The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

Abstract

Background: Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.

Results: The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation.

Conclusion: tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor, N-acetyl glucosamine reduces nod gene expression and nodulation efficiency when present at millimolar concentrations. A role for Tep1 in the efflux of Nod factor precursors could explain the phenotypes associated with tep1 inactivation.

Figure 1

Effect of different concentrations of chloramphenicol on the growth of S. meliloti GR4 and GR4T1. Growth of GR4 (open symbols) and GR4T1 (tep1 mutant) (closed symbols) was tested in TY broth with 0 μg/ml (triangles), 25 μg/ml (diamonds) or 50 μg/ml (squares) chloramphenicol. A representative example from 3 independent experiments is shown.

Figure 2

Swarming motility of S. meliloti wild type and mutant strains. Swarming motility of GR4 (wt), GR4T1 (tep1 mutant), QS77 (fadD mutant) and QSTR1 (double mutant fadD, tep1) was tested on 0.7% agar minimal medium at 28°C.

Figure 3

Nodulation efficiency of S. meliloti GR4 (open diamonds) and GR4T1 (tep1 mutant) (closed squares). Mean values and standard errors (95% confidence) were calculated from three independent experiments.

Figure 4

Nodulation efficiency upon addition of different concentrations of Nod factor precursors. Just before inoculation with S. meliloti GR4, alfalfa plants were supplemented with 50 μM glucosamine (GA) (open squares), 5 mM glucosamine (filled squares), 50 μM N-acetyl glucosamine (NAGA) (open triangles), 5 mM N-acetyl glucosamine (closed triangles) or without the addition of Nod factor precursors (filled circles). A representative example from 3 independent experiments is shown.

Figure 5

Working model showing possible roles for Tep1 and their substrates. Cm, chloramphenicol; IM, inner membrane; OM, outer membrane.