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

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.

Gene transcription and expression is regulated by transcriptional factors, including sigma factors and twocomponent systems that are essential for stabilizing cell homeostasis (López-Maury et al., 2008). The research on transcription factors in C. glutamicum was mainly focused on the type strain C. glutamicum ATCC 13032. But there are specific regulatory elements in other strains that give those strains different metabolic capabilities. Most C. glutamicum strains cannot use L-arabinose as a carbon source, however, the gene cluster for L-arabinose utilization and its regulation have been reported in C. glutamicum ATCC 31831 (Kuge et al., 2015). Fifteen paa (paaTK, paaABCDEGJFH, paaI, paaY, and paaZ) genes encode the phenylacetic acid degradation pathways that regulated by PaaR in C. glutamicum AS 1.542 (Chen et al., 2012). A shikimate transporter, regulated by ShiR in C. glutamicum R, was identified and a shikimic acid biosensor was constructed from ShiR for monitoring shikimate synthesis in C. glutamicum RES167 (Kubota et al., 2015;Liu et al., 2018a). The regulatory elements in such strains provide more options for synthetic biology.
C. glutamicum can use a wide range of nitrogen compounds as nitrogen sources, and AmtR is a critical component of the nitrogen regulatory system that belongs to the TetR family of transcription regulators (Jakoby et al., 2000). In the type strain C. glutamicum ATCC 13032, there has been much research on AmtR structure, function, and regulons (Jakoby et al., 2000;Bendt et al., 2004;Hasselt et al., 2011). A total of 35 genes are regulated by AmtR, including the genes that encode the ammonium transporters amtA, amtB, the urea uptake system urtABCDE, the L-glutamate uptake system gluABCD, the creatinine transporter crnT, the ABC-type transport systems Ncgl 1915Ncgl -1918 involved in the transport of nitrogen sources. The ureABCEFGD, gltBD, gdh, glnA, and codA encoding proteins are involved in nitrogen metabolism (Hasselt et al., 2011). In addition, the mez gene encoding the malic enzyme involved in carbon metabolism is also regulated by AmtR (Gourdon et al., 2000). The binding of AmtR to its regulon promoter region relies on conserved sequences and no small molecule effectors have been identified. A PII-type signal transduction protein GlnK, adenylylated at Tyr51, has been proposed to derepress expression of the AmtR regulons (Strösser et al., 2004).
C. glutamicum ATCC 14067 is an important industrial workhorse employed as an L-glutamate producer and a parental strain for breeding the producers of L-lysine and L-glutamine (Li et al., 2007;Jianzhong et al., 2014;Lv et al., 2021). In this study, ChIP-seq was performed to identify the regulon of AmtR in the non-type strain C. glutamicum ATCC 14067. Four new target genes (RS_01910, RS_01915, RS_15995, and RS_16000) were determined. The proteins encoded by these new target genes may be related to creatine and creatinine degradation, and these genes were given the names crnA, creT, hyuB, and cshA, respectively. Creatine, a component of the dissolve-free amino acid (DFAA) pool, is a metabolite of metazoans. It is found in the excretions of different animals, including earthworms and birds (Wyss and Kaddurah-Daouk, 2000). In addition, some phytoplankton may also produce creatine via metabolism (Wawrik et al., 2017). It has been reported that C. glutamicum ATCC 13032 cannot utilize creatine as a carbon or nitrogen source. We investigated the abilities of C. glutamicum ATCC 14067 to degrade creatine. The result show C. glutamicum ATCC 14067 could grow in CGXII medium with creatine as a sole nitrogen source, and CreT is related to creatine transport. Real-time quantitative PCR (RT-qPCR) analysis and sfGFP (superfolder green fluorescent protein) analysis reveal that AmtR directly and negatively regulate the transcription and expression of crnA, creT, hyuB, and cshA.

