DNA Microarray-Based Identification of Genes Regulated by NtrC in Bradyrhizobium japonicum

Abstract

eThe Bradyrhizobium japonicum NtrBC two-component system is a critical regulator of cellular nitrogen metabolism, including the acquisition and catabolism of nitrogenous compounds. To better define the roles of this system, genome-wide transcriptional profiling was performed to identify the NtrC regulon during the response to nitrogen limitation. Upon cells perceiving low intracellular nitrogen, they stimulate the phosphorylation of NtrC, which induces genes responsible for alteration of the core glutamine synthetase/glutamate synthase nitrogen assimilation pathway, including the genes for the glutamine synthetases and PII proteins. In addition, genes responsible for the import and utilization of multiple nitrogen sources, specifically nitrate and nitrite, were upregulated by NtrC activation. Mutational analysis of a candidate nitrite reductase revealed a role for NtrC in regulating the assimilation of nitrite, since mutations in both ntrC and the gene encoding the candidate nitrite reductase abolished the ability to grow on nitrite as a sole nitrogen source.

An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum

“…The second core cytoplasmic enzyme component of the NO 3 − assimilation pathway is NirA, which is required for growth on either NO 3 − or NO 2 − as sole N-source. Consistent with our findings, it has recently been demonstrated that NirA (encoded by bll4571) is required for utilization of NO 3 − or NO 2 − as sole N-source in B. japonicum [ 46 ]. NO 3 − -dependent induction of nasC (as part of the narK operon) and nirA expression is mediated by the two-component regulator NasS-NasT, an observation that is consistent with the role of this system in other α-proteobacteria [ 21 ].…”

Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens

“…With respect to nitrite-dependent growth, the ntrC and rpoN¡/s mutants exhibited a strong delay in growth kinetics, but were nevertheless able to reach WT growth rates after 10 days of incubation. This pattern is in contrast to recent studies where a B. diazoefficiens ntrC mutant was unable to grow with nitrite as the only N source (Franck et al 2015). This apparent discrep ancy could be explained by the different growth conditions used by Frank and colleagues from those used in this work.…”

“…It has been previously demonstrated the involvement of NtrC on nirA expression as well as the inability of a B. diazoefficiens ntrC mutant to grow on nitrite as sole N source (Franck et al 2015). Our biochemical results confirm the NtrC control over nirA and demonstrate for the first time the involvement of NtrC as a transcriptional regulator of the nasC gene encoding the assimilatory nitrate reductase as well as in the ability of B. diazoefficiens to grow with nitrate as the sole nitrogen source.…”

“…In contrast, very little information is available on the function of NtrC in the control of nitrate assimilation genes expression in rhizobia (Szeto et al 1987;Martin et al 1988). Within this context, recent DNA microarray-based transcriptional profiling has revealed a NtrC-dependent regulon operating in response to nitrogen limitation in B. diazoefficiens and the role of NtrC in regulating the utilization of nitrite as a sole N source (Franck et al 2015). However, the involvement of NtrC on the control of assimilatory nitrate reduction to nitrite has not been reported so far.…”

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

“…Though this system has been widely used in a variety of bacterial species, it is prone to yield significant numbers of false-positive, sucrose-resistant clones in some hosts (2)(3)(4)(5)(6). This prompted the development of alternative systems, which, however, may have other limitations, such as species specificity or dependence on additional functions which must be provided in trans (e.g., I-SceI-or cre-and loxP-based systems) (7)(8)(9)(10)(11)(12)(13)(14).…”