A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a ⌬relA ⌬spoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an ⍀ element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-␤-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.Rhizobia are soil bacteria able to colonize the roots of compatible legumes under conditions of nitrogen limitation (31,39,46). This symbiotic interaction leads to the formation of organelle-like structures called nodules in the plant roots, in which these bacteria differentiate into N 2 -fixing forms known as bacteroids (31,46). Bacteroids in the nodules are surrounded by the plant cell membrane, called the peribacteroidal membrane (31,42). Bacteroids, the peribacteroidal space, and the peribacteroidal membrane are also referred to as symbiosomes (38). In the process of symbiosome formation, freeliving rhizobia move from a variable environment to a more controlled one inside the plant cells by adapting in succession to three different environments: the rhizosphere, the infection thread (IT), and the plant cell cytoplasm (31, 39, 42). Bacteroid differentiation is accompanied by the loss of bacterial cell division and by a switch from a metabolism dedicated to ammonium assimilation to one dedicated to nitrogen fixation (33, 44).The study of rhizobial metabolic networks that lead to productive nodules is clearly of importance for understanding the symbiotic process. Diverse studies have implicated amino acid metabolism in the bacterial adaptation to nodule conditions, as well as in the metabolic interchange between plant and bacteroids in fully developed nodules (7,16,20,33,34). The rhizobial metabolic adaptations required for using amino acids inside the IT and ammonium excretion in these circumstances could function as a signal to uncouple ammonium assimilation a...

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K m , V max , and k cat for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 o C, but the optimal temperature of activity was 37 o C. It also showed maximal and optimal activities at pH 9.0. The values of K m , V max , k cat , and k cat /K m were 8.9 ± 0.967 × 10-3 M, 128 ± 2.8 U/mg protein, 106 ± 2 s-1 , and 1.2 ± 0.105 × 10 4 M-1 s-1 , respectively. The L-asparaginase activity was reduced in the presence of Mn 2+ , Zn 2+ , Ca 2+ , and Mg 2+ metal ions for about 52% to 31%. In addition, we found that + NH 4 , L-Asp, D-Asn, and β-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.

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