Group I Self-Splicing Intron in the recA Gene of Bacillus anthracis

Abstract

Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the recA gene of Bacillus anthracis which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the recA precursor mRNA for RecA that was being translated in E. coli. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of B. anthracis expressed in E. coli was functional in its ability to complement a recA defect. When recA-negative E. coli cells were irradiated with UV, the Bacillus RecA reduced the UV susceptibility of the recA mutant, regardless of the presence of intron.While most of the introns found in eukaryotes and archaebacteria require functions of several proteins for their processing, introns found in eubacterial species are removed by selfsplicing. There are two types of self-splicing introns, classified according to their structures and splicing mechanisms. The processing of group I introns is initiated by a transesterification reaction mediated by an exogenous guanosine cofactor, generating ligated exon with a free intron bearing the guanosine at its 5Ј end (2, 32). The splicing of group II intron is similar to that of eukaryotic pre-mRNA, in which the 5Ј splice site is attacked by a 2Ј OH group of a bulged nucleotide, resulting in a lariat structure joined with a 2Ј-5Ј bond. Subsequently, the cleaved 5Ј exon attacks the 3Ј splice site, resulting in exon ligation and release of the intron lariat (14, 32).Despite a lack of overall primary sequence conservation, the group I introns contain blocks of conserved nucleotide sequence elements (P, Q, R, and S), forming an active center with a secondary structure consisting of characteristic stemloops P1 through P10 (2,22,23). There are also extra sequences, often including open reading frames (ORFs), which are located in loops peripheral to the catalytic intron core. The group II introns contain a different secondary structure, consisting of six conserved pairings emanating from a central wheel (24). In both introns, sequence conservations are confined to small regions of nucleotide residues. The products of internal ORFs of these introns usually play a role in splicing (in which case they are called maturases) or mobility of the intron (also referred to as "homing") (17, 32).The bacterial group I introns are found in several tRNA genes of cyanobacteria and purple proteobacteria (16,28,38).

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

“…AJ511368). Except for recA of B. anthracis, which contains an intron (Ko et al, 2002), all other known recA genes of Bacillus species look very similar. With a length of 1038 bp, both B. megaterium genes are average in size compared to those of other Bacillus species.…”

“…Eventually, low-copy plasmid pACYC177 in combination with the native B. megaterium recA promoters facilitated cloning in a RecA null mutant of E. coli. Due to the high conservation of RecA proteins, such interspecies complementation approaches have been successfully applied not only for several recA genes of Gram-negative bacteria but also for some from Gram-positive species such as Clostridium perfringens (Johnston et al, 1997) and B. anthracis (Ko et al, 2002). The resulting vectors pACYCrecA1 and pACYCrecA2 (for cloning strategies see Methods) were used in parallel with pACYC177 (as the control) for transformation of the RecA null mutant E. coli JM109.…”

“…However, except for the recA gene of Bacillus anthracis, which contains a Group I self-splicing intron (Ko et al, 2002), essentially nothing is known about recA expression in representatives of the genus other than B. subtilis. Thus, we looked for recA and its function in DNA repair in Bacillus megaterium.…”

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic BacteriumGeobacillus stearothermophilus

“…13) However, no intein or group II introns, much less IVS harboring a HEG, were found in flagellin genes, unlike the previously reported bacterial group I introns in proteincoding regions. 4,5,[13][14][15][16] Group I intron-like sequences were also found in locations immediately after flagellin genes in A. flavithermus and Exiguobacterium sibiricum, although E. sibiricum has no intron in the flagellin gene ( Fig. 2A).…”

“…In bacteria, however, only two genes, both highly conserved housekeeping genes, recA and nrdE, have been reported to contain functional group I introns in protein-coding regions. 4,5) Geobacillus stearothermophilus is a Bacillus related, rod-shaped, spore-forming, motile, thermophilic, bacterium that is aerobic or facultatively anaerobic and has an optimal growth temperature of 50 to 60 C. Most bacterial cells, including G. stearothermophilus, swim by rotating their flagella filaments as a propeller.…”