Gram-positive bacteria with a high G+C content are currently recognized as a distinct phylum,
Actinobacteria, on the basis of their branching in 16S rRNA trees. Except for an insert in the 23S rRNA, there are no unique biochemical or molecular characteristics known at present that can distinguish this group from all other bacteria. In this work, three conserved indels (i.e. inserts or deletions) are described in three widely distributed proteins that are distinctive characteristics of the Actinobacteriaand are not found in any other groups of bacteria. The identified signatures are a 2 aa deletion in cytochrome- coxidase subunit 1 (Cox1), a 4 aa insert in CTP synthetase and a 5 aa insert in glutamyl-tRNA synthetase (GluRS). Additionally, the actinobacterial specificity of the large insert in the 23S rRNA was also tested. Using primers designed for conserved regions flanking these signatures, fragments of most of these genes were amplified from 23 actinobacterial species, covering many different families and orders, for which no sequence information was previously available. All the 61 sequenced fragments, except two in GluRS, were found to contain the indicated signatures. The presence of these signatures in various species from 20 families within this phylum provides evidence that they are likely distinctive characteristics of the entire phylum, which were introduced in a common ancestor of this group. The absence of all four of these signatures in Symbiobacterium thermophilumsuggests that this species, which is distantly related to other actinobacteria in 16S rRNA and CTP synthetase trees, may not be a part of the phylum Actinobacteria. The identified signatures provide novel molecular means for defining and circumscribing the phylum Actinobacteria. Functional studies on them should prove helpful in understanding novel biochemical and physiological characteristics of this group of bacteria.