Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.     

Phylogenetic placement of the Spirosomaceae

Comparative analysis of 16S rRNA sequences shows that the family Spirosomaceae belongs within the eubacterial phylum defined by the flavobacteria and bacteriodes. Its constituent genera, Spirosoma, Flectobacillus, and Runella form a monophyletic grouping therein. The phylogenetic assignment is based not only upon evolutionary distance analysis, but also upon sequence signatures and higher order structural synapomorphies in 16S rRNA. Another genus peripherally associated with the Spirosomaceae, Ancylobacter ("Microcyclus"), does not cluster with the flavobacteria and their relatives, but rather belongs to the alpha subdivision of the purple bacteria.     

Phylogenetically distant symbiotic microorganisms reside in Glossina midgut and ovary tissues

Abstract. Many blood-feeding insects, including tsetse flies (Diptera: Glossinidae), harbour intracellular bacterial symbionts. Using isolates from tissues of several Glossina species and diagnostic DNA oligonucleotide primers, a polymerase chain reaction (PCR) based assay was designed to identify symbiotic bacteria. Those inhabiting the midgut of Glossina were found to belong to the gamma subdivision, whereas ovarian Proteobacteria were of the alpha subdivision - probably genus Wolbachia (Rickettsiaceae). The presence of Wolbachia-like Rickettsia in the ovaries of G. morsitans subspecies may help to explain the maternally inherited incompatibility of some crosses within this species.     

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.     

Infectivity, transmission and 16S rRNA sequencing of a rickettsia, Coxiella cheraxi sp. nov., from the freshwater crayfish Cherax quadricarinatus

A rickettsia-like organism isolated from infected, farm-reared Cherax quadricarinatus was cultured in the yolk sac of developing chicken eggs, but could not be cultured in 3 continuous cell lines, bluegill fry (BF-2), fathead minnow (FHM), and Spodoptera frugiperda (Sf-9). The organism was confirmed by fulfilling Koch's postulates as the aetiological agent of mortalities amongst C. quadricarinatus. When C. quadricarinatus was inoculated with the organism, mortality was 100% at 28 degrees C and 80% at an ambient temperature of 24 degrees C. Horizontal transmission with food and via the waterborne route was demonstrated, but mortalities were lower at 30 and 10% respectively over a 4 wk period. The 16S rRNA sequence of 1325 base pairs of the Gram-negative, obligate intracellular organism was 95.6% homologous to Coxiella burnetii. Of 18 species compared to this rickettsia, the next most closely related bacterium was Legionella pneumophila at 86.7%. The suggested classification of this organism is Order Rickettsiales, family Rickettsiaceae, tribe Rickettsieae, within the genus Coxiella. We suggest it should be named Coxiella cheraxi sp. nov.     

Phylogenetic analysis based on groE shows the closest evolutionary relationship between mitochondria and Rickettsia]

The nucleotide sequence of the groE operon of Rickettsia prowazekii, the obligate intracellular parasite of eukaryotes, was determined. The alignment of DNA-inferred amino acid sequences of the Hsp10 and Hsp60 heat-shock proteins with bacterial and mitochondrial homologues revealed the presence within Hsp60 of signatures shared by mitochondria and rickettsiae. Phylogenetic analysis demonstrated that heat-shock proteins of R. prowazekii are the earliest and the least diverging homologues within the family Rickettsiaceae--a sister group to the monophyletic clade of mitochondria. These results are in good agreement with the data obtained when using other molecular chronometers and show the closest relationship between mitochondria and Rickettsia. The possible nature of obligate intracellular parasitism of rickettsiae has been considered on the basis of the assumption that they and mitochondria could have a common evolutionary origin.     

Bacterial genome chimaerism and the origin of mitochondria

Many studies have sought to determine the origin and evolution of mitochondria. Although the Alphaproteobacteria are thought to be the closest relatives of the mitochondrial progenitor, there is dispute as to what its particular sister group is. Some have argued that mitochondria originated from ancestors of the order Rickettsiales, or more specifically of the Rickettsiaceae family, while others believe that ancestors of the family Rhodospirillaceae are also equally likely the progenitors. To resolve some of these disputes, sequence similarity searches and phylogenetic analyses were performed against mitochondria-related proteins in Saccharomyces cerevisiae. The 86 common matches of 5 Alphaproteobacteria (Rickettsia prowazekii, Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Ochrobactrum anthropi) to yeast mitochondrial proteins were distributed fairly evenly among the 5 species when sorted by highest identity or score. Moreover, exploratory phylogenetic analyses revealed that among these common matches, 44.19% (38) had branched most closely with O. anthropi, while only 34.88% (30) corresponded with Rickettsia prowazekii. More detailed phylogenetic analyses with additional Alphaproteobacteria and including genes from the mitochondria of Reclinomonas americana found matches of mitochondrial genes to those of members of the Rickettsiaceae, Anaplasmataceae, and Rhodospirillaceae families. The results support the idea that notable bacterial genome chimaerism has occurred en route to the formation of mitochondria.     

