Scientific Reports

AbstractAdvances in culture-independent methods have meant that we can more readily detect and diagnose emerging infectious disease threats in humans and animals. Metagenomics is fast becoming a popular tool for detection and characterisation of novel bacterial pathogens in their environment, and is particularly useful for obligate intracellular bacteria such as Chlamydiae that require labour-intensive culturing. We have used this tool to investigate the microbial metagenomes of Chlamydia-positive cloaca and choana samples from snakes. The microbial complexity within these anatomical sites meant that despite previous detection of chlamydial 16S rRNA sequences by single-gene broad-range PCR, only a chlamydial plasmid could be detected in all samples, and a chlamydial chromosome in one sample. Comparative genomic analysis of the latter revealed it represented a novel taxon, Ca. Chlamydia corallus, with genetic differences in regards to purine and pyrimidine metabolism. Utilising statistical methods to relate plasmid phylogeny to the phylogeny of chromosomal sequences showed that the samples also contain additional novel strains of Ca. C. corallus and two putative novel species in the genus Chlamydia. This study highlights the value of metagenomics methods for rapid novel bacterial discovery and the insights it can provide into the biology of uncultivable intracellular bacteria such as Chlamydiae.

AbstractProduction of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-β-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-β-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-β-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB.

AbstractGenital mycoplasmas, which can be vertically transmitted, have been implicated in preterm birth, neonatal infections, and chronic lung disease of prematurity. Our prior work uncovered 16S rRNA genes belonging to a novel, as-yet-uncultivated mycoplasma (lineage ‘Mnola’) in the oral cavity of a premature neonate. Here, we characterize the organism’s associated community, growth status, metabolic potential, and population diversity. Sequencing of genomic DNA from the infant’s saliva yielded 1.44 Gbp of high-quality, non-human read data, from which we recovered three essentially complete (including ‘Mnola’) and three partial draft genomes (including Trichomonas vaginalis). The completed 629,409-bp ‘Mnola’ genome (Candidatus Mycoplasma girerdii str. UC-B3) was distinct at the strain level from its closest relative, vaginally-derived Ca. M. girerdii str. VCU-M1, which is also associated with T. vaginalis. Replication rate measurements indicated growth of str. UC-B3 within the infant. Genes encoding surface-associated proteins and restriction-modification systems were especially diverse within and between strains. In UC-B3, the population genetic underpinnings of phase variable expression were evident in vivo. Unique among mycoplasmas, Ca. M. girerdii encodes pyruvate-ferredoxin oxidoreductase and may be sensitive to metronidazole. This study reveals a metabolically unique mycoplasma colonizing a premature neonate, and establishes the value of genome-resolved metagenomics in tracking phase variation.

AbstractFree-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as ‘Candidatus Cochliophilus cryoturris’. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

Abstract‘Candidatus Liberibacter asiaticus’ (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the α-proteobacteria. In this study serologically based methods for the detection of CaLas were developed. An anti-outer membrane protein A (OmpA) polyclonal antibody previously produced (in our laboratory) was highly effective for the detection of CaLas from citrus tissues in a simple tissue printing format. The antibody was also used to capture bacteria from periwinkle extracts. About 80% of all field samples analyzed tested positive with both immune tissue printing and qPCR; whereas 95% were positive with at least one of these two methods. When asymptomatic citrus tissues were tested, the tissue printing method gave a higher rate of detection (83%) than the qPCR method (64%). This is consistent with a lower concentration of CaLas DNA, but a higher proportion of viable cells, in the asymptomatic tissues. The immune tissue printing method also highlights the detail of the spatial distribution of ‘Ca. Liberibacter asiaticus’ in diseased citrus tissues. Both the immune capture PCR and immune tissue printing methods offer the advantages of low cost, high throughput, ease of scaling for multiple samples and simplicity over current PCR-based methods for the detection of ‘Ca. Liberibacter asiaticus’.

AbstractCandidatus Liberibacter solanacearum (Lso) is bacterium transmitted by psyllids to Solanaceae and Apiaceae plants. So far, Lso is found in Europe affecting Apiaceae. In the Mediterranean region, Bactericera trigonica is the only known vector of Lso, but the leek-onion psyllid Bactericera tremblayi is another widespread psyllid and potential vector of Lso. Commonly, carrot, leek and potato are cultivated in the same zones and it is uncertain if these psyllid species are able to transmit Lso to potato plants. Here, we assessed the transmission of Lso by B. trigonica and B. tremblayi to potato plants. B. trigonica showed preference to ingest from the phloem, settle and oviposit on carrot and celery but not on potato. This was correlated with high Lso transmission rates to both carrot (80%) and celery (70%) but very low to potato (≤3%). B. tremblayi preferred leek over carrot and potato, the latter being the less preferred host. B. tremblayi readily ingested from the phloem of infected carrots but failed to transmit Lso from carrot to carrot. Our study shows that the risk of Lso transmission from Apiaceae to potato by B. trigonica is very low, and that B. tremblayi is not a likely vector of Lso.

