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Journal of Bacteriology, December 2001, p. 7241-7252, Vol. 183, No. 24
Instituto de Bioquímica y
Biología Molecular, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, La Plata, Argentina
Received 23 January 2001/Accepted 18 September 2001
In this study, we addressed the effects of N limitation in
Bradyrhizobium japonicum for its association with
soybean roots. The wild-type strain LP 3001 grew for six generations
with a growth rate of 1.2 day
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7241-7252.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Improved Soybean Root Association of N-Starved
Bradyrhizobium japonicum
1 in a minimal medium with
28 mM mannitol as the carbon source and with the N source
[(NH4)2SO4] limited to only 20 µM. Under these conditions, the glutamine synthetase (GS) activity
was five to six times higher than in similar cultures grown with 1 or
0.1 mM (NH4)2SO4. The
NtrBC-inducible GSII form of this enzyme accounted for 60% of the
specific activity in N-starved rhizobia, being negligible in the other
two cultures. The exopolysaccharide (EPS) and capsular polysaccharide
(CPS) contents relative to cell protein were significantly higher in
the N-starved cultures, but on the other hand, the
poly-3-hydroxybutyrate level did not rise in comparison with
N-sufficient cultures. In agreement with the accumulation of CPS in
N-starved cultures, soybean lectin (SBL) binding as well as stimulation
of rhizobial adsorption to soybean roots by SBL pretreatment were
higher. The last effect was evident only in cultures that had not
entered stationary phase. We also studied nodC gene
induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573,
nodC gene expression was induced by genistein 2.7-fold
more in N-starved young cultures than in nonstarved ones. In
stationary-phase cultures, nodC gene expression was
similarly induced in N-limited cultures, but induction was negligible
in cultures limited by another nutrient. Nodulation profiles obtained
with strain LP 3001 grown under N starvation indicated that these
cultures nodulated faster. In addition, as culture age increased, the
nodulation efficiency decreased for two reasons: fewer nodules were
formed, and nodulation was delayed. However, their relative importance
was different according to the nutrient condition: in older cultures
the overall decrease in the number of nodules was the main effect in
N-starved cultures, whereas a delay in nodulation was more responsible
for a loss in efficiency of N-sufficient cultures. Competition for
nodulation was studied with young cultures of two wild-type strains
differing only in their antibiotic resistance, the N-starved cultures
being the most competitive.
*
Corresponding author. Mailing address: Instituto de
Bioquímica y Biología Molecular, Facultad de Ciencias
Exactas, Universidad Nacional de La Plata, Calles 47 y 115 (1900),
La Plata, Argentina. Phone: 54-221-425-0497, ext. 31. Fax:
54-221-422-3409, ext. 56. E-mail:
lodeiro{at}biol.unlp.edu.ar.
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