RESUMO
A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Enzimas de Restrição do DNA/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo I/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Genes BacterianosRESUMO
The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genes Bacterianos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Bacterianos , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Escherichia coli/genética , Biblioteca Genômica , Metilação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Mapeamento por Restrição , Transcrição GênicaRESUMO
The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system.