Phylogenetics and evolution of the aphid genus Uroleucon based on mitochondrial and nuclear DNA sequences

The genus Uroleucon, and the related genus Macrosiphoniella, represent a large Tertiary radiation of aphids, with a total of about 300 species distributed throughout the world, primarily on host plant species in the family Asteraceae. A molecular phylogenetic study was conducted to identify major clades within Uroleucon and to address the cladistic validity of current subgeneric categories, the evolution of host plant associations, the age of origin, and intercontinental movements in this genus. The seventeen study species included members of the three major subgenera of Uroleucon, species from Europe and North America, one member of Macrosiphoniella, and two outgroups. Data consisted of DNA sequences for three mitochondrial regions and the nuclear gene EF1alpha, for a total of 4287 sites. Nodes supported strongly in both parsimony and maximum likelihood analyses suggest that: (1) Nearctic Uromelan are a monophyletic group branching near the base of the genus and not related to European Uromelan, (2) the New World subgenus Lambersius is possibly monophyletic but is not a tightly related group and is not closely related to other North American species, and (3) Nearctic members of subgenus Uroleucon are a closely related monophyletic group not allied with Nearctic Uromelan or Lambersius. Instead they represent a separate colonization by an Old World ancestor, as they are nested within a strongly supported clade containing European members of both subgenera Uroleucon and Uromelan. Neither of these subgenera is monophyletic. Molecular clock calculations, based on calibrations of mitochondrial divergences from other insects, suggest that Uroleucon + Macrosiphoniella is a relatively recent radiation, probably no more than 5–10 million years old. Although largely confined to Asteraceae, this clade did not radiate in parallel with its host plants. Rather, lateral movement between lineages of Asteraceae must have occurred repeatedly.     

Retention and attempted mechanical transmission of Ehrlichia risticii by Stomoxys calcitrans

Abstract. The ability of adult Stomoxys calcitrans (L.) (Diptera: Muscidae) to retain viable Ehrlichia risticii (Rickettsiaceae), the aetiologic agent of Potomac horse fever (PHF), and mechanically transmit the pathogen from citrated bovine blood artificially infected with E.risticii to susceptible mice was studied. Viable E. risticii were found in the digestive tract of S.calcitrans 3h after the flies had engorged to repletion on infected blood; however, no E. risticii were detected in flies ≥2 days after feeding. Subsequently, groups of S.calcitrans were fed for 20 s on infected blood, then fed to repletion on mice 30, 60,130, 180 or 220 min after having feeding interrupted. Mice displayed no clinical signs of PHF and did not produce anti- E. risticii antibodies when assayed 30 days after S. calcitrans had fed. Although S.calcitrans were able to harbour viable E.risticii for at least 3h, transmission of the disease agent to susceptible mice during interrupted feeding was not demonstrated under these experimental conditions.     

A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast

The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts. Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C. burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria. The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.     

Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution

The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris'', a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris'' genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

Molecular link of different stages of the trematode host of Neorickettsia risticii to Acanthatrium oregonense

Neorickettsia risticii , the obligatory intracellular bacterium that causes Potomac horse fever, has been detected in various developmental stages of digenetic trematodes in the environment. Neorickettsia risticii -infected gravid trematodes were identified as Acanthatrium oregonense , based on morphologic keys. However, whether immature trematodes harbouring N. risticii are also A. oregonense was unknown. The objective of this study was to infer the life cycle of N. risticii -positive trematode hosts and transstadial transmission of the bacterium by molecularly characterizing the relationship among adult and immature stages of trematodes confirmed infected with N. risticii . Sequences of 18S ribosomal RNA genes up to 1922 bp in size were obtained from infected adult gravid trematodes, sporocysts and cercariae, and metacercariae. The sequences from the different immature stages of trematode are closely related to those of adult trematodes, some with 100% sequence identity; thus, they likely are life stages of A. oregonense . Comparisons with known 18S ribosomal RNA gene sequences of other digenetic trematodes indicated that all tested stages of the N. risticii -positive trematodes belong to the family Lecithodendriidae, supporting the morphological identification.     

Evolutionary relationship of Rickettsiae and mitochondria.

Phylogenetic data support an origin of mitochondria from the alpha-proteobacterial order Rickettsiales. This high-rank taxon comprises exceptionally obligate intracellular endosymbionts of eukaryotic cells, and includes family Rickettsiaceae and a group of microorganisms termed Rickettsia-like endosymbionts (RLEs). Most detailed phylogenetic analyses of small subunit rRNA and chaperonin 60 sequences consistently show the RLEs to have emerged before Rickettsiaceae and mitochondria sister clades. These data suggest that the origin of mitochondria and Rickettsiae has been preceded by the long-term mutualistic relationship of an intracellular bacterium with a pro-eukaryote, in which an invader has lost many dispensable genes, yet evolved carrier proteins to exchange respiration-derived ATP for host metabolites as envisaged in classic endosymbiont theory.     

