Despite recent advances in commercially optimized identification systems, bacterial identification
remains a challenging task in many routine microbiological laboratories, especially in situations
where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively
for this task when coupled with a well-curated database, such as EzTaxon, containing sequences
of type strains of prokaryotic species with validly published names. Although the EzTaxon
database has been widely used for routine identification of prokaryotic isolates, sequences from
uncultured prokaryotes have not been considered. Here, the next generation database, named
EzTaxon-e, is formally introduced. This new database covers not only species within the formal
nomenclatural system but also phylotypes that may represent species in nature. In addition to an
identification function based on Basic Local Alignment Search Tool (BLAST) searches and pairwise
global sequence alignments, a new objective method of assessing the degree of completeness in
sequencing is proposed. All sequences that are held in the EzTaxon-e database have been
subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification
system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the
identification of cultured and uncultured prokaryotes and offers a valuable means of
communication among microbiologists who routinely encounter taxonomically novel isolates. The
database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.