Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

Journal Article · Mon Apr 01 00:00:00 EST 1985 · J. Bacteriol.; (United States)

OSTI ID:5558755

Sankar, P; Lee, J H; Shanmugam, K T

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.

• Research Organization: Univ. of Florida, Gainesville
• OSTI ID: 5558755
• Journal Information: J. Bacteriol.; (United States), Vol. 162:1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ESCHERICHIA COLI
GENETIC MAPPING
GENES
CLONING
HYDROGEN
METABOLISM
HYDROGENASES
ENZYME ACTIVITY
CHROMOSOMES
DNA
ENZYME INHIBITORS
LYASES
MOLECULAR STRUCTURE
MUTANTS
MUTATIONS
PLASMIDS
BACTERIA
CELL CONSTITUENTS
ELEMENTS
ENZYMES
MAPPING
MICROORGANISMS
NONMETALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
550200* - Biochemistry