NAD(P)
+
-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N
2
Fixation Capabilities
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abstract
ABSTRACT
C
4
-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N
2
-fixing bacteria (bacteroids) within legume nodules. In
Sinorhizobium meliloti
bacteroids from alfalfa, NAD
+
-malic enzyme (DME) is required for N
2
fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont
Rhizobium leguminosarum
, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that
dme
mutants of the broad-host-range
Sinorhizobium
sp. strain NGR234 formed nodules whose level of N
2
fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single
pckA
and single
dme
mutants fixed N
2
at reduced rates, a
pckA dme
double mutant had no N
2
-fixing activity (Fix
−
). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix
−
phenotype of
S. meliloti dme
mutants may be specific to the alfalfa-
S. meliloti
symbiosis. We therefore examined the ME-like genes
azc3656
and
azc0119
from
Azorhizobium caulinodans
, as
azc3656
mutants were previously shown to form Fix
−
nodules on the tropical legume
Sesbania rostrata
. We found that purified AZC3656 protein is an NAD(P)
+
-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N
2
fixation in
A. caulinodans
and
S. meliloti
, in other rhizobia this activity can be bypassed via another pathway(s).