AbstractThe olive fruit fly,Bactrocera oleae, is the most destructive pest of olive orchards worldwide. The monophagous larva has the unique capability of feeding on olive mesocarp, coping with high levels of phenolic compounds and utilizing non-hydrolyzed proteins present, particularly in the unripe, green olives. On the molecular level, the interaction betweenB. oleaeand olives has not been investigated as yet. Nevertheless, it has been associated with the gut obligate symbiotic bacteriumCandidatus Erwinia dacicola. Here, we used aB.oleaemicroarray to analyze the gene expression of larvae during their development in artificial diet, unripe (green) and ripe (black) olives. The expression profiles ofCa. E. dacicolawere analyzed in parallel, using the Illumina platform. Several genes were found overexpressed in the olive fly larvae when feeding in green olives. Among these, a number of genes encoding detoxification and digestive enzymes, indicating a potential association with the ability ofB. oleaeto cope with green olives. In addition, a number of biological processes seem to be activated inCa. E. dacicoladuring the development of larvae in olives, with the most notable being the activation of amino-acid metabolism.

AbstractThe Candidate Phyla Radiation (CPR) is a large group of bacteria, the scale of which approaches that of all other bacteria. CPR organisms are inferred to depend on other community members for many basic cellular building blocks and all appear to be obligate anaerobes. To date, there has been no evidence for any significant respiratory capacity in an organism from this radiation. Here we report a curated draft genome for ‘Candidatus Parcunitrobacter nitroensis’ a member of the Parcubacteria (OD1) superphylum of the CPR. The genome encodes versatile energy pathways, including fermentative and respiratory capacities, nitrogen and fatty acid metabolism, as well as the first complete electron transport chain described for a member of the CPR. The sequences of all of these enzymes are highly divergent from sequences found in other organisms, suggesting that these capacities were not recently acquired from non-CPR organisms. Although the wide respiration-based repertoire points to a different lifestyle compared to other CPR bacteria, we predict similar obligate dependence on other organisms or the microbial community. The results substantially expand the known metabolic potential of CPR bacteria, although sequence comparisons indicate that these capacities are very rare in members of this radiation.

AbstractGut-associated microbiota of ants include Rhizobiales bacteria with affiliation to the genus Bartonella. These bacteria may enable the ants to fix atmospheric nitrogen, but no genomes have been sequenced yet to test the hypothesis. Sequence reads from a member of the Rhizobiales were identified in the data collected in a genome project of the ant Harpegnathos saltator. We present an analysis of the closed 1.86 Mb genome of the ant-associated bacterium, for which we suggest the species name Candidatus Tokpelaia hoelldoblerii. A phylogenetic analysis reveals a relationship to Bartonella and Brucella, which infect mammals. Novel gene acquisitions include a gene for a putative extracellular protein of more than 6,000 amino acids secreted by the type I secretion system, which may be involved in attachment to the gut epithelium. No genes for nitrogen fixation could be identified, but genes for a multi-subunit urease protein complex are present in the genome. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but not in Bartonella, which use blood-borne transmission pathways. We hypothesize that the gain and loss of the urease function is related to transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales.

Abstract“Candidatus Liberibacter asiaticus” (CLas), a non-culturable α-proteobacterium, is associated with citrus Huanglongbing (HLB, yellow shoot disease) currently threatening citrus production worldwide. Here, the whole genome sequence of CLas strain A4 from Guangdong of China was analyzed. Five copies of nrdB, encoding β-subunit of ribonucleotide reductase (RNR), a critical enzyme involving bacterial proliferation, were found. Three nrdB copies were in long form (nrdBL, 1,059 bp) and two were in short form (nrdBS, 378 bp). nrdBS shared >99% identity to 3′ end of nrdBL and had no active site. Sequences of CLas nrdB genes formed a distinct monophyletic lineage among eubacteria. To make use of the high copy number feature, a nrdB-based primer set RNRf/RNRr was designed and evaluated using real-time PCR with 262 HLB samples collected from China and USA. Compared to the current standard primer set HLBas/HLBr derived from the 16S rRNA gene, RNRf/RNRr had Ct value reductions of 1.68 (SYBR Green PCR) and 1.77 (TaqMan PCR), thus increasing the detection sensitivity three-fold. Meanwhile, RNRf/RNRr was more than twice the stability of primer set LJ900f/LJ900r derived from multi-copy prophage. The nrdB-based PCR thereby provides a sensitive and reliable CLas detection with broad application, especially for the early diagnosis of HLB.