism for horizontal transfer is largely unknown. Here we show that newly introduced Wolbachia cross several tissues and infect the germline of the adult Drosophila melanogaster female. Through investigation of bacterial migration patterns during the course of infection, we found that Wolbachia reach the germline through the somatic stem cell niche in the D. melanogaster germarium. In addition, our data suggest that Wolbachia are highly abundant in the somatic stem cell niche of long-term infected hosts, implying that this location may also contribute to efficient vertical transmission. This is, to our knowledge, the first report of an intracellular parasite displaying tropism for a stem cell niche.     

蚜虫次级内共生菌的多样性和功能

内共生菌与其寄主之间的关系一直都是生物学研究的热点。蚜虫类昆虫的内共生菌分为初级内共生菌(Buchnera)和一些次级内共生菌。Buchnera与蚜虫寄主之间有密切的营养共生关系,对蚜虫寄主的生长发育至关重要。虽然次级内共生菌在蚜虫体内含量相对较少,但是随着研究的不断深入,次级内共生菌无论对蚜虫寄主的生态行为(抵御寄生蜂、耐高温特性、体色形成等)还是物种形成和进化的影响都不容忽视。此外,次级内共生菌基因组序列的测定,也促进了功能基因学的发展。在系统梳理已发表文献的基础上,本文主要综述了蚜虫次级内共生菌的基本特征、多样性、及其对蚜虫寄主的生态影响,进而对未来值得关注的问题进行了展望。     

菌丝内生细菌及其宿主真菌共生体系研究进展

微生物共生普遍存在于自然界中，真菌-细菌联合体能以多种方式相互作用，共同发挥各种生态功能。有些细菌驻留在真菌菌丝内部，借以调控真菌的生长、发育、分布和次级代谢过程，这些细菌被称为菌丝内生细菌（endohyphal bacteria, EHB）。EHB的研究揭开了微生物生态学的一个新篇章，是真菌与细菌共生关系中最紧密的代表。在逆境条件下，EHB可以调节寄主生殖机制相关的关键成分或步骤，诱导植物激素类物质的产生，对寄主真菌具有辅助性保护作用。研究最深入的真菌-EHB共生体系是植物致病性根霉菌Rhizopus sp.与伯克霍尔德氏菌Burkholderia sp.，引起水稻幼苗枯萎病所必需的植物毒素——根霉素是由伯克霍尔德氏菌所产生的，而非寄主根霉菌本身产生的。EHB也会影响定殖于高等植物的内生真菌的生态和多样性。在某些情况下，EHB还有助于激活参与识别、转录调节和初级代谢蛋白合成过程的相关基因。目前已开发出了无菌培养分离EHB的方法，然而对真菌-EHB共生体系的研究尚不够深入。综述了菌丝内生细菌EHB及其与宿主真菌的共生体系，阐述这些伴侣之间复杂微妙的相互关系，以及EHB对宿主真菌和宿主植物生长和发育的影响，并对该领域的研究方向提出了建议。     

Ligand-induced rapid redistribution of lysosomes is temporally distinct from endosome translocation

When many ligands bind to cell-surface receptors, ligand-receptor complexes are internalized via clathrin coated pits by a process called receptor-mediated endocytosis. The cytoplasmic fate of ligands internalized within endocytic vesicles or endosomes is variable. For example, maternal immunoglobulins are transported through the cytoplasm of neonatal intestinal epithelial cells and are exocytosed at the basolateral surface. However, other ligands are degraded as a result of their delivery to the lysosomal compartment of cells. Although the translocation of endosomes to the Golgi region in the cell centre seems to be a general phenomenon presumably coupled to ligand degradation by lysosomes and endosomes and lysosomes undergo saltatory movements within the cytoplasm, the spatial control of interaction between the two structures is not understood. To address this problem we have begun to examine the spatial and temporal intracellular distribution of endosomes and lysosomes. Utilizing a new fluorescent microscopic approach, we have now been able simultaneously to visualize endosome and lysosome populations in living cells. Our results suggest that a specific relocation of lysosomes is rapidly induced upon binding of different types of ligands to the cell surface; this migration of lysosomes to the Golgi region of the cell precedes the translocation of endosomes into the same area.     

A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12

For many species of pathogenic bacteria, invasion and survival within animal cells is central to establishing a successful host-parasite relationship. Localization within host cells protects the microorganism from host defences, or permits it to cross epithelial barriers and subsequently become systemically distributed. The precise mechanisms that permit entry of bacteria into host tissues are unclear, therefore we have been studying the invasion of epithelial cells by Yersinia pseudotuberculosis. As a first step towards identifying the factors required for this process, we report here the identification of a single genetic locus from this organism that is sufficient to convert the innocuous Escherichia coli K-12 strain into an organism capable of invading cultured animal cells.     

菌丝内生细菌及其宿主真菌共生体系研究进展

微生物共生普遍存在于自然界中，真菌-细菌联合体能以多种方式相互作用，共同发挥各种生态功能。有些细菌驻留在真菌菌丝内部，借以调控真菌的生长、发育、分布和次级代谢过程，这些细菌被称为菌丝内生细菌（endohyphal bacteria, EHB）。EHB的研究揭开了微生物生态学的一个新篇章，是真菌与细菌共生关系中最紧密的代表。在逆境条件下，EHB可以调节寄主生殖机制相关的关键成分或步骤，诱导植物激素类物质的产生，对寄主真菌具有辅助性保护作用。研究最深入的真菌-EHB共生体系是植物致病性根霉菌Rhizopus sp.与伯克霍尔德氏菌Burkholderia sp.，引起水稻幼苗枯萎病所必需的植物毒素——根霉素是由伯克霍尔德氏菌所产生的，而非寄主根霉菌本身产生的。EHB也会影响定殖于高等植物的内生真菌的生态和多样性。在某些情况下，EHB还有助于激活参与识别、转录调节和初级代谢蛋白合成过程的相关基因。目前已开发出了无菌培养分离EHB的方法，然而对真菌-EHB共生体系的研究尚不够深入。综述了菌丝内生细菌EHB及其与宿主真菌的共生体系，阐述这些伴侣之间复杂微妙的相互关系，以及EHB对宿主真菌和宿主植物生长和发育的影响，并对该领域的研究方向提出了建议。