The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

Abstract

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 DeltarelA DeltaspoT 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 Omega 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-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

FIG. 1.

The Rsh proteins of the Rhizobiaceae are highly conserved. Shown is an alignment of partial Rsh sequences against motifs I, II, and V of the HD domain of metal-dependent phosphohydrolases. Sequences correspond to Rsh sequences of Rhizobium etli (R.etl), Agrobacterium tumefaciens (A.tum), Sinorhizobium meliloti (S.mel), Mesorhizobium loti (M.lot), Brucella melitensis (B.mel), Brucella suis (B.sui), and Bradyrhizobium japonicum (B.jap) and to E. coli SpoT and RelA. The conserved H and D residues of the second motif are shown in boldface. Asterisks under the alignment indicate conserved amino acids.

FIG. 2.

The rsh gene of R. etli produces a functional protein in E. coli cells. One-dimensional TLC analysis of total intracellular nucleotides extracted from E. coli cultures uniformly labeled with [³²P]H₃PO₄. (A) Cells were starved for amino acids in MOPS-MM plus 500 μg/ml serine hydroxamate and 300 μg/ml valine. Lane 1, CF1648 (wild type); lane 2, CF1652 ΔrelA; lane 3, CF1693 ΔrelA ΔspoT; lane 4, CF1652/pMGD44; lane 5, CF1693/pMGD44. (B) E. coli cells were carbon starved in MOPS medium with 0.02% glucose. Lane 1, CF1648; lane 2, CF1652; lane 3, CF1693; lane 4, CF1693/pMGD44.

FIG. 3.

R. etli rsh mutants are affected in (p)ppGpp synthesis during carbon and nitrogen starvation. Shown is a one-dimensional TLC analysis of total intracellular nucleotides extracted from R. etli cultures uniformly labeled with [³²P]H₃PO₄. Cells were grown in MOPS-MM with 1 mM ammonium and 0.02% glucose. Lane 1, CE3 (wild type); lane 2, LM01 (rsh::Tn5); lane 3, AC1 (rsh::Ω); lane 4, LM01/pMGD44; lane 5, AC1/pMGD44.

FIG. 4.

R. etli rsh mutants are affected in nodulation. Ultrastructural differentiation of bacteroids elicited by wild-type CE3 (A and C) and mutant LM01 (B and D) strains. Bars: A and B, 500 nm; C and D, 2 μm. Note that LM01 mutant bacteroids are pleomorphic and devoid of poly-β-hydroxybutyrate (PHB) granules (indicated by an arrow).

FIG. 5.

Transcription of nodA gene is constitutive in R. etli rsh mutants. A nodA-lacZ transcriptional fusion, carried by plasmid pRP30 (47), was used to examine the effect of rsh mutations on nodA gene expression. Cultures were grown for 12 h in MM with 5 mM ammonium. Naringenin was used as inducer at 1.2 μM; values are expressed as Miller units ± standard deviation. (1) CE3 (wild type); (2) LM01 (rsh::Tn5); (3) LM01/pMGD44; (4) AC1 (rsh::Ω).

FIG. 6.

Nod factor production is constitutive in the R. etli LM01 mutant. One-dimensional TLC was performed to determine the presence of Nod factors (which migrate at the position shown by two arrows) in supernatants of R. etli cultures grown in MM with 5 mM ammonium plus [1-¹⁴C]glucosamine. (1) CE3 (wild type); (2) LM01 (rsh::Tn5); (3) LM01/pMGD44. −, without inducer; +, with 1.2 μM naringenin.