Mutants of Escherichia coli with Altered Hydrogenase Activity

Abstract

Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H 2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H 2 . They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.

Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2

“…It is generally accepted that HYD2 is associated with hydrogen uptake (23,25), and in agreement with this role AP3 failed to grow in H2-fumarate medium (Table 2). H2-dependent fumarate reduction activity was also undetectable in AP3.…”

“…An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4). Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40).…”

“…The highly oxygen-labile nature of HYD3 has precluded detailed biochemical characterization. HYD2 is involved in H2 uptake and can be differentially induced to high levels when cells are grown in medium containing H2 as an electron donor and fumarate as an electron acceptor (3,23,25,39). An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4).…”

Biochemical and genetic analysis of hydrogen metabolism in Escherichia coli: the hydB gene

“…This medium also contained 200 ,ug of freshly prepared cycloserine per ml. The flask was shaken (150 rpm) at 37°C for 16 h. In the absence of shaking, the cells settled at the bottom of the flask, resulting in survival of cycloserine-sensitive cells. Under anaerobic conditions, E. coli cultures starting at this high cell density normally double twice.…”

“…A mutation in any one of six genes (hydA, -B, -C, -D, -E, and -F) abolishes the expression of all three hydrogenase activities (7,15,16,18,25,27,32,33). These genes map at two different locations in the chromosome.…”