Bas E. Dutilh

Nitrite-driven anaerobic methane oxidation by oxygenic bacteria

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named ‘Candidatus Methylomirabilis oxyfera’, was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that ‘M. oxyfera’ bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycles major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organisms special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

Metagenomics and future perspectives in virus discovery.

Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. Identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. Traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. However, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. Viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity. In the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. Phylogenetically, some of these viruses are distantly related to previously discovered viruses. In addition, 60–99% of the sequences generated in different viral metagenomic studies are not homologous to known viruses. In this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. We discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. In addition, we propose a new model of the Kochs postulates named the ‘Metagenomic Kochs Postulates’. Unlike the original Kochs postulates and the Molecular Kochs postulates as formulated by Falkow, the metagenomic Kochs postulates focus on the identification of metagenomic traits in disease cases. The metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen.

Towards the human colorectal cancer microbiome

Multiple factors drive the progression from healthy mucosa towards sporadic colorectal carcinomas and accumulating evidence associates intestinal bacteria with disease initiation and progression. Therefore, the aim of this study was to provide a first high-resolution map of colonic dysbiosis that is associated with human colorectal cancer (CRC). To this purpose, the microbiomes colonizing colon tumor tissue and adjacent non-malignant mucosa were compared by deep rRNA sequencing. The results revealed striking differences in microbial colonization patterns between these two sites. Although inter-individual colonization in CRC patients was variable, tumors consistently formed a niche for Coriobacteria and other proposed probiotic bacterial species, while potentially pathogenic Enterobacteria were underrepresented in tumor tissue. As the intestinal microbiota is generally stable during adult life, these findings suggest that CRC-associated physiological and metabolic changes recruit tumor-foraging commensal-like bacteria. These microbes thus have an apparent competitive advantage in the tumor microenvironment and thereby seem to replace pathogenic bacteria that may be implicated in CRC etiology. This first glimpse of the CRC microbiome provides an important step towards full understanding of the dynamic interplay between intestinal microbial ecology and sporadic CRC, which may provide important leads towards novel microbiome-related diagnostic tools and therapeutic interventions.

A bacterial driver–passenger model for colorectal cancer: beyond the usual suspects

Cancer has long been considered a genetic disease. However, accumulating evidence supports the involvement of infectious agents in the development of cancer, especially in those organs that are continuously exposed to microorganisms, such as the large intestine. Recent next-generation sequencing studies of the intestinal microbiota now offer an unprecedented view of the aetiology of sporadic colorectal cancer and have revealed that the microbiota associated with colorectal cancer contains bacterial species that differ in their temporal associations with developing tumours. Here, we propose a bacterial driver–passenger model for microbial involvement in the development of colorectal cancer and suggest that this model be incorporated into the genetic paradigm of cancer progression.

A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes

Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the majority of published human faecal metagenomes, which we refer to as crAssphage. Its ~97 kbp genome is six times more abundant in publicly available metagenomes than all other known phages together; it comprises up to 90% and 22% of all reads in virus-like particle (VLP)-derived metagenomes and total community metagenomes, respectively; and it totals 1.68% of all human faecal metagenomic sequencing reads in the public databases. The majority of crAssphage-encoded proteins match no known sequences in the database, which is why it was not detected before. Using a new co-occurrence profiling approach, we predict a Bacteroides host for this phage, consistent with Bacteroides-related protein homologues and a unique carbohydrate-binding domain encoded in the phage genome.

Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus.

Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting ‘global ocean virome’ dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Discovery of a hapE Mutation That Causes Azole Resistance in Aspergillus fumigatus through Whole Genome Sequencing and Sexual Crossing

Azole compounds are the primary therapy for patients with diseases caused by Aspergillus fumigatus. However, prolonged treatment may cause resistance to develop, which is associated with treatment failure. The azole target cyp51A is a hotspot for mutations that confer phenotypic resistance, but in an increasing number of resistant isolates the underlying mechanism remains unknown. Here, we report the discovery of a novel resistance mechanism, caused by a mutation in the CCAAT-binding transcription factor complex subunit HapE. From one patient, four A. fumigatus isolates were serially collected. The last two isolates developed an azole resistant phenotype during prolonged azole therapy. Because the resistant isolates contained a wild type cyp51A gene and the isolates were isogenic, the complete genomes of the last susceptible isolate and the first resistant isolate (taken 17 weeks apart) were sequenced using Illumina technology to identify the resistance conferring mutation. By comparing the genome sequences to each other as well as to two A. fumigatus reference genomes, several potential non-synonymous mutations in protein-coding regions were identified, six of which could be confirmed by PCR and Sanger sequencing. Subsequent sexual crossing experiments showed that resistant progeny always contained a P88L substitution in HapE, while the presence of the other five mutations did not correlate with resistance in the progeny. Cloning the mutated hapE gene into the azole susceptible akuB KU80 strain showed that the HapE P88L mutation by itself could confer the resistant phenotype. This is the first time that whole genome sequencing and sexual crossing strategies have been used to find the genetic basis of a trait of interest in A. fumigatus. The discovery may help understand alternate pathways for azole resistance in A. fumigatus with implications for the molecular diagnosis of resistance and drug discovery.

Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.)

The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.