Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification

Abstract

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification

“…chelated, Cu-L), standard Cu (0.02 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mu$\end{document} M, Cu-S), or high Cu (13 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mu$\end{document} M, Cu-H) BVMN media. As Cu concentration did not affect WT growth under oxic conditions (Pacheco et al 2022 ), Cu-S oxic growth of each strain was used as control in our experiments. It is important to note that, as shown in Fig.…”

“…The specific Cu concentrations assayed in this study are extensively described in Pacheco et al ( 2022 ). Briefly, the standard Cu concentration of the BVMN medium (0.02 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mu$\end{document} M) is denoted as Cu-S. A concentration of 13 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mu$\end{document} M is referred in this manuscript as high Cu, denoted as Cu-H.…”

“…Besides O 2 and NO, copper (Cu) is an emerging candidate in the regulation of denitrification, since it is an essential cofactor in two enzymes involved in this process, NirK and NosZ. Pacheco et al ( 2022 ) recently proposed that Cu represents an essential factor not only for denitrifying enzymatic activities, but also for the regulation of gene expression, as well as protein synthesis and maturation. Particularly, the expression of nirK, nor , and nos genes, but not nap genes diminished under Cu-limitation in cells grown under NO 3 − -amended O 2 -limiting conditions, which suggests the involvement of a specific Cu-responsive regulator in this control.…”

Molecular and Dual-Isotopic Profiling of the Microbial Controls on Nitrogen Leaching in Agricultural Soils under Managed Aquifer Recharge

“…As for the effects of infiltration rates, several previous studies summarized that NO 3 – removal only occurred when vertical infiltration rates were <0.7 ± 0.2 m/day in native soils with high removal efficiency falling into a range of 0.2–0.4 m/day. , Schmidt et al inferred that the redox conditions at very high infiltration rates were not conducive to denitrification, and this may be particularly true for our soil system, which was dominated by the strictly anaerobic denitrifiers ( Bradyrhizobium ). Nevertheless, we still cannot exclude other factors that impact nitrate removal, for example changes in trace metal availability during flooding, which has been shown to significantly affect denitrification in both laboratory cultures − and environmental samples. − Our study represents initial and novel efforts to understand the factors controlling NO 3 – removal during Ag-MAR events. We recommend future research to investigate these complex interplays related to microbial controls on NO 3 – leaching with Ag-MAR application.…”

Soil pH moderates the resistance and resilience of C and N cycling to transient and persistent stress

“…The soil at pH 7.1 which harboured greater abundances of denitrifiers are more likely to contain species that can cope with Cu and maintain functioning even if others fail (Yachi and Loreau, 1999). Given that Cu is a co-factor in Cu-containing nitrite reductase (CuNIR) which is encoded by nirK, a deficiency in Cu could inhibit the expression of nirK and the transport of nitrite reductase to the periplasm (Pacheco et al, 2022).…”