Heterologous ABC exporter-based cloning of gram-negative bacterial type I secretion pathway-dependent metalloproteases from an Erwinia genomic DNA library in Escherichia coli

Abstract

The gene cluster encoding AprDEF, three-component ABC exporter apparatus of type I secretion pathway of Pseudomonas aeruginosa lipase, was coexpressed with the genomic DNA library of Erwinia chrysanthemi KCTC 2569 in Escherichia coli. Among about 1500 clones of ABC exporter-coexpressed genomic DNA library, an E. coli clone showing the extracellular protease activity was isolated on skim milk agar plates. The sequence analyses of the isolated Erwinia genomic DNA fragment revealed three open reading frames, eprB, eprC and eprF. The predicted EprB and EprC proteins were significantly similar to the serralysin-like zinc metalloproteases that had been known to be secreted via the gram-negative bacterial type I pathway. The EprF protein was predicted to be a putative outer membrane protein as a component of unidentified ABC exporter. When each novel metalloprotease gene was coexpressed with the gene cluster encoding the heterologous AprDEF exporter, it was confirmed that these type I secretion pathway-dependent metalloproteases were successfully secreted and thus their genes could be cloned by virtue of the introduced ABC exporter apparatus. These results suggest that our cloning strategy of using the heterologous ABC exporter apparatus will provide a valuable opportunity of obtaining more genes encoding other type I secretion pathway-related extracellular proteins as well as serralysin-like metalloproteases. (c) 2006 Elsevier Inc. All rights reserved.