Utilization of creatinine as an alternative nitrogen source in Corynebacterium glutamicum

Abstract

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, the use of creatinine as an alternative nitrogen source in Corynebacterium glutamicum, the identification of a membrane protein involved in creatinine uptake, the transcriptional regulation of the corresponding gene, and expression regulation of the gene encoding the creatinine deaminase are reported. As shown by mutant analyses, RNA hybridization experiments and real-time PCR, the expression of two genes, crnT and codA, is increased in response to nitrogen limitation, and regulation depends on the global nitrogen regulator AmtR. In addition, synthesis of creatinine deaminase during nitrogen starvation was shown by two-dimensional gel electrophoresis and MALDI-TOF-MS followed by peptide mass fingerprint analysis.

Overexpression and one‐step renaturation‐purification of the tagged creatinine deiminase of Corynebacterium glutamicum in Escherichia coli cells

“…Digestion of purified active CDI with enterokinase produced a protein of the expected molecular weight (~47 kDa; Figure 2C). The obtained results well correlated with the published data regarding the C. glutamicum ATCC 13032 CDI molecular weight (Bendt et al, 2004). Treatment with enterokinase was used in preliminary analytical experiments only.…”

“…The identity of the isolated protein with C. glutamicum CDI was additionally proved by mass‐spectrometry of the random set of tryptic peptides of the enzyme, as described for arginase (Zakalskiy et al, 2012). On the basis of the results of two‐dimensional gel electrophoresis, the molecular weight of the CDI subunit is 46.3 kDa (Bendt et al, 2004). Using the sedimentation equilibrium method, the molecular weight of purified CDI of C. glutamicum ATCC 15590 was shown to be 195 kDa (Uwajima and Terada, 1980).…”

Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase

“…Dominant in this class are genes involved in nitrogen metabolism. Namely these are amt (encoding ammonia permease; [ 26 ]), codA (creatinine deaminase; [ 27 ]), ureA (urease γ -subunit; [ 28 , 29 ]), gltB (glutamine 2-oxoglutarate aminotransferase large subunit; [ 30 ]), glnA (glutamine synthetase; [ 31 ]), glnK (nitrogen regulatory protein PII; [ 32 ]), urtBC ([ 33 ]) and gdh (glutamate dehydrogenase; [ 34 ]). In E. coli , an increased transcription of the glnK gene was observed under stringent conditions [ 24 ], probably reflecting the fact that E. coli has nitrogen sensing and signal transduction mechanisms quite different to those of C. glutamicum [ 35 ].…”

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

“…For this purpose, urea and creatinine were chosen and added to nitrogen-starved cells. These two nutrients are not extracellularly degraded but are transported into the cell and decomposed intracellularly either by urease leading to ammonium and carbon dioxide (Siewe et al, 1998) or by creatinine deaminase (Bendt et al, 2004) leading to methylhydantoin and ammonium inside the cell. As a marker for nitrogen limitation, gltB expression was monitored (Fig.…”