SummaryP II -type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P II -type protein in Corynebacterium glutamicum , is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnKglnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F 1 b b b b , are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.

SummaryAmtR, the master regulator of nitrogen control in Corynebacterium glutamicum , represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pulldown assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR.The AmtR regulon was characterized in this study by a combination of bioinformatics, transcriptome and proteome analyses. At least 33 genes are directly controlled by the repressor protein including those encoding transporters and enzymes for ammonium assimilation ( amtA , amtB , glnA , gltBD ), urea and creatinine metabolism ( urtABCDE , ureABCEFGD , crnT , codA ), a number of biochemically uncharacterized enzymes and transport systems (NCgl1099, NCgl1100, NCgl 1915-1918) as well as signal transduction proteins ( glnD , glnK ). For the AmtR regulon, an AmtR box has been defined which comprises the sequence tttCTATN 6 AtAGat/aA. Furthermore, the transcriptional organization of AmtR-regulated genes and operons was characterized.

No abstract

The influence of glutamate dehydrogenase activity on nitrogen regulation in Corynebacterium glutamicum was investigated. As shown by RNA hybridization experiments deletion of the gdh gene results in a rearrangement of nitrogen metabolism. Even when sufficiently supplied with nitrogen sources, a gdh deletion strain showed the typical nitrogen starvation response of C. glutamicum. These changes in transcription correlate with distinct alterations of intracellular metabolite pattern. Metabolite analyses of different mutant strains and the wild type indicated that ammonium and 2-oxoglutarate might influence the nitrogen regulation system of C. glutamicum cells.

No abstract

No abstract