We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3 ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC-and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNT) and poly(A) polymerases (PAP) are members of a superfamily of RNA nucleotidyltransferases (13, 39). tRNA nucleotidyltransferases add CCA residues to the 3Ј ends of immature tRNAs and repair the 3Ј ends of damaged tRNAs (36). tRNA nucleotidyltransferases (TNTs) are found in bacteria, archaeae, and eukaryotes, suggesting conservation of structure and function of this class of enzymes over the course of evolution (39). Polyadenylation of RNA 3Ј ends, once thought to occur exclusively in eukaryotic cells, has now been shown definitively to occur in bacterial systems as well (7,25,30,31). Escherichia coli cells contain at least two enzymes capable of adding poly(A) tails to the 3Ј ends of RNA molecules. The major polyadenylating enzyme, poly(A) polymerase I (PAP I), was originally identified by virtue of its role in regulating plasmid copy number and is a product of the pcnB gene (6, 12). PAP I can utilize any of the three nucleoside triphosphates as substrates in vitro, but only A residues are added by the enzyme to RNA 3Ј ends in vivo (38). Messenger RNAs and 16S and 23S ribosomal RNAs are all polyadenylated in E. coli, and this modification is an important element of the RNA degradation pathway in bacterial systems (7,25,30,31). RNA 3Ј ends are generated in E. coli via endonucleolytic cleavage by RNase E and RNase III, thes...