Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis

Catriona J Harper,Ian Wiid,Martin Kidd,Paul Van Helden,Don Hayward

doi:10.1186/1471-2180-10-138

Catriona J Harper, Ian Wiid + Show 3 more

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https://doi.org/10.1186/1471-2180-10-138

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Journal: BMC microbiology	Publication Date: Jan 1, 2010
Citations: 111	License type: cc-by

Affiliation: Stellenbosch University

Abstract

BackgroundThe assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS) have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH), in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH) under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria.ResultsIt was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions.ConclusionsThe physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is it's activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.

Highlights

The assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria
glutamate dehydrogenase (GDH) specific activity in response to ammonium limitation and excess To investigate the effect of nitrogen availability on GDH activity, M. smegmatis was cultured in minimal medium containing a limited amount of ammonium (3 mM (NH4)2SO4)
The specific activity of both the aminating and deaminating reactions catalysed by NAD+- and NADP+-GDH was determined from M. smegmatis whole cell lysates sampled at 0; 0.5; 2 and 4 hour intervals

Summary

Introduction

The assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. Under conditions of nitrogen excess, glutamine synthetase activity is reduced via adenylylation by the adenylyltransferase GlnE [3,4] and under these conditions, the low ammonium affinity glutamate dehydrogenase (GDH) pathway plays a major assimilatory role with a comparatively low associated energy cost [5]. GDH enzymes catalyse the reversible amination of α-ketoglutarate to form glutamate (see Figure 1B) with concomitant reduction of NAD(P)H They serve as metabolic branch enzymes as the GDH enzymes are involved in anapleurotic processes which regulate the flux of intermediates such as αketoglutarate between the Krebs cycle and nitrogen metabolism [6]. NADP+-specific enzymes are normally involved in the assimilation of nitrogen via amination of α-ketoglutarate [7] and may be transcriptionally regulated by a variety of growth conditions, including carbon and nitrogen limitation [8,9,10,11]. A third class of oligomeric NAD+-specific GDH enzymes was defined whose subunits are approximately 180 kDa in size [18,19,20]

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