Alistair C. Darby

Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance

Abstract.  1. Pea aphids, Acyrthosiphon pisum, harbour a range of facultative accessory bacteria (secondary symbionts), including those informally known as PASS (R‐type), PAR, PABS (T‐type), and PAUS (U‐type).

The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans

The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.

The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host–cell interaction genes

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host–cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host–cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

Application of next-generation sequencing technologies in virology

The progress of science is punctuated by the advent of revolutionary technologies that provide new ways and scales to formulate scientific questions and advance knowledge. Following on from electron microscopy, cell culture and PCR, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. Possibilities for these methodologies are only limited by our scientific imagination and, to some extent, by their cost, which has restricted their use to relatively small numbers of samples. Challenges remain, including the storage and analysis of the large amounts of data generated. As the chemistries employed mature, costs will decrease. In addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. An exciting era of viral exploration has begun, and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health.

Diversity of Bacteria Associated with Natural Aphid Populations

ABSTRACT The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, theγ -proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of γ-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n= 74], Amphorophora rubi [n= 109], Aphis sarothamni [n= 42], and Microlophium carnosum [n= 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Antioxidants promote establishment of trypanosome infections in tsetse.

Efficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling

BackgroundIn the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.ResultsWe assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases.ConclusionWe have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

Elucidation of the Transmission Patterns of an Insect-Borne Bacterium

ABSTRACT Quantitative data on modes of transmission are a crucial element in understanding the ecology of microorganisms associated with animals. We investigated the transmission patterns of a γ-proteobacterium informally known as pea aphid Bemisia-like symbiont (PABS), also known as T-type, which is widely but not universally distributed in natural populations of the pea aphid, Acyrthosiphon pisum. The vertical transmission of PABS to asexual and sexual morphs and sexually produced eggs was demonstrated by a diagnostic PCR-based assay, and the maximum estimated failure rate was 2%. Aphids naturally lacking PABS acquired PABS bacteria administered via the diet, and the infection persisted by vertical transmission for at least three aphid generations. PABS was also detected in two of five aphid honeydew samples tested and in all five siphuncular fluid samples tested but in none of 15 samples of salivary secretions from PABS-positive aphids. However, PABS-negative aphids did not acquire PABS when they were cocultured with PABS-positive aphids; the maximal estimated level of horizontal transmission was 18%. A deterministic model indicated that the force of infection by a horizontal transmission rate of 3% is sufficient to maintain a previously described estimate of the prevalence of PABS-positive aphids (37%), if the vertical transmission rate is 98%. We concluded that PABS infections in A. pisum can be maintained by high vertical transmission rates and occasional horizontal transmission, possibly via the oral route, in the absence of selection either for or against aphids bearing this bacterium.

Comparative Genomics and Transduction Potential of Enterococcus faecalis Temperate Bacteriophages

To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, PhiFL1 to 3, were found to be sequence related, with PhiFL1A to C and PhiFL2A and B sharing the greatest identity (87 to 88%), while PhiFL3A and B share 37 to 41% identity with PhiFL1 and 2. PhiFL4A shares 3 to 12% identity with the phages PhiFL1 to 3. The PhiFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.