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Journal of Bacteriology, November 1998, p. 5529-5539, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Oxygen-Insensitive Nitroreductases: Analysis of the Roles of
nfsA and nfsB in Development of Resistance to
5-Nitrofuran Derivatives in Escherichia coli
J.
Whiteway,
P.
Koziarz,
J.
Veall,
N.
Sandhu,
P.
Kumar,
B.
Hoecher, and
I. B.
Lambert*
Biology Department, Carleton University,
Ottawa, Ontario, Canada K1S 5B6
Received 9 April 1998/Accepted 17 August 1998
Nitroheterocyclic and nitroaromatic compounds constitute an
enormous range of chemicals whose potent biological activity has significant human health and environmental implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of
nitroreductase activities. Resistance of bacteria to nitro-substituted compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the
nfsA and nfsB genes of a large number of
nitrofuran-resistant mutants of Escherichia coli and have
correlated mutation with cell extract nitroreductase activity. Our
studies demonstrate that first-step resistance to furazolidone or
nitrofurazone results from an nfsA mutation, while the
increased resistance associated with second-step mutants is a
consequence of an nfsB mutation. Inferences made from
mutation about the structure-function relationships of NfsA and NfsB
are discussed, especially with regard to the identification of flavin
mononucleotide binding sites. We show that expression of
plasmid-carried nfsA and nfsB genes in
resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and
49%, respectively, contained insertion sequence elements in
nfsA and nfsB. IS1 integrated in
both genes, while IS30 and IS186 were found
only in nfsA and IS2 and IS5 were
observed only in nfsB. Insertion hot spots for
IS30 and IS186 are indicated in
nfsA, and a hot spot for IS5 insertion is
evident in nfsB. We discuss potential regional and
sequence-specific determinants for insertion sequence element
integration in nfsA and nfsB.
*
Corresponding author. Mailing address: Biology
Department, Carleton University, 1125 Colonel By Dr., Ottawa, Ontario,
Canada K1S 5B6. Phone: (613) 520-2600, ext. 2755. Fax: (613) 520-4497. E-mail: ilambert{at}ccs.carleton.ca.
Journal of Bacteriology, November 1998, p. 5529-5539, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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