Whole genome-based assessment of the taxonomic position of the arthropod pathogenic bacterium Rickettsiella grylli

Rickettsiella grylli is an intracellular bacterial pathogen of aquatic and terrestrial arthropods. Previous determination of its 16S rRNA-encoding sequence has led to the taxonomic classification of the genus Rickettsiella in the class Gammaproteobacteria, order Legionellales, family Coxiellaceae, i.e. in close vicinity to vertebrate pathogenic bacteria of the genera Coxiella and Legionella. Here we use the additional information available from the recently published first whole genome sequence from this genus to evaluate critically the taxonomic classification of R. grylli beyond the 16S rRNA gene level. Using phylogenetic reconstruction, together with significance testing on a data basis defined by a core set of 211 previously identified families of protein-encoding genes, together with a reanalysis of 16S rRNA gene data, the present study firmly corroborates the assignment of this species to both the class Gammaproteobacteria and the order Legionellales. However, the results obtained from concatenated and single protein, single protein-encoding gene, and 16S rRNA gene data demonstrate a similar phylogenetic distance of R. grylli to both the Coxiellaceae and the Legionellaceae and are, therefore, inconsistent with its current family-level classification. Consequently, a respective reorganization of the order Legionellales is proposed.     

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.     

A Rickettsiella bacterium from the hard tick, Ixodes woodi: molecular taxonomy combining multilocus sequence typing (MLST) with significance testing

Hard ticks (Acari: Ixodidae) are known to harbour intracellular bacteria from several phylogenetic groups that can develop both mutualistic and pathogenic relationships to the host. This is of particular importance for public health as tick derived bacteria can potentially be transmitted to mammals, including humans, where e.g. Rickettsia or Coxiella act as severe pathogens. Exact molecular taxonomic identification of tick associated prokaryotes is a necessary prerequisite of the investigation of their relationship to both the tick and possible vertebrate hosts. Previously, an intracellular bacterium had been isolated from a monosexual, parthenogenetically reproducing laboratory colony of females of the hard tick, Ixodes woodi Bishopp, and had preliminarily been characterized as a "Rickettsiella-related bacterium". In the present molecular taxonomic study that is based on phylogenetic reconstruction from both 16 S ribosomal RNA and protein-encoding marker sequences complemented with likelihood-based significance testing, the bacterium from I. woodi has been identified as a strain of the taxonomic species Rickettsiella grylli. It is the first time that a multilocus sequence typing (MLST) approach based on a four genes comprising MLST scheme has been implemented in order to classify a Rickettsiella-like bacterium to this species. The study demonstrated that MLST holds potential for a better resolution of phylogenetic relationships within the genus Rickettsiella, but requires sequence determination from further Rickettsiella-like bacteria in order to complete the current still fragmentary picture of Rickettsiella systematics.     

'Candidatus Rhabdochlamydia crassificans', an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea)

The genus Rickettsiella comprises various intracellular bacterial pathogens of arthropods, exhibiting a chlamydia-like developmental cycle. Species may be divided into two main groups, the R. popilliae-R. grylli group and the R. chironomi group. Previous phylogenetic studies based on the 16S ribosomal RNA encoding gene showed that two Rickettsiella species, one from each group, belong in reality to two distantly related lineages, the gamma-Proteobacteria (R. grylli) and the Chlamydiales ('Candidatus Rhabdochlamydia porcellionis', a pathogen of terrestrial isopods). In the present work, the 16S rDNA sequence of another Rickettsiella-like species, causing abdominal swelling to its cockroach host Blatta orientalis, was determined and phylogenetic analysis performed. Identical 16S rDNA sequences of 1495 nucleotides were obtained from fat body and ovary tissues of both healthy and diseased cockroach individuals. The sequence shared only 73% of similarity with R. grylli, but 82-87% with most Chlamydiales, and even 96.3% with 'Candidatus Rhabdochlamydia porcellionis'. Phylogenetic analyses confirmed the affiliation of the cockroach pathogen within the order Chlamydiales, and based on ultrastructural characteristics and genetic analyses, we propose its inclusion in the 'Candidatus Rhabdochlamydia' as a distinct taxon, 'Candidatus Rhabdochlamydia crassificans'. These results extend our knowledge of the phylogenetic diversity of the Chlamydiales.     

