Differential Regulation of Rhizobium etli rpoN2 Gene Expression during Symbiosis and Free-Living Growth

Abstract

The Rhizobium etli rpoN1 gene, encoding the alternative sigma factor ς54 (RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D’hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729–1740, 1998). We identified a second rpoN gene copy in R. etli,rpoN2, encoding a 54.0-kDa protein which displays 59% amino acid identity with the R. etli RpoN1 protein. TherpoN2 gene is cotranscribed with a short open reading frame, orf180, which codes for a protein with a size of 20.1 kDa that is homologous to several prokaryotic and eukaryotic proteins of similar size. In contrast to the R. etli rpoN1mutant strain, inactivation of the rpoN2 gene did not produce any phenotypic defects during free-living growth. However, symbiotic nitrogen fixation was reduced by approximately 90% in therpoN2 mutant, whereas wild-type levels of nitrogen fixation were observed in the rpoN1 mutant strain. Nitrogen fixation was completely abolished in the rpoN1 rpoN2 double mutant. Expression of rpoN1 was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. In contrast, rpoN2-gusA and orf180-gusA fusions were not expressed aerobically but were strongly induced at low oxygen tensions or in bacteroids. Expression of rpoN2 andorf180 was abolished in R. etli rpoN1 rpoN2 andnifA mutants under all conditions tested. Under free-living microaerobic conditions, transcription of rpoN2 andorf180 required the RpoN1 protein. In symbiosis, expression of rpoN2 and orf180 occurred independently of the rpoN1 gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.

The four different σ54 factors of Rhodobacter sphaeroides are not functionally interchangeable

“…In almost every bacterial chromosome sequenced, at least one copy of the rpoN gene has been found (Mittenhuber, 2002). In bacteria, in which two copies of this gene are present, the different RpoN proteins can essentially substitute for each other (Kullik et al ., 1991; Michiels et al ., 1998). It has also been observed that an rpoN gene from one species is able to complement an rpoN mutation in another species, even across different phylogenetic groups within the domain Bacteria (Leary et al ., 1998).…”

Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512

“…Propane was used as an internal standard. For stress experiments, bacteroids were purified from the nodules by differential centrifugation [ 49 ] and subsequently resuspended in 10 mM MgSO 4 .…”

“…For bacteroid experiments, three samples were tested for each strain. To prepare a sample, nodules from 3 different inoculated plants were isolated, weight was determined and bacteroids were isolated [ 49 ] and suspended in 4.5 ml 10 mM MgSO 4 (approximately equal numbers). Stress survival of bacteroids in the presence of 10 mM H 2 O 2 or 2.5 M NaCl or at 45°C was determined as described for free-living bacteria.…”

Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere

“…Upregulation of rpoN was also observed in Azoarcus sp. BH72 during growth in nitrogen fixation conditions and in Rhizobium etli during symbiosis (Michiels et al, 1998;Sarkar and Reinhold-Hurek, 2014). Indeed, most nif genes ( Fig.…”