Single mutation makes Escherichia coli an insect mutualist.

Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host's high adult emergence and improved body colour; these were called 'mutualistic' E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of 'mutualistic' E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host-microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Internal Jugular and Subclavian Vein Thrombosis in a Case of Ovarian Cancer.

Central venous catheter insertion and cancer represent some of the important predisposing factors for deep venous thrombosis (DVT). DVT usually develops in the lower extremities, and venous thrombosis of the upper extremities is uncommon. Early diagnosis and treatment of deep venous thrombosis are of importance, because it is a precursor of complications such as pulmonary embolism and postthrombotic syndrome. A 47-year-old woman visited our department with painful swelling on the left side of her neck. Initial examination revealed swelling of the region extending from the left neck to the shoulder without any redness of the overlying skin. Laboratory tests showed a white blood cell count of 5,800/mm3 and an elevated serum C-reactive protein of 4.51 mg/dL. Computed tomography (CT) of the neck revealed a vascular filling defect in the left internal jugular vein to left subclavian vein region, with the venous lumina completely occluded with dense soft tissue. On the basis of the findings, we made the diagnosis of thrombosis of the left internal jugular and left subclavian veins. The patient was begun on treatment with oral rivaroxaban, but the left shoulder pain worsened. She was then admitted to the hospital and treated by balloon thrombectomy and thrombolytic therapy, which led to improvement of the left subclavian venous occlusion. Histopathologic examination of the removed thrombus revealed adenocarcinoma cells, indicating hematogenous dissemination of malignant cells.

Obligate bacterial mutualists evolving from environmental bacteria in natural insect populations.

Diverse organisms are associated with obligate microbial mutualists. How such essential symbionts have originated from free-living ancestors is of evolutionary interest. Here we report that, in natural populations of the stinkbug Plautia stali, obligate bacterial mutualists are evolving from environmental bacteria. Of six distinct bacterial lineages associated with insect populations, two are uncultivable with reduced genomes, four are cultivable with non-reduced genomes, one uncultivable symbiont is fixed in temperate populations, and the other uncultivable symbiont coexists with four cultivable symbionts in subtropical populations. Symbiont elimination resulted in host mortality for all symbionts, while re-infection with any of the symbionts restored normal host growth, indicating that all the symbionts are indispensable and almost equivalent functionally. Some aseptic newborns incubated with environmental soils acquired the cultivable symbionts and normal growth was restored, identifying them as environmental Pantoea spp. Our finding uncovers an evolutionary transition from a free-living lifestyle to obligate mutualism that is currently ongoing in nature.

Draft Genome Sequence of "Candidatus Phytoplasma asteris" Strain OY-V, an Unculturable Plant-Pathogenic Bacterium.

Phytoplasmas are unculturable plant-pathogenic bacteria causing devastating damage to agricultural production worldwide. Here, we report the draft genome sequence of "Candidatus Phytoplasma asteris" strain OY-V. Most of the known virulence factors and host-interacting proteins were conserved in OY-V. This genome furthers our understanding of genetic diversity and pathogenicity of phytoplasmas.

Diversity of bacterial endosymbionts associated with Macrosteles leafhoppers vectoring phytopathogenic phytoplasmas.

Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola," and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. "Ca. Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, "Candidatus Phytoplasma asteris," associated with aster yellows and related plant diseases, and "Candidatus Phytoplasma oryzae," associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.

New ex vivo reporter assay system reveals that σ factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants.

Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. "Candidatus Phytoplasma asteris", onion yellows strain M (OY-M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ~850 kb genome. The OY-M genome encodes two sigma (σ) factors, RpoD and FliA, that are homologous to Escherichia coli σ(70) and σ(28) , respectively. Previous studies show that gene expression of OY-M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two σ factors and gene regulatory systems in OY-M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli-based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY-M-derived σ factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma-derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria.

Dramatic transcriptional changes in an intracellular parasite enable host switching between plant and insect.

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner.

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

Process of reductive evolution during 10 years in plasmids of a non-insect-transmissible phytoplasma.

