Transcriptional Regulation of the Creatine Utilization Genes of Corynebacterium glutamicum ATCC 14067 by AmtR, a Central Nitrogen Regulator

Abstract

In the genus Corynebacterium, AmtR is a key component of the nitrogen regulatory system, and it belongs to the TetR family of transcription regulators. There has been much research on AmtR structure, functions, and regulons in the type strain C. glutamicum ATCC 13032, but little research in other C. glutamicum strains. In this study, chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq) was performed to identify the AmtR regulon in C. glutamicum ATCC 14067. Ten peaks were obtained in the C. glutamicum ATCC 14067 genome including two new peaks related to three operons (RS_01910-RS_01915, RS_15995, and RS_16000). The interactions between AmtR and the promoter regions of the three operons were confirmed by electrophoretic mobility shift assays (EMSAs). The RS_01910, RS_01915, RS_15995, and RS_16000 are not present in the type strain C. glutamicum ATCC 13032. Sequence analysis indicates that RS_01910, RS_01915, RS_15995, and RS_16000, are related to the degradation of creatine and creatinine; RS_01910 may encode a protein related to creatine transport. The genes RS_01910, RS_01915, RS_15995, and RS_16000 were given the names crnA, creT, cshA, and hyuB, respectively. Real-time quantitative PCR (RT-qPCR) analysis and sfGFP (superfolder green fluorescent protein) analysis reveal that AmtR directly and negatively regulates the transcription and expression of crnA, creT, cshA, and hyuB. A growth test shows that C. glutamicum ATCC 14067 can use creatine or creatinine as a sole nitrogen source. In comparison, a creT deletion mutant strain is able to grow on creatinine but loses the ability to grow on creatine. This study provides the first genome-wide captures of the dynamics of in vivo AmtR binding events and the regulatory network they define. These elements provide more options for synthetic biology by extending the scope of the AmtR regulon.

Keywords: AmtR; ChIP-seq; Corynebacterium glutamicum; MFS transporter; creatine; creatinine.

FIGURE 1

Genome-wide in vivo identification of the AmtR binding sites in C. glutamicum ATCC 14067. AmtR-3Flag IP reads were plotted against the number of reads from the Negative IP. Black arrows show the verified genes; red arrows show the new target genes. In AmtR-3Flag IP, AmtR-3Flag production was induced using 0.5 mM IPTG for 8 h; AmtR-3Flag production was not induced with 0.5 mM IPTG as the negative control.

FIGURE 2

AmtR ChIP-seq peaks of selected genes. The putative palindrome sequence is shown in red; the AmtR binding sequences are predicted by MEME and underlined. +1 indicates the transcription start site (TSS), and putative -35, -10 elements and TSSs in the promoters of creT-crnA, cshA, and hyuB were predicted using the online website (http://www.fruitfly.org/seq_tools/promoter.html) (Reese, 2001). (A) Putative -35 and -10 elements in the promoter of creT-crnA enclosed by black rectangles. (B) Putative -35 and -10 elements in the promoter of cshA enclosed by black rectangles, -35 and -10 elements in the promoter of hyuB enclosed by red rectangles.

FIGURE 3

AmtR interacts in vitro with different DNA fragments. Minus represents that AmtR was not added, the lower band is the free DNA that is unbound with AmtR. Black triangle represents that AmtR was gradually increasing. The DNA fragments were annealed using two complementary single-stranded oligonucleotides. The positive control is the protomer region of amtA reported, and the negative control is the protomer region of hyuB that does not contain the AmtR binding sequence. Increasing amounts of AmtR (0, 0.1, 0.2, 0.4, and 0.8 μg) and 100 ng DNA fragments were used.

FIGURE 4

Growth curve of C. glutamicum ATCC 14067 wild-type, the △creT, and C. glutamicum ATCC 13032 strains with a different nitrogen sources. (A) C. glutamicum ATCC 14067 grows in CGXII medium with ammonium and urea, 10 mM creatine or 10 mM creatinine as nitrogen sources. (B) C. glutamicum ATCC 14067-△creT grows in CGXII medium with ammonium and urea, 10 mM creatine or 10 mM creatinine as nitrogen sources. (C) C. glutamicum ATCC 13032 grows in CGXII medium with ammonium and urea, 10 mM creatine or 10 mM creatinine as nitrogen sources.

FIGURE 5

AmtR regulates creatine degradation in C. glutamicum ATCC 14067. (A) Relative expression of creT, crnA, cshA, and hyuB of C. glutamicum ATCC 14067 wild-type and ΔamtR strains grown in CGXII medium with creatine or ammonium and urea as nitrogen sources. (B) Promoter activity of creT, cshA, and hyuB of C. glutamicum ATCC 14067 wild-type and ΔamtR strains grown in CGXII medium with creatine or ammonium and urea as nitrogen sources.

FIGURE 6

Schematic representation of the creatine and creatinine degradation gene cluster in C. glutamicum and related bacteria. Black bold italics indicate locus_tag prefix; homologous genes are presented in the same color. Red represents the gene encoding creatinase (CreA). Navy blue represents the gene encoding the protein related to creatine transport. In C. glutamicum ATCC 13032, soxA is interrupted by tnp13b. In P. putida NBRC 14164, P. putida B1, Arthrobacter sp. AQ5-05 and R. denitrificans Och114, sarcosine oxidase is a heterotetrameric; hydantoinase is a heterodimer. Double slashes indicate that the genes are not continuous. (A) The genes related to creatine and creatinine degradation are regulated by AmtR in these strains. (B) Homologs of AmtR are not present in these strains.

FIGURE 7

Model of the AmtR regulating creatine and creatinine degradation in C. glutamicum ATCC 14067. Blue dotted line: transcriptional repression by AmtR; black arrows: creatine and creatinine degradation pathway; red arrows: the transport of creatine and creatinine; dotted arrow: creatinase (CreA) is not present in C. glutamicum ATCC 14067.