Genetic analysis of the chitinase system of Serratia marcescens 2170.
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.
Watanabe
T
T
Department of Biosystem Science, Graduate School of Science and Technology, Niigata University, Ikarashi, Japan. wata@agr.niigata-u.ac.jp
Kimura
K
K
Sumiya
T
T
Nikaidou
N
N
Suzuki
K
K
Suzuki
M
M
Taiyoji
M
M
Ferrer
S
S
Regue
M
M
D000595:Amino Acid Sequence; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000032:analysis / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D017077:Culture Media, Conditioned; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D002686:Chitin; D002688:Chitinases