A non-insect-transmissible phytoplasma strain (OY-NIM) was obtained from insect-transmissible strain OY-M by plant grafting using no insect vectors. In this study, we analyzed for the gene structure of plasmids during its maintenance in plant tissue culture for 10 years. OY-M strain has one plasmid encoding orf3 gene which is thought to be involved in insect transmissibility. The gradual loss of OY-NIM plasmid sequence was observed in subsequent steps: first, the promoter region of orf3 was lost, followed by the loss of then a large region including orf3, and finally the entire plasmid was disappeared. In contrast, no mutation was found in a pseudogene on OY-NIM chromosome in the same period, indicating that OY-NIM plasmid evolved more rapidly than the chromosome-encoded gene tested. Results revealed an actual evolutionary process of OY plasmid, and provide a model for the stepwise process in reductive evolution of plasmids by environmental adaptation. Furthermore, this study indicates the great plasticity of plasmids throughout the evolution of phytoplasma.

In the non-insect-transmissible line of onion yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region.

'Candidatus Phytoplasma asteris', onion yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium.

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

Cloning of immunodominant membrane protein genes of phytoplasmas and their in planta expression.

Phytoplasmas are plant pathogenic bacteria that cause devastating yield losses in diverse crops worldwide. Although the understanding of the pathogen biology is important in agriculture, the inability to culture phytoplasmas has hindered their full characterization. Previous studies demonstrated that immunodominant membrane proteins could be classified into three types, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp), and they are nonhomologous to each other. Here, cloning and sequencing of imp-containing genomic fragments were performed for several groups of phytoplasma including the aster yellows and rice yellow dwarf groups, for which an imp sequence has not previously been reported. Sequence comparison analysis revealed that Imps are highly variable among phytoplasmas, and clear positive selection was observed in several Imps, suggesting that Imp has important roles in host-phytoplasma interactions. As onion yellows (OY) phytoplasma was known to have Amp as the immunodominant membrane protein, the protein accumulation level of Imp in planta was measured compared with that of Amp. The resulting accumulation of Imp was calculated as approximately one-tenth that of Amp, being consistent with the immunodominant property of Amp in OY. It is suggested that an ancestral type of immunodominant membrane protein could be Imp, and subsequently the expression level of Amp or IdpA is increased in several phytoplasma groups.

Heterogeneic dynamics of the structures of multiple gene clusters in two pathogenetically different lines originating from the same phytoplasma.

Phytoplasmas are phloem-limited plant pathogens that are transmitted by insect vectors and are associated with diseases in hundreds of plant species. Despite their small sizes, phytoplasma genomes have repeat-rich sequences, which are due to several genes that are encoded as multiple copies. These multiple genes exist in a gene cluster, the potential mobile unit (PMU). PMUs are present at several distinct regions in the phytoplasma genome. The multicopy genes encoded by PMUs (herein named mobile unit genes [MUGs]) and similar genes elsewhere in the genome (herein named fundamental genes [FUGs]) are likely to have the same function based on their annotations. In this manuscript we show evidence that MUGs and FUGs do not cluster together within the same clade. Each MUG is in a cluster with a short branch length, suggesting that MUGs are recently diverged paralogs, whereas the origin of FUGs is different from that of MUGs. We also compared the genome structures around the lplA gene in two derivative lines of the 'Candidatus Phytoplasma asteris' OY strain, the severe-symptom line W (OY-W) and the mild-symptom line M (OY-M). The gene organizations of the nucleotide sequences upstream of the lplA genes of OY-W and OY-M were dramatically different. The tra5 insertion sequence, an element of PMUs, was found only in this region in OY-W. These results suggest that transposition of entire PMUs and PMU sections has occurred frequently in the OY phytoplasma genome. The difference in the pathogenicities of OY-W and OY-M might be caused by the duplication and transposition of PMUs, followed by genome rearrangement.

Presence of two glycolytic gene clusters in a severe pathogenic line of Candidatus Phytoplasma asteris.

SUMMARY: Phytoplasmas are plant-pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY-M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30-kb region including the glycolytic genes was tandemly duplicated in the genome of OY-W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop-codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full-length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6-phosphofructokinase is known as a rate-limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY-W and OY-M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY-W was higher than that of OY-M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.