Requirement of Nitric Oxide for Induction of Genes Whose Products Are Involved in Nitric Oxide Metabolism in Rhodobacter sphaeroides 2.4.3

Adam V Kwiatkowski,James P Shapleigh

doi:10.1074/jbc.271.40.24382

Abstract

During denitrification, freely diffusible nitric oxide (NO) is generated for use as a terminal electron acceptor. NO is produced by nitrite reductase (Nir) and reduced to nitrous oxide by nitric oxide reductase (Nor). Using Nir and Nor-deficient mutants of Rhodobacter sphaeroides 2.4.3, we have shown that the endogenous production of NO or the addition of exogenous NO induces transcription of certain genes encoding Nir and Nor. A Nor-deficient strain was found to be capable of expressing wild type levels of nirK-lacZ and norB-lacZ fusions in medium unamended with nitrogen oxides. When this experiment is performed in the presence of hemoglobin, fusion expression is eliminated. NO and the NO-generator, sodium nitroprusside, can induce expression of both fusions in a strain lacking Nir and the consequent ability to produce NO. Sodium nitroprusside cannot induce expression of nirK-lacZ in a strain lacking the transcriptional activator NnrR (nitrite and nitric oxide reductase regulator). Addition of the cyclic nucleotides cAMP and 8-bromoguanosine-cGMP does not result in expression of either fusion. These results demonstrate that denitrifying bacteria produce NO as a signal molecule to activate expression of the genes encoding proteins required for NO metabolism.

Highlights

Many bacteria have the capacity to use compounds other than oxygen as terminal electron acceptors during respiration
Expression of nirK and norB Fusions in a Nitric oxide reductase (Nor)-deficient Strain—Experiments with a nitrite reductase (Nir)-deficient mutant of R. sphaeroides 2.4.3, strain 11.10, demonstrated that Nir activity is an obligate requirement for the transcriptional activation of nirK.2
No induction of norBlacZ was observed in any of the 11.10 cultures with either cAMP or the cGMP analog. Taking advantage of both Nir and Nor-deficient strains of R. sphaeroides 2.4.3 we have provided clear evidence that freely diffusible Nitric oxide (NO) must be present for the induction of nirK and the nor operon in the denitrifier R. sphaeroides 2.4.3

Summary

Introduction

Many bacteria have the capacity to use compounds other than oxygen as terminal electron acceptors during respiration. Nitrite is generated by the two electron reduction of nitrate, the initial reaction in denitrification. The expression of genes whose protein products are involved in denitrification requires both low oxygen and the presence of N-oxides [1]. How the cell recognizes these environmental signals is not well understood It is not clear how the cell regulates those enzymes directly involved in NO metabolism to mitigate the accumulation of this toxic intermediate. Given the indirect evidence suggesting that NO production is required for expression of the genes encoding Nir and Nor in denitrifiers, as well as the established role of NO as modulator. NO-dependent Gene Expression in R. sphaeroides of biological activities in eukaryotes, we have carried out a series of experiments to determine if NO is directly involved in regulation of genes involved in denitrification. The nor operon includes norC and norB, which encode the structural subunits, and two other genes likely required for Nor assembly. Through monitoring the expression of reporter gene fusions in denitrification mutants under various conditions, we have provided evidence that clearly shows the role of NO in modulating nirK and nor operon expression

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Results

Conclusion