Bacterial Strains, Media, and Culture Conditions
The strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli DH5α was used for gene cloning, and BL21 (DE3) was used for protein expression. E. coli strains were grown in Luria-Bertani (LB) (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride) medium at 37°C or 16°C. C. glutamicum strains were grown overnight in BHI medium (37 g/L brain-heart infusion) (Becton, Dickinson and Co.) and then cultured in fresh BHI medium or CGXII medium (Keilhauer et al., 1993) at 30°C. Cells grown in the CGXII medium, were centrifuged and washed twice with CGXII and then cultured in fresh CGXII medium. If necessary, CGXII medium was supplemented with 10 mM creatine or creatinine as a nitrogen and carbon source. Strain growth was monitored by measuring the optical density (OD) of the cultures at 600 nm. The medium was supplemented with antibiotics at the following concentrations: kanamycin (Kan): 50 μg/ml for E. coli and 25 μg/ml for C. glutamicum, chloramphenicol (Chl): 15 μg/ml for E. coli and 7.5 μg/ml for C. glutamicum.
Construction of the amtR, creT Deletion Mutants and amtR-3Flag Complementation in C. glutamicum ATCC 14067 Standard DNA cloning, Gibson assembly, and transformation procedures were employed (Sambrook and Russell, 2001;Gibson et al., 2009). The amtR and creT deletion mutants (△amtR and △creT) in C. glutamicum ATCC 14067 were constructed using homologous recombination (Huang et al., 2017). The DNA fragments of the upstream and downstream homology arms of amtR and creT were amplified by PCR using the primers amtR-L/ amtR-L-lox71 and amtR-R/amtR-R-lox66, creT-L/creT-L-lox71 and creT-R/creT-R-lox66 overlapping PCR with a Cre-kan cassette to construct a self-excisable cassette, respectively. The self-excisable cassettes were introduced into C. glutamicum ATCC 14067 carrying the recombinase-exonuclease pairs by electroporation (Takano et al., 2008). BHI solid medium supplemented with Kan 25 μg/ml and Chl 7.5 μg/ml was used for recombinant selection. Theophylline (1 mM) was used for inducing Cre expression that mediated intermolecular excision. The DNA fragments for amtR and creT deletion strains were confirmed by DNA sequencing (Sangon Biotech, China). A 3Flag tag was added to the C-terminal of AmtR, and the amtR-3Flag fragment was amplified using the primers amtR-3Flag-S/amtR-3Flag-A. A plasmid backbone was amplified from plasmid pEC-XK99E using the primers 99E-S/99E-A. Then the plasmid backbone and the amtR-3Flag fragment were assembled by Gibson assembly to construct the plasmid pEC-XK99E-amtR-3Flag, which was introduced into the △amtR strains by electroporation. The primers used in this study are listed in Supplementary Table S2.

Western Blot Analysis
Complemented strain C. glutamicum 14067-△amtR:amtR-3Flag was grown in BHI medium, and AmtR-3Flag production was induced using 0.5 mM IPTG (isopropyl -β-D-thiogalactopyranoside) for 8 h. AmtR-3Flag production was not induced with 0.5 mM IPTG as the negative control. The cells were harvested by centrifugation and washed with PBS buffer (150 mM NaCl, 3 mM KCl, 10 mM Na 2 PO 4 , 3 mM KH 2 PO 4 , pH 7.5), suspended in PBS buffer to normalize the culture densitiy based on the OD 600 value, and disintegrated with silica beads (0.1 mm) for 12 cycle of 30 s at a speed rating of 6.0 with 3 min resting intervals by Bead Ruptor 12 (OMNI International, United States). Soluble extracts were fractionated on a 12% denaturing polyacrylamide gel before being transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, United States). The membrane was blocked with 3% bovine serum albumin and incubated overnight at 4°C with a 1:2,000 dilution of Flag-specific (Sigma-Aldrich, St. Louis, MO, United States) mouse antiserum, and incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, United States). Finally, the immunoreactive protein bands were visualized with an ECL reagent (Thermo Fisher Scientific Inc., Waltham, MA).