Multilocus Sequence Analysis (MLSA) of ‘Rickettsiella agriotidis’, an Intracellular Bacterial Pathogen of Agriotes Wireworms

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as ‘Rickettsiella agriotidis’. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of ‘R. agriotidis’ to the pathotypes ‘Rickettsiella melolonthae’ and ‘Rickettsiella tipulae’, i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. ‘R. agriotidis’ forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of ‘R. agriotidis’ and further R. popilliae synonyms has been critically evaluated.     

Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa

Rickettsiella tipulae is an intracellular bacterial pathogen of larvae of the crane fly, Tipula paludosa (Diptera: Tipulidae) and has previously been claimed to represent an independent species within the genus Rickettsiella. Recently, this taxon has been reorganized and transferred as a whole from the α-proteobacterial order Rickettsiales to the γ-proteobacterial order Legionellales. Here we present the electron-microscopic identification of this rickettsial pathogen together with the first DNA sequence information for R. tipulae. The results of our 16S rDNA-based phylogenetic analysis demonstrate that the transfer to the order Legionellales is justified for R. tipulae. However, there is no phylogenetic basis to consider R. tipulae an independent species, but instead conclusive evidence substantiating its species level co-assignment with Rickettsiella melolonthae. Furthermore, implications of our results for a possible reorganization of the internal structure of the genus Rickettsiella are discussed.     

Molecular characterization and evolution of arthropod-pathogenic Rickettsiella bacteria

We determined the 16S rRNA gene sequences of three crustacean "Rickettsiella armadillidii" strains. Rickettsiella bacteria overall appear to form a monophyletic group that diverged from Coxiella bacteria approximately 350 million years ago. Therefore, the genus Rickettsiella as a whole (not just Rickettsiella grylli) should be classified among the Gammaproteobacteria instead of the Alphaproteobacteria.     

嗜麦芽寡养单胞菌D2株2套Ⅱ型分泌系统

嗜麦芽寡养单胞菌D2株经前期研究发现有2套Ⅱ型分泌系统（T2SS）,根据嗜麦芽寡养单胞菌K279a、R551—3、JV3、D457的T2SS基因簇序列,以及D2株的部分测序结果设计引物,采用基因移步法逐一扩增2套T2SS基因序列,产物连接至T载体,经酶切鉴定后测序、拼接。发现有22个完整的开放多码框架（ORF）,比对分析后发现T2SSl的基因簇与同种属细菌相应基因簇序列同源性均在80％以上,对应氨基酸序列同源性均能达到97％以上,而T2SS2基因簇与K279a、R551—3对应基因序列和氨基酸序列的同源性均不及T2SS1高。2套T2SS的GspE、F、I基因同源性在50％以上,其他对应基因同源性在35％～68％之间不等,氨基酸序列同源性则为15％～61％。2套他ss与铜绿假单胞菌PA01的同源性高于不同科的小肠结肠炎耶尔森菌。T2SS的序列测定及同源性分析为进一步研究细菌蛋白分泌机制奠定基础。     

Light and electron microscopic study of a Rickettsiella species from the cockroach Blatta orientalis

An infection with Rickettsiella sp. was responsible for an illness causing heavy body swelling in the Oriental cockroach Blatta orientalis. Reproduction of the colony stagnated. Vacuoles with parasitic bacteria occurred mainly in the fat body, but also in nearly all other organs, such as gut epithelium, Malpighian tubules, blood cells, and ovarioles. The parasites clearly differed from the symbiotic bacteria of the genus Blattabacterium, which regularly occur in the mycetocytes of B. orientalis. The vacuoles contained four stages of Rickettsiella: (1) infectious, electron-dense, rod-like elementary bodies (mean size 300 x 145 nm); (2) an electron-dense, flat intermedium stage, called flat body (mean size 515 x 255 x 125 nm); (3) an electron-light, spherical intermedium stage, called condensing sphere (mean size 340 nm); portions of cytoplasm condensed crescent-like at the border or in the center of the cell; and (4) large, spherical, electron-light initial bodies that multiplied by binary fission (mean size 600 nm). The initial bodies had a three-layered cell boundary, but all other stages had a five-layered cell boundary. Elementary and flat bodies contained an electron-light, oblique lamella and an oval structure with an array of ribosome-like granules, respectively. In contrast to other species of Rickettsiella, crystal formation or multiple division did not occur. The described species of Rickettsiella is different from "R. blattae," which belongs to the R. popilliae group. Instead, it shares more similarities with the R. chironomi group. To avoid confusion, it was provisionally named "R. crassificans."     

