Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses.

The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences. Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion. We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level. The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and γ-proteobacteria) were screened for prophage sequences. The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria. The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities. The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and γ-proteobacteria