Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli.

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as 'attributive tags' for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

Cloning and characterization of the yjeA gene, encoding a novel deoxyribonuclease, from Bacillus subtilis.

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 C-terminal His(6)-tag, was purified from Escherichia coli for functional studies. YjeA-H was shown to be an endonuclease, which hydrolyses both single-stranded and double-stranded DNA, but not RNA. Covalently closed circular pBR322 DNA incubated with YjeA-H was shown by gel electrophoresis to be first nicked to an open circular form, and then to a linearized structure on a background of DNA smear, and finally to small species of linear molecules that accumulated gradually. When (32)P-labelled pBR322 DNA was used as substrate, YjeA-H was shown to progressively nick both DNA strands in a random fashion, creating intermediates of various structures, as well as DNA smears comprising linear molecules of different sizes. The final products were found to consist essentially of degraded species of DNA. 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 E. 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.