The global decline of coral reefs heightens the need to understand how corals respond to changing environmental conditions. Corals are metaorganisms, so-called holobionts, and restructuring of the associated bacterial community has been suggested as a means of holobiont adaptation. However, the potential for restructuring of bacterial communities across coral species in different environments has not been systematically investigated. Here we show that bacterial community structure responds in a coral host-specific manner upon cross-transplantation between reef sites with differing levels of anthropogenic impact. The coral Acropora hemprichii harbors a highly flexible microbiome that differs between each level of anthropogenic impact to which the corals had been transplanted. In contrast, the microbiome of the coral Pocillopora verrucosa remains remarkably stable. Interestingly, upon cross-transplantation to unaffected sites, we find that microbiomes become indistinguishable from back-transplanted controls, suggesting the ability of microbiomes to recover. It remains unclear whether differences to associate with bacteria flexibly reflects different holobiont adaptation mechanisms to respond to environmental change.

The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPβ and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.

Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.

Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.

Endosymbiosis is an important evolutionary event for organisms, and there is widespread interest in understanding the evolution of endosymbiosis establishment. Hydra is one of the most suitable organisms for studying the evolution of endosymbiosis. Within the genus Hydra, H. viridissima and H. vulgaris show endosymbiosis with green algae. Previous studies suggested that the endosymbiosis in H. vulgaris took place much more recently than that in H. viridissima, noting that the establishment of the interaction between H. vulgaris and its algae is not as stable as in H. viridissima. To investigate the on-going process of endosymbiosis, we first compared growth and tolerance to starvation in symbiotic and aposymbiotic polyps of both species. The results revealed that symbiotic H. viridissima had a higher growth rate and greater tolerance to starvation than aposymbiotic polyps. By contrast, growth of symbiotic H. vulgaris was identical to that of aposymbiotic polyps, and symbiotic H. vulgaris was less tolerant to starvation. Moreover, our gene expression analysis showed a pattern of differential gene expression in H. viridissima similar to that in other endosymbiotically established organisms, and contrary to that observed in H. vulgaris. We also showed that H. viridissima could cope with oxidative stress that caused damage, such as cell death, in H. vulgaris. These observations support the idea that oxidative stress related genes play an important role in the on-going process of endosymbiosis evolution. The different evolutionary stages of endosymbiosis studied here provide a deeper insight into the evolutionary processes occurring toward a stable endosymbiosis.

Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management.

Epulopiscium is a group of giant bacteria found in high abundance in intestinal tracts of herbivorous surgeonfish. Despite their peculiarly large cell size (can be up to 600 μm), extreme polyploidy (some with over 100,000 genome copies per cell) and viviparity (whereby mother cells produce live offspring), details about their diversity, distribution or their role in the host gut are lacking. Previous studies have highlighted the existence of morphologically distinct Epulopiscium cell types (defined as morphotypes A to J) in some surgeonfish genera, but the corresponding genetic diversity and distribution among other surgeonfishes remain mostly unknown. Therefore, we investigated the phylogenetic diversity of Epulopiscium, distribution and co-occurrence in multiple hosts. Here, we identified eleven new phylogenetic clades, six of which were also morphologically characterized. Three of these novel clades were phylogenetically and morphologically similar to cigar-shaped type A1 cells, found in a wide range of surgeonfishes including Acanthurus nigrofuscus, while three were similar to smaller, rod-shaped type E that has not been phylogenetically classified thus far. Our results also confirmed that biogeography appears to have relatively little influence on Epulopiscium diversity, as clades found in the Great Barrier Reef and Hawaii were also recovered from the Red Sea. Although multiple symbiont clades inhabited a given species of host surgeonfish and multiple host species possessed a given symbiont clade, statistical analysis of host and symbiont phylogenies indicated significant cophylogeny, which in turn suggests co-evolutionary relationships. A cluster analysis of Epulopiscium sequences from previously published amplicon sequencing dataset revealed a similar pattern, where specific clades were consistently found in high abundance amongst closely related surgeonfishes. Differences in abundance may indicate specialization of clades to certain gut environments reflected by inferred differences in the host diets. Overall, our analysis identified a large phylogenetic diversity of Epulopiscium (up to 10% sequence divergence of 16S rRNA genes), which lets us hypothesize that there are multiple species that are spread across guts of different host species.