100th anniversary of the death of Ricketts: Howard Taylor Ricketts (1871-1910). The namesake of the Rickettsiaceae family

The US American pathologist and microbiologist Howard Taylor Ricketts died 100 years ago. He is renowned for discovering the causative organism and the transmission route of Rocky Mountain spotted fever and of tabardillo--an epidemic louse-borne typhus occurring especially in Mexico. He also found that both diseases were caused by related infectious agents (Rickettsia rickettsii and Rickettsia prowazekii). The scientific community therefore named both a taxonomic family (Rickettsiaceae) and an order (Rickettsiales) after the scientist. Ricketts' work on immunity and serums became the basis for further advances in vaccine development.     

Characterization and localization of Rickettsia sp. in water beetles of genus Deronectes (Coleoptera: Dytiscidae)

In the present study, Rickettsia sp. was detected in four water beetles of the genus Deronectes ( Dytiscidae ) for the first time. Rickettsiae were found in 100% of examined specimens of Deronectes platynotus (45/45), 39.4% of Deronectes aubei (28/71), 40% of Deronectes delarouzei (2/5) and 33.3% of Deronectes semirufus (1/3). Analysis of 16S rRNA gene sequences revealed a phylogenetic relationship with rickettsial isolates of Limonia chorea ( Diptera ), tentatively classified as members of the basal ancestral group. Phylogenetic analysis of the gltA (citrate synthase) gene sequences showed that Deronectes symbionts were closest to bacterial symbionts from spiders. Ultrastructural examinations revealed typical morphological features and intracellular arrangements of rickettsiae. The distribution, transmission and localization of Rickettsia sp. in D. platynotus were studied using a diagnostic PCR assay and FISH. Eggs from infected females of D. platynotus were all Rickettsia -positive, indicative of a vertical transmission.     

A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data

The cestode family Taeniidae consists of 2 genera, Taenia and Echinococcus, which both have been the focus of intensive taxonomic and epidemiological studies because of their zoonotic importance. However, a comprehensive molecular phylogeny of this family has yet to be reconstructed. In this study, 54 isolates representing 9 Taenia species were characterized using DNA sequences in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Phylogenetic relationships within the family Taeniidae were inferred by combining cox1 and nad1 sequence data of the present and previous studies. In the phylogenetic analysis, the genus Echinococcus was shown to be monophyletic, but Taenia proved to be paraphyletic due to the position of T. mustelae as a probable sister taxon of Echinococcus. This indicates that T. mustelae should form a genus of its own. Taenia ovis krabbei was placed distant from T. ovis ovis, as a sister taxon of T. multiceps, supporting its recognition as a distinct species, T. krabbei. High intraspecific sequence variation within both T. polyacantha and T. taeniaeformis suggests the existence of cryptic sister species.     

Cellular tropism, population dynamics, host range and taxonomic status of an aphid secondary symbiont, SMLS (Sitobion miscanthi L type symbiont)

SMLS (Sitobion miscanthi L type symbiont) is a newly reported aphid secondary symbiont. Phylogenetic evidence from molecular markers indicates that SMLS belongs to the Rickettsiaceae and has a sibling relationship with Orientia tsutsugamushi. A comparative analysis of coxA nucleotide sequences further supports recognition of SMLS as a new genus in the Rickettsiaceae. In situ hybridization reveals that SMLS is housed in both sheath cells and secondary bacteriocytes and it is also detected in aphid hemolymph. The population dynamics of SMLS differ from those of Buchnera aphidicola and titer levels of SMLS increase in older aphids. A survey of 13 other aphids reveals that SMLS only occurs in wheat-associated species.     

Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach

The diversity of purple and green sulfur bacteria in the multilayered sediments of the Ebro Delta was investigated. Specific oligonucleotide primers for these groups were used for the selective amplification of 16S rRNA gene sequences. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis and sequenced, which yielded a total of 32 sequences. Six of the sequences were related to different cultivated members of the green sulfur bacteria assemblage, whereas seven fell into the cluster of marine or halophilic Chromatiaceae. Six sequences were clustered with the family Ectothiorhodospiraceae, three of the six being closely related to chemotrophic bacteria grouped together with Halorhodospira genus, and the other three forming a group related to the genus Ectothiorhodospira. The last thirteen sequences constituted a cluster where no molecular isolate from microbial mats has so far been reported. Our results indicate that the natural diversity in the ecosystem studied has been significantly underestimated in the past and point out the presence of novel species not related to all known purple sulfur bacteria. Furthermore, the detection of green sulfur bacteria, after only an initial step of enrichment, suggests that -- with the appropriate methodology -- several genera, such as Prosthecochloris, could be established as regular members of marine microbial mats.