Regulation of chitinase production by the 5'-untranslated region of the ybfM in Serratia marcescens 2170.

Serratia marcescens 2170 produces three chitinases and the chitin-binding protein CBP21, and efficiently degrades insoluble and crystalline chitin. The three chitinases and CBP21 are induced by N,N'-diacetylchitobiose [(GlcNAc)2], the major product of chitin hydrolysis by S. marcescens chitinases. We have found that uptake of both (GlcNAc)2 and N-acetylglucosamine (GlcNAc) is important for the efficient utilization of (GlcNAc)2 because (GlcNAc)2 is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. In order to determine the mechanism of utilization of the degradation products of chitin by S. marcescens, chitobiase deficient transposon mutants were screened. A transposon present in chitobiase-deficient mutants was inserted into the ybfMN-ctb cluster. The mutants produced chitinases, except for TT327, in which a transposon was inserted into the 5'-untranslated region (5'-UTR) of ybfM. Ectopic expression of this region in TT327 restored chitinase production. These results indicate that the 5'-UTR of ybfM is important for chitinase induction in S. marcescens.

The importance of chitobiase and N-acetylglucosamine (GlcNAc) uptake in N,N'-diacetylchitobiose [(GlcNAc)2] utilization by Serratia marcescens 2,170.

N,N'-diacetylchitobiose [(GlcNAc)(2)] is the main degradation product from chitin by the action of chitinases of Serratia marcescens 2170. Uptake of (GlcNAc)(2) via a (GlcNAc)(2)-specific enzyme II permease by this bacterium has been demonstrated previously. Here, we report the contribution of chitobiase and N-acetylglucosamine (GlcNAc) uptake to the utilization of (GlcNAc)(2). When S. marcescens 2170 was cultivated in a medium containing chitin, chitobiase activity was detected both inside and outside the cells; intracellular chitobiase was more abundant and suggested to be mainly located in the periplasm. Production of chitobiase was induced by GlcNAc and more effectively by (GlcNAc)(2). For induction of chitobiase, uptake of (GlcNAc)(2) was essential but ChiR, an essential regulator of chitinase induction, was not required. S. marcescens 2170 grew well on both GlcNAc and (GlcNAc)(2) but mutants defective in either chitobiase or NagE, the GlcNAc-specific enzyme II permease, showed reduced growth on (GlcNAc)(2). These results suggest that, in addition to uptake as (GlcNAc)(2), a proportion of the (GlcNAc)(2) is converted to GlcNAc by chitobiase, mainly in the periplasm, and incorporated into the cytoplasm by NagE. The mutant defective in chitobiase grew more slowly on (GlcNAc)(2) than on GlcNAc, indicating that (GlcNAc)(2) is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. Therefore, uptake as both (GlcNAc)(2) and GlcNAc is important for efficient utilization of (GlcNAc)(2) in S. marcescens.

Structure of full-length bacterial chitinase containing two fibronectin type III domains revealed by small angle X-ray scattering.

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 consists of an N-terminal catalytic domain, two fibronectin type III domains (FnIIIDs), and a C-terminal chitin-binding domain. The full-length structure of ChiA1 was studied by small angle X-ray scattering. The obtained low-resolution structure showed that ChiA1 is an elongated molecule with a length of approximately 145 A composed of a large globular head and a rod-like tail. Combination with known high-resolution structures of individual ChiA1 domains provided a model of the domain arrangement. In this model, two FnIIIDs connect to each other in an extended rod-like shape without large bending between the FnIIIDs, and contribute largely to the length of ChiA1.

Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin.

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.