A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases

Abstract

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.

FIG. 1.

Neighbor-joining phylogenetic tree relating the proteins listed in Table 2. The tree was constructed using PHYLIP (11), as indicated in Materials and Methods. Bootstrap scores are shown at each node. The bar at the bottom of the figure represents the number of amino acid substitutions per site. The numbers in brackets identify the NTSFs from B. halodurans [1], Bdellovibrio bacteriovorus [2], D. vulgaris [3], and G. sulfurreducens [4].

FIG. 2.

Sequence alignment of Bacillus NTSFs. The alignment was performed using ClustalX as described in Materials and Methods. Identical residues are indicated by an asterisk. Similar residues are indicated by one or two dots, depending on the degree of similarity of the relevant amino acids. BAN, Bacillus anthracis; BTH, Bacillus thuringiensis; BCE, Bacillus cereus; BST, B. stearothermophilus; BSU, B. subtilis; BHA, B. halodurans.

FIG. 3.

RT-PCR of B. halodurans total RNA. Fifteen microliters of nested PCR products, prepared as described in Materials and Methods, were run on a 1% agarose gel. Lane 1, size standards; lanes 2 and 4, NTSFI and NTSFII RT-PCR products; lanes 3 and 4, parallel RT-PCRs run without reverse transcriptase.

FIG. 4.

Measurement of the lengths of oligo(A) and poly(A) stretches found in the 3′ tails of RNAs from E. coli DH5α (lane 2) and B. halodurans C-125 (lane 3). Lane 1 contains an oligo(dT)₁₈ size standard. Labeled 3′ tails were fractionated on a 12% denaturing polyacrylamide gel.

FIG. 5.

Location and composition of 3′ tails of cDNA clones derived from B. halodurans NTSFI, NTSFII, and 16S rRNA. The position of each tail is reckoned from the translation start codon of mRNAs or the mature 5′ end of the 16S rRNA transcript and is indicated by the number on the line preceding the cDNA tail sequence. The number of bases to the translation stop codon or the mature 3′ end of rRNA is indicated by the number at the top right hand end of each gene box. The method used to create the cDNAs [either oligo(dT) or polyguanylation-poly(G)] is indicated to the right. Genes and tail locations are not drawn to scale.

FIG. 6.

pH dependency of the tRNA nucleotidyltransferase assays of B. halodurans NTSFI and NTSFII. Left panel, NTSFI assayed with ATP and CTP; right panel, NTSFII assayed with ATP and CTP. Assays were performed as described in Materials and Methods, and results were corrected by subtracting a zero enzyme control value from each experimental value. Results are expressed as picomoles of AMP or CMP incorporated in a 60-μl reaction mixture.

FIG. 7.

Autoradiogram of a 10% denaturing polyacrylamide gel of the reaction products from assays of SCO3896, B. halodurans NTSFI and NTSFII, and E. coli PAP I. Lane 1 contains end-labeled RNA size standards of 400, 300, 200, and 100 bases. Lanes 2 and 6, products of reactions with no added enzyme; lanes 3 and 7, products of assays of SCO3896 with ATP and CTP, respectively; lanes 4 and 8, products of assays of NTSFI with ATP and CTP, respectively; lanes 5 and 9, products of assays of NTSFII with ATP and CTP, respectively; lane 10, products of the PAP I assay. Other conditions were as described in Materials and Methods.