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Journal of Bacteriology, August 1998, p. 3853-3863, Vol. 180, No. 15
Mikrobiologisches Institut,
Eidgenössische Technische Hochschule, CH-8092 Zürich,
Switzerland
Received 2 February 1998/Accepted 27 May 1998
Many nitrogen fixation-associated genes in the soybean symbiont
Bradyrhizobium japonicum are regulated by the
transcriptional activator NifA, whose activity is inhibited by
aerobiosis. NifA is encoded in the fixR-nifA
operon, which is expressed at a low level under aerobic
conditions and induced approximately fivefold under low-oxygen tension.
This induction depends on a
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression of the fixR-nifA Operon in
Bradyrhizobium japonicum Depends on a New Response
Regulator, RegR
24/
12-type promoter
(fixRp1) that is recognized by the
54 RNA polymerase and activated by NifA. Low-level
aerobic expression and part of the anaerobic expression originates from
a second promoter (fixRp2) that overlaps with
fixRp1 and depends on an upstream DNA region
(UAS) located around position
68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760-1765, 1995). A protein
binding to the UAS was previously postulated to act as an activator.
This protein has now been purified, and the corresponding gene
(regR) has been cloned. On the basis of the
predicted amino acid sequence, RegR belongs to the family of
response regulators of two-component regulatory systems. We identified
upstream of the regR gene an additional gene
(regS) encoding a putative sensor kinase. A
regR mutant was constructed in which neither a specific
UAS-binding activity nor fixRp2-dependent transcript formation and fixR'-'lacZ expression was
detected in aerobically grown cells. Anaerobic fixR'-'lacZ
expression was also decreased in regR mutants to about 10%
of the level observed in the wild type. Similarly, regR
mutants showed only about 2% residual nitrogen fixation activity, but
unlike nodules induced by nifA mutants, the morphology of
those nodules was normal, displaying no signs of necrosis. While
regR mutants grew only slightly slower in free-living,
aerobic conditions, they displayed a strong growth defect under
anaerobic conditions. The phenotypic properties of regS
mutants differed only marginally, if at all, from those of the wild
type, suggesting the existence of a compensating sensor activity in
these strains. The newly identified RegR protein may be regarded as a
master regulator in the NifA-dependent network controlling
nif and fix gene expression in B. japonicum.
*
Corresponding author. Mailing address:
Mikrobiologisches Institut, Eidgenössische Technische Hochschule,
Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland. Phone:
41-1-632-3318. Fax: 41-1-632-1382. E-mail:
hennecke{at}micro.biol.ethz.ch.
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