Culture-independent metagenomics supports discovery of uncultivable bacteria within the genus Chlamydia

Abstract

Advances 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.

Figure 1

Phylogenetic position of the novel taxon, Ca. Chlamydia corallus G3/2742-324 within the Chlamydiaceae. Phylogenetically informative marker genes were retrieved from each genome, concatenated and aligned using MAFFT, prior to tree construction using FastTree. Numbers on the branches indicate support values.

Figure 2

Plasmid sequence-based phylogenetic analysis of all samples sequenced in this study and other chlamydial plasmid sequences. Nucleotide sequences were aligned using MAFFT and tree was constructed using FastTree. Support values are shown on the branches.

Figure 3

Comparison of plasticity zone regions encoded by Ca. Chlamydia corallus and related chlamydial species. Figure constructed using Easy Fig. Grey shading represents tBLASTx matches (see BLAST identity scale). Coloured arrows represent coding regions (see legend).