The yjeA gene, encoding a secreted protein, YjeA, of Bacillus subtilis, was cloned and characterized. A derivative of YjeA, the recombinant YjeA-H, which contained a His₆-tag at the C-terminus of the protein, was purified from Escherichia coli and employed for functional studies. YjeA-H was shown to possess an endonuclease activity, which utilizes both dsDNA and ssDNA, but not RNA, as substrates. Employing ³²P-labeled pBR322 DNA as the substrate, YjeA-H was demonstrated to progressively nick both DNA strands in a random fashion, creating intermediates with various structural conformations, as well as DNA smears comprising linear molecules of different sizes. The detection of a putative signal peptide at the N-terminus of YjeA, together with the purification of YjeA-H from the culture su...[ Read more ]

The yjeA gene, encoding a secreted protein, YjeA, of Bacillus subtilis, was cloned and characterized. A derivative of YjeA, the recombinant YjeA-H, which contained a His₆-tag at the C-terminus of the protein, was purified from Escherichia coli and employed for functional studies. YjeA-H was shown to possess an endonuclease activity, which utilizes both dsDNA and ssDNA, but not RNA, as substrates. Employing ³²P-labeled pBR322 DNA as the substrate, YjeA-H was demonstrated to progressively nick both DNA strands in a random fashion, creating intermediates with various structural conformations, as well as DNA smears comprising linear molecules of different sizes. The detection of a putative signal peptide at the N-terminus of YjeA, together with the purification of YjeA-H from the culture supernatants of Escherichia coli yjeA-H clones, and the identification of YjeA in the culture medium of Bacillus subtilis, supports the conclusion that YjeA is a secretory protein of Bacillus subtilis.

We have constructed various pBR322-based shuttle vectors for secretory expression of heterologous proteins in Bacillus subtilis. When a portion of one of these plasmids, pM2, comprising pBR322 DNA responsible for antibiotic resistance and plasmid replication in Escherichia coli was replaced by a pACYC184 DNA sequence encoding equivalent activities, the resulting construct, p184110, showed a remarkable 15-fold increase in the efficiency of transforming Bacillus subtilis. Furthermore, a newly designed co-transformation assay revealed that the number of Escherichia coli co-transformants was substantially lower in cells over-expressing YjeA than in the ones lacking such an expression. The effect of yjeA expression on transformation of Bacillus subtilis with plasmid pM2 was then studied. The results showed that YjeA-deficient cells were transformed at least twice as efficient as those wild type cells expressing YjeA. The functional activity of YjeA identified in this research has laid the cornerstone for further studies into the enzymatic properties of this novel endonuclease as well as the biological role it plays in Bacillus subtilis.