Pulsed-field gel electrophoresis determination of the genome size of obligate intracellular bacteria belonging to the genera Chlamydia, Rickettsiella, and Porochlamydia.

The chromosome length of obligate intracellular procaryotes was determined by pulsed-field gel electrophoresis of intact or NotI- and SfiI-restricted genomes. Sizes averaged 2,100, 1,720, 1,550, 2,650, and 1,450 kilobases for Rickettsiella grylli, Rickettsiella melolonthae, Porochlamydia buthi, Porochlamydia chironomi, and Chlamydia psittaci and Chlamydia trachomatis, respectively. An SfiI restriction map of the R. melolonthae genome was derived.     

Complete genome sequence of Rhodospirillum rubrum type strain (S1)

Rhodospirillum rubrum (Esmarch 1887) Molisch 1907 is the type species of the genus Rhodospirillum, which is the type genus of the family Rhodospirillaceae in the class Alphaproteobacteria. The species is of special interest because it is an anoxygenic phototroph that produces extracellular elemental sulfur (instead of oxygen) while harvesting light. It contains one of the most simple photosynthetic systems currently known, lacking light harvesting complex 2. Strain S1(T) can grow on carbon monoxide as sole energy source. With currently over 1,750 PubMed entries, R. rubrum is one of the most intensively studied microbial species, in particular for physiological and genetic studies. Next to R. centenum strain SW, the genome sequence of strain S1(T) is only the second genome of a member of the genus Rhodospirillum to be published, but the first type strain genome from the genus. The 4,352,825 bp long chromosome and 53,732 bp plasmid with a total of 3,850 protein-coding and 83 RNA genes were sequenced as part of the DOE Joint Genome Institute Program DOEM 2002.     

Granulosicoccaceae fam. nov., to include Granulosicoccus antarcticus gen. nov., sp. nov., a non-phototrophic, obligately aerobic chemoheterotroph in the order Chromatiales, isolated from Antarctic seawater

A Gram-negative, motile by tuft flagella, obligately aerobic chemoorganoheterotrophic, sphere-form bacterium, designated IMCC3135(T), was isolated from the Antarctic surface seawater of King George Island, West Antarctica. The strain was mesophilic, neutrophilic, and requiring NaCl for growth, but neither halophilic nor halotolerant. The 16S rRNA gene sequence analysis indicated that the strain was most closely related to genera of the order Chromatiales in the class Gammaproteobacteria. The most closely related genera showed less than 90% 16S rRNA gene sequence similarity and included Thioalkalispira (89.9%), Thioalkalivibrio (88.0%-89.5%), Ectothiorhodospira (87.9%-89.3%), Chromatium (88.3%-88.9%), and Lamprocystis (87.7%-88.9%), which represent three different families of the order Chromatiales. Phylogenetic analyses showed that this Antarctic strain represented a distinct phylogenetic lineage in the order Chromatiales and could not be assigned to any of the defined families in the order. Phenotypic characteristics, including primarily non-phototrophic, non-alkaliphilic, non-halophilic, and obligately aerobic chemoheterotrophic properties, differentiated the strain from other related genera. The very low sequence similarities (<90%) and distant relationships between the strain and members of the order suggested that the strain merited classification as a novel genus within a novel family in the order Chromatiales. On the basis of these taxonomic traits, a novel genus and species is proposed, Granulosicoccus antarcticus gen. nov., sp. nov., in a new family Granulosicoccaceae fam. nov. Strain IMCC3135(T) (=KCCM 42676(T)=NBRC 102684(T)) is the type strain of Granulosicoccus antarcticus.     

Genetic and Electron-Microscopic Characterization of ‘<Emphasis Type="Italic">Rickettsiella agriotidis</Emphasis>’, a new <Emphasis Type="Italic">Rickettsiella</Emphasis> Pathotype Associated with Wireworm, <Emphasis Type="Italic">Agriotes</Emphasis> sp. (Coleoptera: Elateridae)

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae), are severe and widespread agricultural pests affecting numerous crops. A previously unknown intracellular bacterium has been identified in a diseased Agriotes larva. Microscopic studies revealed the subcellular structures characteristic of Rickettsiella infections. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the wireworm pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the new pathotype designation ‘R. agriotidis’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that—on the basis of the currently accepted organization of the genus Rickettsiella—this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.     

