Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status

Abstract

The Corynebacterium glutamicum gltB and gltD genes, encoding the large (α) and small (β) subunit of glutamate synthase (GOGAT), were investigated in this study. Using RT-PCR, a common transcript of gltB and gltD was shown. Reporter gene assays and Northern hybridization experiments revealed that transcription of this operon depends on nitrogen starvation. The expression of gltBD is under control of the global repressor protein AmtR as demonstrated by gel shift experiments and analysis of gltB transcription in an amtR deletion strain. In contrast to other bacteria, in C. glutamicum GOGAT plays no pivotal role ; e.g. gltB and gltD inactivation did not result in growth defects when cells were grown in standard minimal medium and only a slight increase in the doubling time of the corresponding mutant strains was observed in the presence of limiting amounts of ammonia or urea. Additionally, mutant analyses revealed that GOGAT has no essential function in glutamate production by C. glutamicum.

Ammonium Toxicity in Bacteria

“…Strain MJ6-18 carries a genomic amtR deletion [13], which leads to the lack of the master regulator of nitrogen control, AmtR. As a consequence, genes encoding the enzymes of the glutamine synthetase-glutamate synthase pathway (GS/GOGAT) [3,24] and ammonium carriers AmtA and AmtB [23,31] are constitutively expressed. Double deletion strain TMDgdhDglnA is lacking the ammonium assimilation enzymes glutamate dehydrogenase and glutamine synthetase (GS I).…”

“…As in C. glutamicum, this is caused by osmotic or ionic stress rather than by ammonium toxicity, as shown by the addition of sodium sulfate to cells, which led to even severe effects (Table 2). It might be speculated that the less drastic effect of ammonium compared to sodium is the result of a faster equilibration due to diffusion of NH 3 and resulting smaller osmotic gradients between medium and cytoplasm.…”

“…Ammonium (in this communication, the term ''ammonium'' is used in a general sense, including NH 4 + and NH 3 ; distinct ammonium species are indicated by chemical formula) is the preferred nitrogen source for most bacteria and fungi, and also plants acquire ammonium as a nitrogen source from the soil [35]. When present in high concentrations, diffusion of NH 3 , which is in equilibrium with NH 4 + , across the cytoplasmic membrane into the cell is sufficient to promote growth.…”

“…When present in high concentrations, diffusion of NH 3 , which is in equilibrium with NH 4 + , across the cytoplasmic membrane into the cell is sufficient to promote growth. In a situation of ammonium limitation, diffusion becomes negligible and special carriers are synthesized in bacteria, fungi, and plants (for a recent review, see [36]).…”

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

“…It has also been suggested that extracellular GS may manipulate phagosome pH modulation and consequently prevent phagosome-lysosome fusion (27,26). Furthermore, extracellular GS1 has been implicated in the synthesis of poly-L-glutamine-glutamate, a cell wall constituent of pathogenic mycobacteria (27)(28)(29), which may play a role in cell wall integrity. M tuberculosis GS1 lacks a leader peptide sequence and is considered to be an intracellular protein in most other bacteria, thus its abundance in the extracellular environment of M. tuberculosis and other pathogenic mycobacteria (26) has become a focus of study.…”