Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe

Journal Article · Tue Jul 01 00:00:00 EDT 1986 · Appl. Environ. Microbiol.; (United States)

OSTI ID:5018389

Wortman, A T; Somerville, C C; Colwell, R R

To initiate study of the genetic control of chitinolytic activity in vibrios, the chitobiase gene was isolated by cloning chromosomal DNA prepared from Vibrio vulnificus. Chimeric plasmids were constructed from Sau3A I partial digests of chromosomal DNA by ligating 5 to 15-kilobase fragments into the BamHI site, i.e., in the Tc/sup r/ gene, of pBR322 (Am/sup r/Tc/sup r/). The resulting plasmids were transformed into Escherichia coli DH1. Chitobiase activity of the insert-bearing clones was detected by using a chromogenic substrate, p-nitrophenyl-N-acetyl-..beta..,D-glucosaminide, and confirmed by the appearance of a fluorescent end product from the hydrolysis of 4-methylumbelliferyl-..beta..,D-N-N'-diacetylchitiobiose. Endochitinase activity was demonstrated by liberation of water-soluble products produced by the degradation of (/sup 3/H)chitin. Transformation of E. coli Y10R (lacY) with plasmids from chitinase-positive clones restored the lactose-positive phenotype, suggesting the presence of a permease associated with chitinase activity. Physical mapping of plasmids containing the chitinase determinants indicate that transcription of these genes in E. coli may be initiated at a V. vulnificus promoter.

• Research Organization: Univ. of Maryland, College Park
• OSTI ID: 5018389
• Journal Information: Appl. Environ. Microbiol.; (United States), Vol. 52:1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BACTERIA
ENZYME ACTIVITY
CHITIN
ENZYMATIC HYDROLYSIS
AQUATIC ORGANISMS
CLONING
ESCHERICHIA COLI
GENES
GENETICS
PLASMIDS
TRACER TECHNIQUES
TRANSCRIPTION
TRITIUM COMPOUNDS
AMINES
BIOLOGY
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL REACTIONS
DECOMPOSITION
HYDROLYSIS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYSIS
MICROORGANISMS
MUCOPOLYSACCHARIDES
ORGANIC COMPOUNDS
POLYSACCHARIDES
SACCHARIDES
SOLVOLYSIS
550701* - Microbiology- Tracer Techniques
550201 - Biochemistry- Tracer Techniques