Publications

4539

ABSTRACTHere we investigated the bacterial endosymbionts of weevils of the genusCurculio. From all four species ofCurculioweevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in theGammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such asNardonellaspp. andSodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont ofCurculioweevils and proposed the designation “CandidatusCurculioniphilus buchneri.” In addition to “Ca.Curculioniphilus,” we identifiedSodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil,Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that suchSodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor ofSitophilusgrain weevils.

Abstract Background Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. Results To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. Conclusion This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task.

The citrus disease Huanglongbing (HLB) is highly destructive in many citrus-growing regions of the world. The putative causal agent of this disease, ‘Candidatus Liberibacter asiaticus’, is difficult to culture, and Koch's postulates have not yet been fulfilled. As a result, efforts have focused on obtaining the genome sequence of ‘Ca. L. asiaticus’ in order to give insight on the physiology of this organism. In this work, three next-generation high-throughput sequencing platforms, 454, Solexa, and SOLiD, were used to obtain metagenomic DNA sequences from phloem tissue of Florida citrus trees infected with HLB. A culture-independent, polymerase chain reaction (PCR)-independent analysis of 16S ribosomal RNA sequences showed that the only bacterium present within the phloem metagenome was ‘Ca L. asiaticus’. No viral or viroid sequences were identified within the metagenome. By reference assembly, the phloem metagenome contained sequences that provided 26-fold coverage of the ‘Ca. L. asiaticus’ contigs in GenBank. By the same approach, phloem metagenomic data yielded less than 0.2-fold coverage of five other alphaproteobacterial genomes. Thus, phloem metagenomic DNA provided a PCR-independent means of verifying the presence of ‘Ca L. asiaticus’ in infected tissue and strongly suggests that no other disease agent was present in phloem. Analysis of these metagenomic data suggest that this approach has a detection limit of one ‘Ca. Liberibacter’ cell for every 52 phloem cells. The phloem sample sequenced here is estimated to have contained 1.7 ‘Ca. Liberibacter’ cells per phloem cell.