The Rhizobium etli rpoN locus: DNA sequence analysis and phenotypical characterization of rpoN, ptsN, and ptsA mutants

Abstract

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.

FIG. 1

Physical map of the R. etli rpoN region. The 1.8-, 4.6-, and 6.1-kb EcoRI fragments hybridizing to the A. tumefaciens probe are shown above the physical map of the 5.6-kb region that was sequenced. Triangles represent insertions of the Ω-Km interposon or the uidA-aphII cassette. The positions and orientations of the identified ORFs are indicated below the restriction map. Restriction sites are abbreviated as follows: B, BstXI; H, HindIII; E, EcoRI; N, NotI; P, PstI.

FIG. 2

Effects of malate and succinate concentrations on the growth curves of the R. etli wild-type strain CNPAF512 (A) and rpoN (B), ptsN (C), and ptsA (D) mutants. Cultures were grown in AMS medium containing NH₄Cl (20 mM) as a nitrogen source and mannitol (20 mM) (▪), succinate (10 mM [▴] or 20 mM [▵]), or malate (10 mM [•] or 20 mM [○]) as a carbon source. OD595, OD at 595 nm.

FIG. 3

Melanin production (A and B) and expression of nifH (C and D) in wild-type R. etli (wt) and rpoN (FAJ1154), ptsN (FAJ1165), and ptsA (FAJ1166) mutants. All data are the means from four independent replicates. Error bars denote the standard deviations. Precultures were grown overnight in TY medium at 30°C, diluted 20-fold in the different media, and incubated overnight with 0.5% oxygen (34). The nitrogen source used was alanine (20 mM). The carbon sources were mannitol (black bars), succinate (stippled bars), and malate (white bars) at 5 mM (A and C) and 20 mM (B and D). To quantify melanin production, cultures were lysed at 37°C in the presence of a solution containing sodium dodecyl sulfate (1%), CuSO₄ (10 μg/ml), and tyrosine (30 μg/ml). The OD of the culture after lysis was measured at 340 nm in a microplate reader after 60 and 120 min of incubation. The difference between the ODs at 340 nm was used to calculate the units. Units are expressed as the ratio of the change in OD at 340 nm to the OD at 595 nm. β-Glucuronidase activities of the translational pnifH-gusA fusion plasmid pFAJ21 are expressed as Miller units.

FIG. 4

DNA sequence flanking the Tn5-mob insertion of R. etli FAJ1166. The deduced amino acid sequence (RE) is compared to that of the E. coli (EC) phosphoenolpyruvate-protein phosphotransferase PtsA (enzyme I; accession no. P32670) from amino acid 111 to 226. The conserved phosphorylation site (H189) is underlined. Identical and similar (S-T-A, L-V-I-M, K-R, Q-N, and F-Y-W) residues are indicated below the amino acid sequences (cons). Stars denote similar residues.

FIG. 5

Toxic effects of malate on cell growth in the presence of mannitol and succinate. The strains tested are the R. etli wild-type strain CNPAF512 (A) and rpoN (B), ptsN (C), and ptsA (D) mutants. Cultures were grown in AMS medium containing alanine (20 mM) as a nitrogen source and mannitol (20 mM) (▪) or succinate (10 mM) (▴) alone or in combination with 20 mM malate (□, mannitol and malate; ▵, succinate and malate). OD595, OD at 595 nm.

FIG. 6

Influence of pH on growth of R. etli wild-type strain CNPAF512 (A) and rpoN (B), ptsN (C), and ptsA (D) mutants. Alanine (20 mM) was used as a nitrogen source. Malate was at 20 mM. Symbols: ▪, pH 5.5; •, pH 6.0; ▴, pH 6.5; ⧫, pH 7.0. OD595, OD at 595 nm.

FIG. 7

Alignment of promoter regions from rhizobial rpoN genes. The aligned rpoN sequences are from R. meliloti (Rm), R. etli (Re), Rhizobium sp. strain NGR234, and B. japonicum rpoN2 (BjrpoN2). The −10 and −35 regions from the R. meliloli rpoN promoter are overlined, and the transcription start site is indicated by an arrow (43). The inverse/complement of the consensus (Cons) sequence of a −24/−12 promoter is indicated; conserved GC and CC residues are in boldface. Nucleotides identical to the consensus −24/−12 sequence are underlined. Ec, E. coli.