AN ALL-PURPOSE CELLULASE REPORTER FOR GENE FUSION STUDIES AND APPLICATION TO THE PARACRYSTALLINE SURFACE (S)-LAYER PROTEIN OF CAULOBACTER-CRESCENTUS

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (DELTACenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of DELTACenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of DELTAcenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of DELTACenA as markers, a simple assay for periplasmic DELTACenA hybrids was developed. This assay indicated that DELTACenA activity was largely independent of cellular location. To facilitate the use of DELTACenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating DELTAcenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:DELTAcenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that DELTACenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because DELTACenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, DELTACenA possessed many of the attributes of an ''all-purpose'' reporter.