Regulation and Symbiotic Role of nirK and norC Expression in Rhizobium etli

Abstract

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria

“…By contrary, in E. meliloti NnrR and FixK are part of two different regulatory pathways (for a review, see Cabrera et al, 2011). Additionally, the N 2 -fixation regulator NifA has a negative effect on the transcription of the nirK operon (Gomez-Hernandez et al, 2011). This finding contradicts those reported in B. japonicum where NifA is involved in maximal expression of nap, nirK and norC denitrification genes (Bueno et al, 2010).…”

“…Rhizobium sullae (formerly R. hedysari) only expresses NirK (Toffanin et al, 1996). The genetic determinants for expression of NirK and cNor are present in R. etli CFN42 (Bueno, Gomez-Hernandez, Girard, Bedmar, & Delgado, 2005;Gomez-Hernandez et al, 2011). E. meliloti (formerly Sinorhizobium meliloti) (Galibert et al, 2001;Holloway, McCormick, Watson, & Chan, 1996;Torres et al, 2011) and B. japonicum (recently reclassified as Bradyrhizobium diazoefficiens USDA 110, Delamuta et al, 2013;Bedmar et al, 2005;Kaneko et al, 2002) contain nap, nirK, nor and nos genes (http://www.kazusa.or.jp/rhizobase).…”

“…Although R. etli is unable to respire nitrate and to perform a complete denitrification pathway, the presence of NirK-and Nor-coding regions in this bacterium suggests an NO-detoxifying role for these enzymes, preventing accumulation of NO inside the free-living cells or in the nodules. In fact, in vivo experiments demonstrated that NirK is required for nitrite reduction to NO and that Nor is required to detoxify NO under free-living conditions (Bueno et al, 2005;Gomez-Hernandez et al, 2011). In R. etli, microaerobic expression of nirK and norC promoters requires a functional FixKf, whereas the response to NO is mediated by NnrR.…”

“…etli nirK and norC denitrification genes are also functional in common bean nodules. NirK is an important contributor to the formation of NO in response to nitrate, since levels of LbNO complexes in nodules exposed to nitrate increased in those produced by the norC mutant, but decreased in nirK nodules compared with LbNO levels detected in wild-type nodules (Gomez-Hernandez et al, 2011;Fig. 11).…”

Control of NO level in rhizobium-legume root nodules

“…11 In Rhizobium etli CFN42, the product of norC is required in vivo to detoxify NO. 27 To assess the involvement of the NO reductase in NO degradation in S. meliloti, we tested the sensitivity to NO of a S. meliloti 2011 norB mutant, grown in culture. 28 The results shown in Figure 1 indicate that the norB mutant is more sensitive to NO as compared with the wt strain, although to a lesser extent than the hmp mutant.…”