The role of GlnD in ammonia assimilation in Mycobacterium tuberculosis

Abstract

SummaryThe control of ammonia assimilation in Mycobacterium tuberculosis is poorly understood. We have been investigating a regulatory cascade predicted to control the activity of glutamine synthetase (GS). We previously demonstrated that the GS-modifying protein, GlnE (an adenylyl transferase), is essential for M. tuberculosis growth. GlnD, a uridylyl transferase, is involved in the control of GlnE activity in other bacteria. In M. tuberculosis, glnD is arranged in an apparent operon with amt and glnB; all three genes are up-regulated in a low-ammonia medium. We constructed an in-frame deletion of glnD by homologous recombination. The mutant had no growth defect in media containing different nitrogen sources. Total GS activity in culture filtrates was markedly reduced in the mutant, although activity in cell-free extracts remained normal. Virulence was unaffected in both in vitro and in vivo model systems of infection, indicating that the presence of extra-cellular GS is not critical for virulence and that the residual intra-cellular GS activity is sufficient. Thus although GlnD does play a role in the control of ammonia assimilation, it is not required for virulence.

Nitrogen Control in Mycobacterium smegmatis : Nitrogen-Dependent Expression of Ammonium Transport and Assimilation Proteins Depends on the OmpR-Type Regulator GlnR

“…Additionally, studies of the glnE gene product adenylyltransferase, which is crucial for posttranslational modification and regulation of glutamine synthetase, and the glnD-encoded uridylyltransferase, which is putatively involved in nitrogen signal transduction, were carried out (11,12,18,20). In this paper, we describe the first in vivo characterization of GlnR as a regulator of nitrogen control in mycobacteria.…”

“…Previous work on nitrogen uptake and assimilation concentrated mainly on the major pathogenic member of the genus, Mycobacterium tuberculosis. While extensive data are available for glutamine synthetases, especially the physiologically crucial GlnA1 enzyme (12), the associated adenylyltransferase GlnE (7,18), and the uridylyl transferase GlnD (20), not much information is available for the transcriptional regulation of nitrogen metabolism in mycobacteria (19). In Mycobacterium smegmatis, homologs of all previously characterized genes encoding glutamine synthetases in M. tuberculosis are present in the genome (GlnA1, MSMEG_ 4290; GlnA2, MSMEG_4294; GlnA3, MSMEG_3561; GlnA4, MSMEG_2595), as are open reading frames encoding additional glutamine synthetase-like proteins with unknown physiological functions (e.g., MSMEG_1116, MSMEG_3827, MSMEG_5374, and MSMEG_6693).…”

Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor

“…The same GlnK-independent regulation of GlnA1 activity in C. glutamicum was also found (1) which could indicate that in these organisms GlnE is posttranslationally modified by an unknown mechanism or that GlnE may play a role as a sensor of cellular nitrogen status since glutamine synthetase activity is regulated by GlnE according to nitrogen availability in the absence of well-known nitrogen sensors. Read et al (34) constructed a glnD mutant strain of M. tuberculosis and showed that there was no difference in GlnA1 activity (in the cell free extract) between the wild-type and mutant bacteria grown on media containing differing nitrogen concentrations. It can thus be concluded that GlnA1 in this bacillus is regulated by GlnE in a GlnK and GlnD independent manner.…”

“…Read et al (34) have shown that amtB, glnK, and glnD are up-regulated under conditions of nitrogen limitation. Searches for the amtR gene and Blast analyses with the C. glutamicum AmtR protein have pointed to the existence of a putative AmtR-type transcriptional regulator (Rv3610c) in M. tuberculosis.…”

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

“…12 In the E. coli GlnK, the Tyr51 residue is uridylylated by the GlnD enzyme, but in the Gram-positive soil bacteria Corynebacterium glutamicum and Streptomyces coelicolor GlnD has been shown to act as an adenylyl transferase, adenylylating instead of uridylylating the GlnK protein at this conserved Tyr51 residue. [13][14][15] Under nitrogen limiting conditions, the GlnK protein is uridylylated/adenylylated at the Tyr51 residue, which prevents its interaction with AmtB allowing an unhindered flow of extracellular ammonium into the cell through AmtB. 16 The AmtB:GlnK complex is also implicated in the membrane sequestration of enzymes involved in nitrogen fixation, such as the glycohydrolase DraG in Azospirillum brasilense.…”