The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.Chitin is an insoluble polysaccharide consisting of ,-(1,4)-linked N-acetylglucosamine (GlcNac) units. Large reservoirs of this polysaccharide in the marine and terrestrial environments are derived from the cell walls of fungi and the cuticles of crustaceans and insects (9, 23). Many bacteria in these environments are able to hydrolyze chitin to GlcNac (16,18,19) by using two enzymes, chitinase and N,N'-diacetylchitobiase (chitobiase). Chitin is first hydrolyzed by chitinase to low-molecular-weight multimers of GlcNac, the dimer N,N'-diacetylchitobiose (chitobiose) being predominant. Chitobiase then hydrolyzes chitobiose to GlcNac.To begin an analysis of these enzymes, we have cloned the genes encoding chitinase and chitobiase from the gramnegative marine bacterium Vibrio harveyi. Chitinase genes have also been cloned from Serratia marcescens (5, 10), in which two chitinase genes encoding two different chitinases have been identified (10). The chitobiase gene has been isolated from S. marcescens (8) and Vibrio vulnificus (25). Here, we show that chitobiase is located in the outer membrane of Escherichia coli cells containing the cloned chitobiase gene (chb). Based on minicell analysis of plasmidencoded proteins, the approximate sizes of the unprocessed and processed proteins are 95.3 kilodaltons (kDa) and 92.1 kDa, respectively. When the first 21 amino acids of lacZ from pUC19 were substituted for the first 22 amino acids of prechitobiase, a hybrid protein resulted that had chitobiase activity, remained unprocessed, and was found only in the cytoplasm, as would be expected if the amino terminus of prechitobiase were involved in processing and translocation to the outer membrane.
MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli K-12 strain LE392 (F-) hsdR lacY galK2 galT22 metBI trpR55 supE44 supF58, E. coli K-12 strain JM109 (F' traD36 proAB+ lacIqZAMJ5) recAl endAl gyrA96 thi hsdR17 supE44 relAl * Corresponding author.A(la...