Nucleotide sequence of a nuc gene encoding the thermonuclease of Staphylococcus intermedius.

A recombinant plasmid, pIP1607, containing the thermonuclease gene (nuc) of S.intermedius was detected in a pUC18 library, constructed by cloning cellular DNA of S.intermedius strain LRA076 partially digested with So«3A in the BamHl site. E.coli strain TGI was used as a recipient for DNA transformation. The screening method consisted in replica-plating the ampicillinresistant colonies on DNA-toluidine blue agar plates (1, 2). Of the 3000 transformants tested, one exhibited a nuclease activity that was further demonstrated as thermostable by boiling 5 nil of a cell suspension (1). The 1.4 kbp insert of the recombinant plasmid pIP1607 was sequenced on both strands by the Sanger dideoxy chain termination method performed on denatured double strand DNA in the presence of SSB protein (3). The G+C content of this DNA fragment (36 mol%) is similar to those observed among staphylococci. Computer-assisted sequence analysis revealed only one potential translational reading frame of 168 amino acids (Figure 1). The free energy of interaction of the most stable structure between the putative RBS sequence, located 4 bp upstream from the ATG codon, and the 3' terminus of the 16S rRNA (4, 5) was calculated according to Tinoco (6) and was evaluated as —55,2 kJ/mol. This value reflects a stable association and falls within those previously determined among Grampositive bacteria (4, 5). The N-terminal region of the predicted amino acid sequence, 'MKKITTGVLILAIAIWLIFQYINGDG, had all the features (7) of a leader peptide: two positively charged residues (lysine) at positions 2 3 , an unbroken stretch of 13 hydrophobic residues at positions 8 to 20, and a 'glycine-aspartic acid-glycine' sequence at positions 25-26—27 which might correspond to the consensus sequence preceding the cleavage site of the signal peptidase. Within the nuc gene product of S.intermedius strain LRA076, the cleavage site would therefore be located between glycine and proline at positions 27—28, yielding a secreted protein of 141 amino acids (Figure 1). The sequence of this putative secreted protein displayed a significant homology with that of the mature S.aureus thermonuclease, also named nuclease A (1). In addition, all the amino acids involved in the biological activity of the nuclease A (8), except one threonine at position 113, matched at the same relative positions (Figure 1).