Chitobiase, a new reporter enzyme.
N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-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 study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases. We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters. In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site. These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region.
Kalabat
D Y
DY
San Diego State University, CA, USA.
Froelich
J M
JM
Phuong
T K
TK
Forsyth
R A
RA
Newman
V G
VG
Zyskind
J W
JW
D000118:Acetylglucosaminidase / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001435:Bacteriophages; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005838:Genotype; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016662:Virus Integration
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011993:Recombinant Fusion Proteins; D000118:Acetylglucosaminidase