Publications

4451

AbstractBackgroundAce Lake is a marine-derived, stratified lake in the Vestfold Hills of East Antarctica with an upper oxic and lower anoxic zone. Cyanobacteria are known to reside throughout the water column. ASynechococcus-like species becomes the most abundant member in the upper sunlit waters during summer while persisting annually even in the absence of sunlight and at depth in the anoxic zone. Here, we analysed ~ 300 Gb of Ace Lake metagenome data including 59Synechococcus-like metagenome-assembled genomes (MAGs) to determine depth-related variation in cyanobacterial population structure. Metagenome data were also analysed to investigate viruses associated with this cyanobacterium and the host’s capacity to defend against or evade viruses.ResultsA singleSynechococcus-like species was found to exist in Ace Lake,CandidatusRegnicoccus frigidus sp. nov., consisting of one phylotype more abundant in the oxic zone and a second phylotype prevalent in the oxic-anoxic interface and surrounding depths. An important aspect of genomic variation pertained to nitrogen utilisation, with the capacity to perform cyanide assimilation and asparagine synthesis reflecting the depth distribution of available sources of nitrogen. Both specialist (host specific) and generalist (broad host range) viruses were identified with a predicted ability to infectCa.Regnicoccus frigidus. Host-virus interactions were characterised by a depth-dependent distribution of virus type (e.g. highest abundance of specialist viruses in the oxic zone) and host phylotype capacity to defend against (e.g. restriction-modification, retron and BREX systems) and evade viruses (cell surface proteins and cell wall biosynthesis and modification enzymes).ConclusionIn Ace Lake, specific environmental factors such as the seasonal availability of sunlight affects microbial abundances and the associated processes that the microbial community performs. Here, we find that the population structure forCa.Regnicoccus frigidus has evolved differently to the other dominant phototroph in the lake,CandidatusChlorobium antarcticum. The geography (i.e. Antarctica), limnology (e.g. stratification) and abiotic (e.g. sunlight) and biotic (e.g. microbial interactions) factors determine the types of niches that develop in the lake. While the lake community has become increasingly well studied, metagenome-based studies are revealing that niche adaptation can take many paths; these paths need to be determined in order to make reasonable predictions about the consequences of future ecosystem perturbations.

Abstract Background The human louse (Pediculus humanus) is a haematophagous ectoparasite that is intimately related to its host. It has been of great public health concern throughout human history. This louse has been classified into six divergent mitochondrial clades (A, D, B, F, C and E). As with all haematophagous lice, P. humanus directly depends on the presence of a bacterial symbiont, known as “Candidatus Riesia pediculicola”, to complement their unbalanced diet. In this study, we evaluated the codivergence of human lice around the world and their endosymbiotic bacteria. Using molecular approaches, we targeted lice mitochondrial genes from the six diverged clades and Candidatus Riesia pediculicola housekeeping genes. Methods The mitochondrial cytochrome b gene (cytb) of lice was selected for molecular analysis, with the aim to identify louse clade. In parallel, we developed four PCR primer pairs targeting three housekeeping genes of Candidatus Riesia pediculicola: ftsZ, groEL and two regions of the rpoB gene (rpoB-1 and rpoB-2). Results The endosymbiont phylogeny perfectly mirrored the host insect phylogeny using the ftsZ and rpoB-2 genes, in addition to showing a significant co-phylogenetic congruence, suggesting a strict vertical transmission and a host–symbiont co-speciation following the evolutionary course of the human louse. Conclusion Our results unequivocally indicate that louse endosymbionts have experienced a similar co-evolutionary history and that the human louse clade can be determined by their endosymbiotic bacteria. Graphical Abstract

Abstract Objective ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.

Abstract Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

Abstract Anaerobic ammonium oxidation (Anammox) bacteria are a group of extraordinary bacteria exerting a major impact on the global nitrogen cycle. Their phylogenetic breadth and diversity, however, are not well constrained. Here we describe a new, deep-branching family in the order of Candidatus Brocadiales, Candidatus Bathyanammoxibiaceae, members of which have genes encoding the key enzymes of the anammox metabolism. In marine sediment cores from the Arctic Mid-Ocean Ridge (AMOR), the presence of Ca. Bathyanammoxibiaceae was confined within the nitrate-ammonium transition zones with the counter gradients of nitrate and ammonium, coinciding with the predicted occurrence of the anammox process. Ca. Bathyanammoxibiaceae genomes encode the core genetic machinery for the anammox metabolism, including hydrazine synthase for converting nitric oxide and ammonium to hydrazine, and hydrazine dehydrogenase for hydrazine oxidation to dinitrogen gas, and hydroxylamine oxidoreductase for nitrite reduction to nitric oxide. Their occurrences assessed by genomes and 16S rRNA gene sequencings surveys indicate that they are present in both marine and terrestrial environments. By introducing the anammox potential of Ca. Bathyanammoxibiaceae and charactering their ideal niche in marine sediments, our findings suggest that the diversity and abundance of anammox bacteria may be higher than previously thought, and provide important insights on cultivating them in the future to not only assess their biogeochemical impacts but also constrain the emergence and evolutionary history of this functional guild on Earth.