Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity

Abstract

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase p subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the j9 subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Characterization of the paramagnetic iron‐containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase

“…The periplasmic nitrate reductase was purified from T pantotropha strain M6 (AnarH:: TnS(Kmy [2]) as previously described [ 1] except that the final gel titration step was omitted. Protein concentration was estimated using the Bradford method [8].…”

“…The enzyme from Thiosphaera pantotropha has been most extensively characterized [1,2] but the enzyme has also been purified from Rhodobacter sphaeroides f.sp. denitrificans [3], Rhodobacter capsulatui [4-61 and Alcaligenes eutrophus [7].…”

The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases

“…Chlorate is an analogue of nitrate and functions as a substrate for respiratory Nars, with toxic chlorite as the product of reduction (Bell et al, 1990(Bell et al, , 1993. We developed an assay to determine the rate of survival after exposure of S. coelicolor to sodium chlorate based on a previously described anaerobic stress survival assay (van Keulen et al, 2007).…”

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures

“…Aerobic nitrate reduction has been shown to occur in a variety of bacteria (Carter et al, 1995). It is for Paracoccus sp., however, that most is known about aerobic nitrate respiration (Robertson & Kuenen, 1984, 1990Bell et al, 1990Bell et al, , 1993Richardson & Ferguson, 1992;Sears et al, 1997). The periplasmic nitrate reductase (Nap) is responsible for the aerobic reduction of nitrate to nitrite.…”