The intein of the Thermoplasma A-ATPase A subunit: structure, evolution and expression in E. coli

Abstract

Background: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation.

Results: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain.T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli.

Conclusions: Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

Figure 1

Alignment of archaeal ATPase A-subunit intein sequences. The large gap in the Thermoplasma sequences indicates that the Thermoplasma acidophilum (Tac) and Thermoplasma volcanium (Tvo) inteins do not contain an endonuclease domain and only consist only of the self splicing domain, while the Pyrococcus abyssi (Pab), P. furiosus (Pfu) and P. horikoshii (Pho) A-ATPase A-subunits inteins are bifunctional. Regions corresponding to conserved blocks [18] are indicated in bold and labeled A-H. Blocks A, B, F and G are part of the splicing domain, whereas blocks C, D, E, H are typical for endonucleases of the LAGDIDAG type [18].

Figure 2

Divergence along the archaeal and vacuolar ATPase A subunits. The diagram depicts the number of substitutions per site calculated for a sliding window of 10 amino acid residues along an alignment of 62 vacuolar and archaeal ATPase catalytic subunits. The ordinate gives the number of substitutions observed in each position in the alignment. Arrows indicate the locations of the inteins. The first (A) gives the location of the inteins in Thermoplasma and Pyrococcus, the second (B) gives the location in the yeasts' vacuolar ATPases.

Figure 3

Comparison of vacuolar/archaeal ATPase A subunit and small subunit ribosomal RNA phylogenies. The phylogeny depicted in (A) was calculated from the extein portions of the genes only; (B) was calculated from small subunit ribosomal RNAs. The trees were calculated as unrooted, but are depicted as rooted using the eukaryotes as an outgroup to the Archaea (A), or the Crenarchaeota as an outgroup to the Euryarchaeota (B). Numbers give bootstrap values calculated from 100 bootstrapped samples analyzed using parsimony analysis. Values are only given for groups with more than 50% support. Asterisks denote branches that in the maximum likelihood evaluation were not at least 3 or 2 times larger, respectively, than their estimated standard error. All other branches were at least three times larger than their standard deviation. See experimental procedures for further details on the phylogenetic reconstruction methods used. Species whose A-subunit contains an intein are indicated by arrows.

Figure 4

SDS polyacrylamide gel electrophoresis of proteins from induced E. coli. Panel A, Lanes 1 and 3: E. coli Bl21-CodonPlus(DE3)-RIL strain transformed with empty pET-11a vector (negative control); lanes 2, 4, 5 and 6: E. coli Bl21-CodonPlus(DE3)-RIL strain transformed with the Thermoplasma A-ATPase cloned into pET-11a; lane 1-4: cells were induced for 4 hours; lane 5: cells were induced at 16°C for 16 hours; lane 6: cells were induced at 42°C for 2 hours; Sup.: supernatant. Panel B depicts the splicing process schematically. The unprocessed T. acidophilum A-ATPase A subunit has a calculated size of 85 kDalton. The expected molecular weight of the intein is 20 kDalton, and the religated host protein weighs approximately 65 kDalton.

Figure 5

Non-denaturing polyacrylamide gel electrophoresis of proteins from induced E. coli. Lanes 1 and 2: non-denaturing polyacrylamide gel electrophoresis of proteins from the supernatant of the cell lysate. Arrows point towards bands that are present in induced E. coli transformed with the Thermoplasma A-ATPase, but absent in the negative control. The sample buffer did not contain DTT or SDS. Lanes 3, 4, 5 and 6 are separations in denaturing SDS polyacrylamide gels. Lane 3: electro-eluted protein from lane 2 (arrow A); lane 4: electro-eluted protein from lane 2 (arrow B); lanes 1 and 5: E. coli Bl21-CodonPlus(DE3)-RIL strain transformed with empty pET-11a vector (negative control); lanes 2 and 6: E. coli Bl21-CodonPlus(DE3)-RIL strain transformed with the Thermoplasma A-ATPase cloned into pET-11a and induced for 8 hours at 37°C.