Overexpression and Purification of AmtR
AmtR-His 6 expression plasmid constructed by PCR using the primers amtR-S/amtR-A was inserted into pET28a, which had a His 6 -tag at its C-terminus. The plasmid was confirmed by DNA sequencing (Sangon Biotech, China). AmtR with a C-terminal His 6 -tag was expressed in E. coli BL21, plasmid carrying cells were grown to an OD 600 of 0.6 at 37°C in LB medium, and protein production was induced using 0.5 mM IPTG at 16°C for 16 h. Cells were harvested by centrifugation and suspended in buffer A (100 mM Tris-HCl, 100 mM NaCl, pH 7.5), then supplemented with Mini protease inhibitor cocktail tablets (Roche, Germany). Cells disrupted by sonication at 4°C, and cell debris was removed by centrifugation at 4°C (15,000 g for 20 min). The AmtR-His 6 was purified by 5 ml nickel affinity chromatography using Ni-NTA agarose (Novagen, United States).

Total RNA Extraction and RT-qPCR Analysis
The C. glutamicum ATCC 14067 wild-type and △amtR strains were grown overnight in BHI medium, inoculated into fresh CGXII medium to an OD 600 of 0.2, and cultured for 8 h before the extraction of RNA. If necessary, CGXII medium was supplemented with 10 mM creatine as a nitrogen source. Total RNA was extracted using an RNA extraction kit (Tiangen, Beijing, China) with on-column DNaseI treatment. The final RNA concentrations and purities were determined on a Thermo Scientific NanoDrop spectrophotometer; equal amounts of RNA (1 μg) were used to generate cDNA (Toyobo, Tsuruga, Japan) using 6-mer random primers. Primers for various genes (Supplementary Table S2) were designed using Primer Premier6 software. Three independent RT-qPCR experiments were performed, and each experiment was run in triplicate. The reactions were run on an Applied Biosystems 7500 real-time system (Applied Biosystems), and the transcript levels were normalized to the 16S rRNA level in each sample using the ΔΔC T method.
The wild-type C. glutamicum ATCC 14067 strain and the △amtR strain with reporter plasmids were grown overnight in BHI medium, the cells washed with CGXII medium, then inoculated into fresh CGXII medium to an OD 600 of 0.2. If necessary, CGXII medium was supplemented with 10 mM creatine as a nitrogen source. After cultivation at 30°C for 8 h, Frontiers in Bioengineering and Biotechnology | www.frontiersin.org February 2022 | Volume 10 | Article 816628 the cells were washed twice with PBS buffer and resuspended in 200 μl PBS buffer, in 96-well plates for measurement of GFP fluorescence. Fluorescence was assessed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm using a multifunctional microplate reader (Infinite M200, Tecan, Switzerland).

ChIP-Seq
The ChIP-seq protocol was based on previous experiments with C. glutamicum (Jungwirth et al., 2013). Strain C. glutamicum ATCC-14067-△amtR:amtR-3Flag was grown at 30°C in BHI medium, and 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. To achieve protein-DNA crosslinking in vivo, a final concentration of 1% formaldehyde was added to the cultures, which was incubated at room temperature for 15 min with gentle shaking. A final concentration of 125 mM glycine was added to stop crosslinking. Cells were harvested by centrifugation, washed twice with a complete protease inhibitor cocktail (Roche) in an ice-cold Tris buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl), and stored at −80°C. To prepare lysates, the pellets were resuspended in FA-1 buffer (HEPES-KOH at 50 mM [pH 7.5], NaCl at 140 mM, EDTA at 1 mM, Triton X-100 at 1%, and complete protease inhibitor cocktail). One ml sample of this cell suspension was mixed with 0.6 g of 0.1 mm silica beads (BioSpec Products, United States), and the cells were disrupted for 12 cycles of 30 s at a speed rating of 6.0 with 3 min resting intervals by Bead Ruptor 12 (OMNI International, United States). The cell debris was removed by centrifugation at 14,000 g for 15 min at 4°C, and the DNA in the supernatant was sheared to an average length of 200-500 bp by sonication in a water bath (Bioruptor, Diagenode). The lysates were pre-cleared with 30 μl of ChIP magnetic A + G beads (Merck Millipore). The rest of the precleared lysates were incubated overnight at 4°C with monoclonal anti-Flag M2 (Sigma-Aldrich  (Zhang et al., 2008).

