Applied and Environmental Microbiology

ABSTRACT In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium “ Candidatus Bealeia paramacronuclearis” occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, “ Candidatus Fokinia cryptica,” whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that “ Candidatus Bealeia paramacronuclearis” clusters with the so-called “basal” Rickettsiales , and “ Candidatus Fokinia cryptica” belongs to “ Candidatus Midichloriaceae.” We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae , Anaplasmataceae , and “ Candidatus Midichloriaceae” (RAM clade), and the other represented by “basal Rickettsiales ,” including “ Candidatus Bealeia paramacronuclearis.” Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise “basal Rickettsiales ” to an independent order, Holosporales ord. nov., inside Alphaproteobacteria , which presently includes four family-level clades. Additionally, we define the family “ Candidatus Hepatincolaceae” and redefine the family Holosporaceae . IMPORTANCE In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host (Ciliophora, Alveolata). Both symbionts belong to “traditional” Rickettsiales , one representing a new species of the genus “ Candidatus Fokinia” (“ Candidatus Midichloriaceae”), and the other representing a new genus of a “basal” Rickettsiales . According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of “traditional” Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.

ABSTRACT The uncultured bacterial symbiont “ Candidatus Endobugula sertula” is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina . The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of “ Ca . Endobugula sertula.” We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS , is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that “ Ca . Endobugula sertula” may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for “ Ca . Endobugula sertula” to be transmitted horizontally. IMPORTANCE The bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for “ Ca . Endobugula sertula” shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously.

ABSTRACT Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer “ Candidatus Nitrosotalea devanaterra” provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of “ Ca . Nitrosotalea devanaterra” reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that “ Ca . Nitrosotalea devanaterra” contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography–mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of “ Ca . Nitrosotalea devanaterra” were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization.

ABSTRACT The Rickettsiales ( Alphaproteobacteria ) are obligate intracellular bacteria that colonize a wide range of eukaryotic hosts, including diverse metazoa and protists. Here, we characterize rickettsial endosymbionts discovered in the cytoplasm of the algivorous amoeboflagellates Viridiraptor invadens and Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria), supplying evidence of free-living, phagotrophic members of the Cercozoa serving as hosts for Rickettsiales . According to 16S rRNA gene phylogenies, the bacteria represent two closely related but distinct genotypes within a deep-branching rickettsial clade, which contains the genera “ Candidatus Odyssella,” “ Candidatus Paracaedibacter,” and “ Candidatus Captivus.” Using the full-cycle rRNA approach, we detected the novel bacteria in four of nine viridiraptorid strains tested. Furthermore, two specific oligonucleotide probes with a single-nucleotide-difference discriminated both bacterial genotypes by fluorescence in situ hybridization (FISH). We establish the candidate species “ Candidatus Finniella inopinata” (found in Viridiraptor invadens ) and “ Candidatus Finniella lucida” (found in Orciraptor agilis ) for the novel bacteria and propose a new, provisional family of Rickettsiales , “ Candidatus Paracaedibacteraceae.”

ABSTRACT Thermoproteales (phylum Crenarchaeota ) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 μM As) and relate this organism to geochemical processes occurring in situ . Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.

ABSTRACT Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “ Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri . We investigated the infection density dynamics of “ Ca . Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “ Ca . Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “ Ca . Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “ Ca . Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10 3 to 10 7 bacteria per insect. In spring, the infection densities were low in March, at ∼10 3 bacteria per insect, increasing up to 10 6 to 10 7 bacteria per insect in April and May, and decreasing to 10 5 to 10 6 bacteria per insect in late May, whereas the infection densities were constantly ∼10 6 to 10 7 bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “ Ca . Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “ Ca . Liberibacter asiaticus”-infected insects suggested that (i) “ Ca . Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10 6 bacteria per insect) of “ Ca . Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “ Ca . Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.

ABSTRACT Large sulfur-oxidizing bacteria in the family Beggiatoaceae are important players in the global sulfur cycle. This group contains members of the well-known genera Beggiatoa , Thioploca , and Thiomargarita but also recently identified and relatively unknown candidate taxa, including “ Candidatus Thiopilula” spp. and “ Ca . Thiophysa” spp. We discovered a population of “ Ca . Thiopilula” spp. colonizing cold seeps near Barbados at a ∼4.7-km water depth. The Barbados population consists of spherical cells that are morphologically similar to Thiomargarita spp., with elemental sulfur inclusions and a central vacuole, but have much smaller cell diameters (5 to 40 μm). Metatranscriptomic analysis revealed that when exposed to anoxic sulfidic conditions, Barbados “ Ca . Thiopilula” organisms expressed genes for the oxidation of elemental sulfur and the reduction of nitrogenous compounds, consistent with their vacuolated morphology and intracellular sulfur storage capability. Metatranscriptomic analysis further revealed that anaerobic methane-oxidizing and sulfate-reducing organisms were active in the sediment, which likely provided reduced sulfur substrates for “ Ca . Thiopilula” and other sulfur-oxidizing microorganisms in the community. The novel observations of “ Ca . Thiopilula” and associated organisms reported here expand our knowledge of the globally distributed and ecologically successful Beggiatoaceae group and thus offer insight into the composition and ecology of deep cold seep microbial communities.

ABSTRACT The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as “ Methanoplasmatales ”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “ Candidatus Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “ Ca . Methanoplasma termitum” lacks the entire pathway for CO 2 reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae . Instead, all strains possess a novel complex that is related to the F 420 :methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F 420 -oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila . Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales , which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.

ABSTRACT “ Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “ Ca . Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “ Ca . Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “ Ca . Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity ( V max of 10 μmol min −1 mg −1 protein, K m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “ Ca . Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.