Articulo Microbiana

JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1291-1297
Copyright © 1969 American Society for Microbiology

Vol. 97, No. 3
Printed In U.S.A.

Nitrate Reductase Complex of Escherichia coli K-12:
Isolation and Characterization of Mutants
Unable to Reduce Nitrate
JOSE RUIZ-HERRERA,1 MICHAEL K. SHOWE,9 AND J. A. DEMOSS
Department of Biology, University of California, La Jolla, California 92037Thirty-eight mutants unable to reduce nitrate were isolated from Escherichia coli
and characterized biochemically and genetically. All of the mutants exhibited reduced or insignificant levels of formate dehydrogenase, nitrate reductase, or various
combinations of these activities and cytochrome bi under conditions which resulted
in the production of high levels of these activities by thewild-type parental strains.
Most of the mutants reverted readily to wild type, and all mapped within a restricted region on the chromosome linked to the tryptophan genes. It was proposed
that nitrate reduction in E. coli was catalyzed exclusively by an organized complex
containing formate dehydrogenase, cytochrome bi, and nitrate reductase.
The reduction of nitrate to nitrite by Escherichia
coli iscatalyzed by a membrane-bound nitrate
reductase (6, 12) which is induced by nitrate and
repressed by oxygen (12). This enzyme has been
extracted in a soluble form from membrane fragments and found to be associated with cytochrome bi (8) and formate dehydrogenase (7). It
has been generally assumed that nitrate acts as a
general electron acceptor in E. coli growing anaerobically, providingenergy through a mechanism of "anaerobic respiration."
Several features of the nitrate reductase system
make it a very convenient system for a study of
the genetic control, the biochemical and physical
organization, and the biogenesis of a subcellular
particle in bacteria. (i) Nitrate reductase is membrane-bound and apparently associated with
other measurable membrane-bound components.
(ii) Itis induced and repressed by controllable
environmental factors. (iii) It is a dispensible
component of cell metabolism, a property which
makes it possible to isolate mutants deficient in
their ability to reduce nitrate. To initiate a study
on the genetic control and biogenesis of this
organized enzyme system, we have developed a
direct procedure for isolating mutants of E. coli
K-12 whichare unable to reduce nitrate under
normal growth conditions. We present here the
results of a biochemical analysis and a preliminary
genetic study of a series of such mutants.
Recently, Piechaud et al. (11) isolated a group
l Present address: Departamento de Microbiologia, Escuela
Nacional de Ciencias Biologicas, I.P.N., Carpio y Plan de Ayala,
Mexico, D.F.
2 Present address: HaverfordCollege, Haverford, Pa. 19041

of mutants from E. coli and other organisms
which are defective in their ability to reduce nitrate by selecting mutants which are resistant to
normally inhibitory levels of chlorate. Results
with a few such mutants (3, 10, 11) suggest that
they correspond to at least some of the mutant
classes which we have observed in the present
study.
MATERIALS AND METHODSE. coli strain HfrH (str, thi-18) was obtained from
D. Helinski. Strains AB2102 (F-, strr, thi, thr-, leuc,
pro-, trp-, hisr, gat-, mtt mat-) and AB1870 (Hfr
(0-32: his-gal-lac) thi-18, stre) were obtained from E.
Adelberg. These strains and the mutants derived from
them were maintained on nutrient agar slants.
The complete medium utilized to cultivate strains
consisted of mineral salts(13) supplemented with 1%
glucose, 0.4% nutrient broth, and 5 jug of thiamine
per ml. Where indicated, the medium was supplemented with 1% potassium nitrate. For aerobic
growth conditions, cultures were sparged with air. For
anaerobic conditions, cultures were sparged with a
mixture of 95% nitrogen and 5% CO2. For biochemical analyses, cells from 75 ml of medium were
centrifuged and washed...