DNA sequence data reveal a subfamily-level divergence within Thamnophilidae (Aves: Passeriformes)

The Thamnophilidae is a diverse radiation of insectivorous passerine birds that comprises nearly 220 species and is mostly restricted to the lowlands and lower montane forests of the Neotropics. Current classification within Thamnophilidae relies primarily on morphological variation, but recent incorporation of molecular and vocal data has promoted changes at various taxonomic levels. Here we demonstrate that the genus Terenura is polyphyletic because Terenura callinota, T. humeralis, T. spodioptila, and T. sharpei are phylogenetically distant from the type species of the genus, Terenura maculata. More importantly, the former four species are not particularly closely related to any other thamnophilids and represent a clade that is sister to all other members of the family. Because no genus name is available for this previously undetected lineage in the Thamnophilidae, we describe the genus Euchrepomis for callinota, humeralis, spodioptila, and sharpei, and erect the subfamily Euchrepomidinae. We discuss the taxonomic and evolutionary significance of this divergent lineage. This study highlights the importance of taxonomic coverage and the inclusion of type taxa to redefine classifications to reflect accurately evolutionary relationships.     

The phylogenetic problem of Huia (Amphibia: Ranidae)

A taxonomic consensus for the diverse and pan-global frog family Ranidae is lacking. A recently proposed classification of living amphibians [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370] included expansion of the Southeast Asian ranid frog genus Huia from seven to 47 species, but without having studied the type species of Huia. This study tested the monophyly of this concept of Huia by sampling the type species and putative members of Huia. Molecular phylogenetic analyses consistently recovered the type species H. cavitympanum as the sister taxon to other Bornean-endemic species in the genus Meristogenys, rendering all previously published concepts of Huia as polyphyletic. Members of Huia sensu [Frost, D.R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006. The amphibian tree of life. B. Am. Mus. Nat. Hist. 297, 1-370.] appear in four places within the family Ranidae. A clade containing the type species of Odorrana is phylogenetically unrelated to the type species of Huia, and Odorrana is removed from synonymy with Huia. These findings underscore the need to include relevant type species in phylogenetic studies before proposing sweeping taxonomic changes. The molecular phylogenetic analyses revealed a high degree of homoplasy in larval and adult morphology of Asian ranid frogs. Detailed studies are needed to identify morphological synapomorphies that unite members in these major clades of ranid frogs.     

Flavobacterium ginsengiterrae sp. nov., isolated from a ginseng field

A novel strain of Flavobacterium, DCY55(T), a Gram-negative, yellow-pigmented, rod-shaped, non-spore-forming and gliding-motile bacterium, was isolated from the soil of a ginseng field in South Korea. Phylogenetic analysis, based on the 16S rRNA sequence, demonstrated that strain DCY55(T) belongs to the genus Flavobacterium within the family Flavobacteriaceae. Strain DCY55(T) showed the highest similarity with F. johnsoniae UW101(T) (97.1%), F. ginsenosidimutans THG 01(T) (96.8%), F. defluvii EMB 117(T) (96.6%), F. banpakuense 15F3(T) (96.3%) and F. anhuiense D3(T) (95.8%). Chemotaxonomic results showed that strain DCY55(T) predominantly contains menaquinone MK-6, that its DNA G+C content is 36.1mol%, and that its major cellular fatty acids are iso-C(15:0), summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) ω 7c) and C(16:0). The chemotaxonomic and genotypic characteristics support the taxonomic classification of strain DCY55(T) to the genus Flavobacterium. The results of physiological and biochemical tests confirmed that strain DCY55(T) is distinct from previously validated species. We conclude that strain DCY55(T) should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacterium ginsengiterrae sp. nov. is proposed, with the type strain DCY55(T) (=KCTC 23319(T) = JCM 17337(T)).     

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae)

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped “giant bodies” either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation ‘Rickettsiella pyronotae’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that - on the basis of the currently accepted organization of the genus Rickettsiella - this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.