Tesis

Copyright 0 1990 by the Genetics Society of America

Structural Genes for Nitrate-Inducible Formate Dehydrogenasein Escherichia coli K-12
Barbara L. Berg and Valley Stewart
Section o Microbiology, Cornell University, Zthaca, New York 14853-8101 f Manuscript received December 10, 1989 Accepted for publication April 17, 1990
ABSTRACT Formate oxidation coupled to nitrate reduction constitutesa major anaerobic respiratory pathway in Escherichia cola. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We haveisolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the threesubunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to thesubunit sizes of purifiedformate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the f d n operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of 9VdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are alsorequired for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

HE facultative aerobe Escherichia coli synthesizes a number of anaerobic respiratory chains. Formate, produced from pyruvate during anaerobiosis, serves as an efficient electron donorfor nitraterespiration. T h e oxidation of formate during nitrate respiration is catalyzed by formate dehydrogenase-N. A major anaerobic respiratory chain consists of formate dehydrogenase-N,cytochrome bj?‘, quinone, cytochrome b:kR, and nitrate reductase (ENOCH and LESTER 1974; RUIZ-HERRERA and DEMOS 1969; reviewed by STEWART 1988). Purified formate dehydrogenase-N consists of three subunits (a,and y) of 110, 32 and 20 kDa, respec/3 tively (ENOCH and LESTER 1975). The a subunit contains selenocysteine and molybdenum cofactor, and is likely to form the active site. T h e function of the p subunit is unknown, and the y subunit is probably cytochrome bi??. Nitratereductase also consists of three subunits and contains molybdenum cofactor and cytochrome b?kR.Formate dehydrogenase-N andnitrate reductase are both cytoplasmic membranebound enzyme complexes (ENOCH and LESTER1974; reviewed by STEWART 1988). T h e structuralgenes are encoded by the narGHJI fornitratereductase operon at 27 min on the E. coli genetic map (reviewed by STEWART 1988). contrast, the structural genes In for formate dehydrogenase-N have not been characterized. Synthesis of formate dehydrogenase-N and nitratereductase is induced by nitrate during anaerobic growth.Figure1 illustrates ourcurrent model for regulation of narGHJI transcription by anaerobiosis and nitrate.Anaerobic induction is mediated by FNR,

T

an activator of anaerobic respiratory genes (LAMBDEN and GUEST1976; NEWMAN and COLE1978; reviewed by STEWART 1988). Induction by nitraterequires NarL, the product the regulatory genenarL(STEWof ART 1982; STEWART and PARALES 1988). Transposon insertions in narX have only subtle effects on the induction of narGHJI by nitrate (STEWART PARand ALES 1988). Mutations in narL and narX also affect nitrate repression of other anaerobic enzymes, including fumarate reductase, dimethylsulfoxide reductase, and pyruvate-formate lyase (COTTER and GUNSALUS 1989; IUCHIand LIN 1987; KALMAN and GUNSALUS1989; SAWERS BOCK 1988; STEWART and and BERG 1988). Comparisons of predicted aminoacid seqences show that NarX (NOHNO al. 1989; STEWART, PARet ALES and MERKEL1989) and NarL (GUNSALUS, KALMAN and STEWART 1989; NOHNO al. 1989; STEWet ART, PARALES and MERKEL1989) are similar to other prokarotic regulatory proteins known as “two component regulatory systems” (STOCK, NINFA and STOCK 1989). A second...