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The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.The number of studies dealing with bacterial chitinasestheir biochemical properties, the structure of the genes encoding them, the catalytic mechanism involved, and their tertiary structures-has been increasing rapidly. The hydrolysis of chitin by chitinases is the most critical step in the degradation and utilization of chitin by bacteria. However, the study of chitinases is not sufficient to elucidate the process by which chitin is degraded and utilized by bacteria. The process involves a number of steps, including the recognition of chitin outside of the cell, the induction of chitinases, the maintenance of proper levels of chitinase production, and the incorporation and catabolism of degradation products. In this study our intent was to identify the genes involved in the degradation and utilization of chitin. Our long-term goal is to answer the following questions. How do bacteria recognize chitin? How is chitinase production induced and regulated? Why do chitinolytic bacteria produce multiple chitinases? How are degradation products processed? Our ultimate goal in these studies is a general understanding of how bacterial cells degrade and utilize chitin.We have studied the chitinase system of Bacillus circulans WL-12 and have provided comprehensive findings on biochemical properties, structure-function relationships, the identification of essential amino acid residues for catalytic activity, and the mechanisms by which multiple chitinases are generated (1, 2, 4, 23, 31-35). However, this bacterium is not a suitable...

Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day −3 to Day −1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.