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J Bacteriol, April 1998, p. 1729-1740, Vol. 180, No. 7
F. A. Janssens Laboratory of Genetics,
K. U. Leuven, B-3001 Heverlee, Belgium
Received 31 October 1997/Accepted 16 January 1998
The rpoN region of Rhizobium etli was
isolated by using the Bradyrhizobium japonicum rpoN1 gene
as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of
this region revealed the presence of four complete open reading frames
(ORFs), ORF258, rpoN, ORF191, and ptsN, coding
for proteins of 258, 520, 191, and 154 amino acids, respectively. The
gene product of ORF258 is homologous to members of the ATP-binding
cassette-type permeases. ORF191 and ptsN are homologous to
conserved ORFs found downstream from rpoN genes in other
bacterial species. Unlike in most other microorganisms,
rpoN and ORF191 are separated by approximately 1.6 kb. The
R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of
nifH, and the metabolism of C4-dicarboxylic
acids and several nitrogen sources (ammonium, nitrate, alanine, and
serine). Expression of the rpoN gene was negatively
autoregulated and occurred independently of the nitrogen source.
Inactivation of the ptsN gene resulted in a decrease of
melanin synthesis and nifH expression. In a search for
additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in
ptsA, a gene homologous to the Escherichia coli
gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA
mutant also displayed reduced expression of nifH. The
ptsN and ptsA mutants also displayed increased
sensitivity to the toxic effects of malate and succinate. Growth of
both mutants was inhibited by these C4-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these
conditions. The effect of malate occurred independently of the nitrogen
source used. Growth inhibition was decreased by lowering the pH of the
growth medium. These results suggest that ptsN and
ptsA are part of the same regulatory cascade, the
inactivation of which renders the cells sensitive to toxic effects of
elevated concentrations of malate or succinate.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Rhizobium etli rpoN Locus: DNA
Sequence Analysis and Phenotypical Characterization of rpoN,
ptsN, and ptsA Mutants

*
Corresponding author. Mailing address: F.A. Janssens
Laboratory of Genetics, K.U. Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. Phone: 32 16 321631. Fax: 32 16 321966. E-mail: jozef.vanderleyden{at}agr.kuleuven.ac.be.
Present address: Laboratoire de Biologie Moléculaire,
Institut Pasteur d'Algérie, Alger, Algeria.
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