Towards uncultured-microbe genomics

The authors constructed two BAC libraries from environmental DNA samples, termed metagenomic libraries. The first library contained a total of approximately 100 Mb of environmental DNA with a mean fragment size of 27 kb, while the second, constructed using a refined technique, contained 24,576 cloned fragments of mean size 44.5 kb - making a total of around 1000 Mb of environmental DNA. Antibacterial, lipase, amylase, nuclease, and hemolytic activity could all be detected from the smaller of these libraries, and DNase and antibacterial genes from two clones were successfully sequenced. Seven of the clones from the smaller library had detectable 16S rRNA genes, which could be used to place the clones in a wide diversity of bacterial phyla - low G+C Gram-positives, cytophagales, proteobacteria and acidobacteria.Environmental DNA was extracted using fairly standard methods, although pulsed-field gel electrophoresis was used to isolate only high-molecular weight DNA for use in the second library. The major advance in this paper is the use of BAC vectors, which allows significant expression of heterologous environmental DNA in Escherichia coli, making it possible to screen the library for novel functional genes using standard, quick biochemical assays. The authors also use an interesting PCR protocol involving competitive oligonucleotides to amplify 16S rRNA genes in the presence of E. coli DNA, which they intend to describe in detail elsewhere.A strategy of BAC cloning of large environmental DNA fragments allows both traditional and functional genomic studies of uncultured microorganisms to be carried out.As with any new technique, there are many unanswered questions about this work. Most important, perhaps, is to investigate how efficiently genes from different bacteria are expressed in BACs. Clearly the efficiency of expression will depend on the size of inserts, on how completely environmental genomic DNA is sampled in the library and on how similar the protein trans