Chitobiase, a new reporter enzyme

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harvey i is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyz es the disaccharide chitobiose to N-acetyl glucosamine. The advantages of t he reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-a cetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii ) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available. The use of chitobiase as a reporter enzyme is generally applicable to the stud y of gene expression in those bacteria that do not contain N-acetyl-beta-D- glucosaminidases. We constructed plasmid vectors containing a multiple clon ing site for producing in-frame fusions to chitobiase, the attP of lambda p hage for movement into the bacterial chromosome for single-copy analysis, t he gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 orig in of replication and the rrnBtlt2 terminator region upstream of the chb ge ne to prevent read-through from other promoters. In-frame fusions between t he dnaA gene and chb were moved to the chromosome by site-specific recombin ation with the chromosomal attB site. These single-copy fusions were assaye d for chitobiase to examine the effects of a deletion in the dnaA regulator y region.