Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation

Abstract

BackgroundOrthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation.ResultsThe activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type.ConclusionsFnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0432-6) contains supplementary material, which is available to authorized users.

The role of Fnr paralogs for controlling anaerobic metabolism in diazotrophPaenibacillus polymyxaWLY78

“…We do not know whether Fnr5 or Fnr7 could bind to the promoter region of glnR. In contrast to our results, the combined deletions in both the fnr1 and fnr3 genes in H. seropedicae led to higher expression of nifA , nifB and nifH , which was probably as a consequence of their influence on respiratory activity in relation to oxygen availability (16). It was shown that Fnr was required for relief of NifL inhibition in K. oxytoca under anaerobic conditions (14).…”

“…Fnr-related transcriptional regulators of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family have been reported to be involved in nitrogen fixation of some Gram-negative diazotrophs (12–15). For example, Fnr proteins are indirectly involved in controlling the activity of NifA in Herbaspirillum seropedicae SmR1 by regulating respiratory activity in relation to oxygen availability (16, 17). Fnr protein of Klebsiella oxytoca is required to relieve inhibition of NifA activity by its partner regulatory protein NifL under anaerobic conditions (14).…”

The Role of Fnr Paralogs in Controlling Anaerobic Metabolism in the Diazotroph Paenibacillus polymyxa WLY78

“…Especially, the quadruple deletion mutation of fnr1, fnr3, fnr5, and fnr7 resulted in the lowest growth rates among all fnr deletion mutants and near loss of the nitrogenase activity and nifH transcript. It was reported previously that there are three fnr-like genes and that Fnr1 and Fnr3 were indirectly involved in controlling the activity of NifA in H. seropedicae (16). Fnr1 and Fnr3 of H. seropedicae directly regulate discrete groups of promoters (groups I and II, respectively), while Fnr3-Fnr1 heterodimers regulate a third group (group III) promoters (23).…”

“…Fnr-related transcriptional regulators of the Crp/Fnr (cyclic AMPbinding protein/fumarate nitrate reduction regulatory protein) family have been reported to be involved in nitrogen fixation of some Gram-negative diazotrophs (12)(13)(14)(15). For example, Fnr proteins are indirectly involved in controlling the activity of NifA in Herbaspirillum seropedicae SmR1 by regulating respiratory activity in relation to oxygen availability (16,17). The Fnr protein of Klebsiella oxytoca is required to relieve inhibition of NifA activity by its partner regulatory protein NifL under anaerobic conditions (14).…”

Hierarchical interactions between Fnr orthologs allows fine-tuning of transcription in response to oxygen in Herbaspirillum seropedicae

“…β-Galactosidase assays were performed as described previously ( 11 , 29 , 30 ). To reach oxygen limiting conditions for assays using the E. coli BTH101 (bacterial two hybrid reporter) strain, we used 6 ml screw cap bijou universals filled to the top with LB medium supplemented with 1% glucose as described elsewhere ( 31 ).…”