Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli

Abstract

Escherichja 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-Ill 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'eptablished 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-kilobasepair DNA (pSE-130) or fragments derived from this DNA in different orientations inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, straip K-10, but only during an early induction period. These results, based on complementation analysjs 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, ffl4 andfdv are also njeded, All four genes map between 58 and 59 min in the E. coli chromosome. SE-5, SE-6, SE-7, and SE-6}, respectively. Table 1 provides the pedigree and hyd genotype of the hydrogenase-defective mutant strains of E. coli used in this study, and these mutant strains were isolated by E$V selection (22).Media and growth conditions. Luria broth, glucose minimal medium, and HF have been described previously (22).Ampicillin and tetracycliqp, when present, were added to the medium after autoclaving to a final concentration of 100 and 15 p.g/ml, respectively. Cultures for enzyme assays were 353 on July 31, 2020 by guest

Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12

“…In our case, however, the mutant patterns of hybridizing bands were identical to that of the parent strain (data not shown). Therefore, it was concluded that the gene carried on pHG1O is not identical to hydL, F, or C. It is probably also not identical to hydA, B, or E, since these genes have been cloned and their physical maps do not show homology (9,16,34,44). The gene carried on pHG1O was apparently a new one and was designated hydG.…”

“…Some (regulatory) genes such as hydA (16,34), hydB (9,34,44), and hydE (9) have been cloned. This approach has been shown to be fruitful in the study of other gene clusters, e.g., the nif (14), nar (40), or frd (15) operons.…”

Nitrate respiration in relation to facultative metabolism in enterobacteria.

“…These mutations were named ant (anaerobic electron transport) because they were thought to be distinct from hyd; however, genetic mapping reveals an order of cysC-mutS-ant-srl-recA-nalB (435). Therefore, ant and hyd are tightly linked, but apparently not identical (335); that the ant insertion mutations span 5 kilobases indicates that this region contains more than one gene (435).…”

“…Genetic mapping and complementation analyses with cloned DNA fragments indicate that these mutations define two linked, yet distinct regions termed hydA and hydB. Additional complementation studies with subclones indicates that hydB may include genes required for formate dehydrogenase-H (fdv) and formate-hydrogen lyase (fh1) activities (335). Alterations of fdv would provide an explanation for the lowered formate dehydrogenase-H activity observed in many hyd mutants.…”

“…Alterations of fdv would provide an explanation for the lowered formate dehydrogenase-H activity observed in many hyd mutants. Indeed, hydB12 (335) and a newly isolated mutation thought to affect fdv (429) both eliminate expression of a fdhF-lacZ operon fusion (429).…”