Regulation of AmtR‐controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon

Abstract

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.

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

“…GlnD is required for the adenylylation of GlnK, without which, the supposed transcriptional repression of nitrogen-controlled genes would not be relieved thereby leading to inadequate uptake and assimilation of nitrogen to support growth. The role of GlnK, with regards to transcription, was verified through site-directed mutagenesis in which the Tyr51 residue was exchanged for Phe (35). These mutants did not respond to nitrogen limitation at the level of transcription, thereby showing that adenylylation at Tyr51 is essential for the interaction of GlnK with AmtR to allow transcription.…”

“…Through identification of these nitrogen-regulated genes; Jakoby et al (6) were able to isolate the transcriptional regulator and identify the DNA target sequence to which it binds. Gel retardation tests carried out with DNA fragments upstream of amtA and deletion analyses revealed a palindromic sequence to which AmtR binds: tttCTATAN 4 AtAGt (bases represented in capital letters are highly conserved and ''N'' refers to any base) (6,35).…”

“…In addition to amtA and amtB, AmtR regulates the expression of at least 33 other genes in C. glutamicum (35). These candidate genes were initially identified through genome screening for sequences to which AmtR may bind and have since been verified through various expression analyses such as microarrays and RT-PCR (6,35,44).…”

“…These candidate genes were initially identified through genome screening for sequences to which AmtR may bind and have since been verified through various expression analyses such as microarrays and RT-PCR (6,35,44). AmtR target sequences have been found not only upstream of amtA but also upstream of the amtB-glnK-glnD operon, the gltBD operon (28), glnA1 (19), and upstream of amtR itself (6) (Refer to Table 1).…”

Mutation-induced metabolite pool alterations in Corynebacterium glutamicum: Towards the identification of nitrogen control signals

“…In C. glutamicum the TetR-family transcriptional regulator AmtR was identified as the master regulator of nitrogen control (Jakoby et al, 2000;Beckers et al, 2005). Under conditions of high nitrogen supply AmtR blocks the transcription of nitrogen-controlled genes, e.g.…”