Publications

4467

Phytopathogenic mollicutes, which include spiroplasmas and phytoplasmas, are cell wall-less bacteria that parasitize plant hosts and insect vectors. Knowledge of the evolution of these agents is important in understanding their biology. The availability of the first complete phytoplasma and several partial spiroplasma and phytoplasma genome sequences made possible an investigation of evolutionary relationships between phytopathogenic mollicutes and other micro-organisms, especially Gram-positive bacteria, using a comparative genomics approach. Genome data from a total of 41 bacterial species were used in the analysis. Sixty-one conserved proteins were selected from each species for the construction of a hypothetical phylogenetic tree. The genes encoding these selected proteins are among a core of genetic elements that constitute a hypothetical minimal genome. The proteins were concatenated into five superproteins according to their functional categories, and phylogenetic trees were reconstructed using distance, parsimony and likelihood methods. Phylogenetic trees based on the five sets of concatenated proteins were congruent in both clade topology and relative branching length.Spiroplasma kunkeliiand phytoplasmas clustered together with other mollicutes, forming a monophyletic group. Phytoplasmas diverged from spiroplasmas and mycoplasmas at early stages in the evolution of mollicutes. Branch lengths on the phylogenetic trees were noticeably longer in theMollicutesclade, suggesting that the genes encoding the five sets of proteins evolved at a greater rate in this clade than in other clades. This observation reinforces the concept that mollicutes have rapidly evolving genomes.

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by ‘Candidatus Liberibacter asiaticus' and in Africa by ‘Candidatus Liberibacter africanus’. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. ‘Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the α-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the α-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus ‘Ca. Liberibacter’. However, while the 16S rRNA gene sequences of ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had 98·4 % similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96·0 % similarity with the 16S rRNA gene sequences of ‘Ca. L. asiaticus' or ‘Ca. L. africanus’. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the ‘Ca. L asiaticus’/‘Ca. L. africanus group’, but as a separate branch. Within the genus ‘Candidatus Liberibacter’ and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of ‘Ca. L. asiaticus’, from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had quite different sequences, with similarity between 66·0 and 79·5 %. These results confirm that the SPS-HLB liberibacter is a novel species for which the name ‘Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the ‘American’ liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of ‘Ca. L. asiaticus’, suggesting that this psyllid is also a vector of ‘Ca. L. americanus' in SPS. ‘Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while ‘Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that ‘Ca. L. americanus' is the major cause of HLB in SPS.

The distinct lifestyle of obligately intracellular bacteria can alter fundamental forces that drive and constrain genome change. In this study, sequencing the 792-kb genome of Blochmannia pennsylvanicus, an obligate endosymbiont of Camponotus pennsylvanicus, enabled us to trace evolutionary changes that occurred in the context of a bacterial–ant association. Comparison to the genome of Blochmannia floridanus reveals differential loss of genes involved in cofactor biosynthesis, the composition and structure of the cell wall and membrane, gene regulation, and DNA replication. However, the two Blochmannia species show complete conservation in the order and strand orientation of shared genes. This finding of extreme stasis in genome architecture, also reported previously for the aphid endosymbiont Buchnera, suggests that genome stability characterizes long-term bacterial mutualists of insects and constrains their evolutionary potential. Genome-wide analyses of protein divergences reveal 10- to 50-fold faster amino acid substitution rates in Blochmannia compared to related bacteria. Despite these varying features of genome evolution, a striking correlation in the relative divergences of proteins indicates parallel functional constraints on gene functions across ecologically distinct bacterial groups. Furthermore, the increased rates of amino acid substitution and gene loss in Blochmannia have occurred in a lineage-specific fashion, which may reflect life history differences of their ant hosts.

ABSTRACT The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “ Candidatus Consessoris aphidicola” for T type and “ Candidatus Adiaceo aphidicola” for U type.

The taxonomic identity of the hereditary prokaryotic symbiont of the olive flyBactrocera oleae(Diptera: Tephritidae) was investigated. In order to avoid superficial microbial contaminants and loosely associated saprophytic biota, flies were surface-sterilized at the larval stage and reared under aseptic conditions until adult emergence.B. oleaeflies originating from different geographical locations and collected at different times of the year were tested. Bacterial isolation was undertaken from the cephalic oesophageal bulb, which is known to be a specific site of accumulation for the hosted microsymbionts in the adult insect. Despite evidence of multiplication cycles taking place within the insect, attempts at cultivation of the isolated bacteriaex situwere not productive at any stage, leading to the choice of unculturable status definition. PCR amplification and nucleotide sequencing of the entire 16S rRNA gene consistently yielded a single sequence that displayed marked similarity with enterobacterial lineages, with closest matches (97 %) toErwinia persicinaandErwinia rhapontici. The novel taxon differs from common intestinal bacterial species of fruit flies and from instances of culturable bacteria previously described inB. oleaeraised without sterility precautions, which we also observed as minority occupants or occasional contaminants. The symbiont's identity is also distinct fromPseudomonas savastanoi. In all observations, the numerically dominant inhabitant of the olive fly oesophageal organ was the same unculturable organism, whose presence at later stages was also regularly observed in the midgut. A novel species is proposed, by virtue of its unique properties, under the designation ‘CandidatusErwinia dacicola’.

Bacteria called ‘Fritschea’ are endosymbionts of the plant-feeding whitefly Bemisia tabaci and scale insect Eriococcus spurius. In the gut of B. tabaci, these bacteria live within bacteriocyte cells that are transmitted directly from the parent to oocytes. Whiteflies cause serious economic damage to many agricultural crops; B. tabaci fecundity and host range are less than those of Bemisia argentifolii, possibly due to the presence of this endosymbiont. The B. tabaci endosymbiont has been characterized using electron microscopy and DNA analysis but has not been isolated or propagated outside of insects. The present study compared sequences for 11 endosymbiont genes to genomic data for chlamydial families Parachlamydiaceae, Chlamydiaceae and Simkaniaceae and to 16S rRNA gene signature sequences from 330 chlamydiae. We concluded that it was appropriate to propose ‘Candidatus Fritschea bemisiae’ strain Falk and ‘Candidatus Fritschea eriococci’ strain Elm as members of the family Simkaniaceae in the Chlamydiales.