Bioinformatics Data Analysis
The gene sequence of C. glutamicum ATCC 14067 used in this study was obtained from the GenBank file for NZ_CP022614. Databank searches were performed using BLAST (https://blast. ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990). The putative -35, -10 elements and transcription start site (TSS) in the promoters were predicted using an online website (http:// www.fruitfly.org/seq_tools/promoter.html) (Reese, 2001). The sequence analysis tool MEME (http://meme-suite.org/) (Bailey et al., 2015) was used for the identification of AmtR binding motif in the genus Corynebacterium.

Genome-Wide Identification of the AmtR Regulon by ChIP-Seq
AmtR acts as a transcriptional regulator and plays an essential role in the genus Corynebacterium. Previous studies have identified the target genes of AmtR by transcriptomics and EMSA (Hasselt et al., 2011;Chen et al., 2013). These studies mainly focused on the type strain C. glutamicum ATCC 13032. Due to the different environments and selection pressure during evolution, various strains have apparent differences in several phenotypic characteristics (Yang and Yang, 2017). To identify the direct binding loci of AmtR in C. glutamicum ATCC 14067, the pEC-XK99E-amtR-3Flag plasmid was used to express the AmtR protein fused with a C-terminal 3Flag-tag and driven by the lac promoter in the ΔamtR strain. Western blotting confirmed the expression of AmtR-3Flag (Supplementary Figure S1). AmtR-3Flag IP and negative IP DNA were pooled and subjected to Illumina sequencing. To identify the AmtR binding regions, ChIP-seq peaks obtained from the AmtR-3Flag IP samples were compared to the negative IP sample. On ChIP-seq maps, the fold change of peaks above 1.5 was fixed as the minimum cutoff value for AmtR peak calling. On this basis, ten AmtR peaks were detected (Figure 1). Nine of the peaks were located in the promoter regions, and related to 26 verified genes and 4 new target genes (Supplementary Table S3). The other one of the peaks was located in the coding region of argR, and the related  Figure S2).

Validation of Novel AmtR Targets
In C. glutamicum ATCC 14067, two new peaks related to three operons were identified using ChIP-seq. (Figure 2). According to a previous prediction by CoryneRegNet 7.0 (Parise et al., 2020), CEY17_RS01915 is the first gene in a two-gene operon encoding putative MFS transporter and cretininase, and CEY17_RS15995 and CEY17_RS16000 are single gene operons encoding putative N-carbamoylsarcosine amidase and hydantoin utilization protein HyuB, respectively. The RS_01910, RS_01915, RS_15995, and RS_16000 were given the names crnA, creT, cshA, and hyuB in C. glutamicum. The putative TSS (Figure 2) of these genes was predicted using the online website (http://www.fruitfly.org/seq_ tools/promoter.html). In C. glutamicum, previous studies have reported that AmtR has a 14-bp palindromic binding sequence consisting of two conserved 4-bp sequences forming an inverted repeat separated by a random 6-bp spacer (CTAT-N6-ATAG) (Hasselt et al., 2011). The DNA sequence analysis revealed that the promoter of creT had two potential AmtR binding sites, PcreT-1 and PcreT-2. The intergenic region of cshA and hyuB had three potential AmtR binding sites, PhyuB-1, PhyuB-2, and PhyuB-3, respectively. PhyuB-2 and PhyuB-3 in the promoter of hyuB are not strictly conserved. To detect the binding of AmtR and potential AmtR binding site, we assayed the purified AmtR binding to the potential AmtR binding sites in vitro. AmtR with a C-terminal His 6 -tag was expressed in E. coli BL21 and purified by affinity nickel affinity chromatography as described in the Materials and Methods. DNA fragments contained the binding motif were annealed using two complementary single-stranded oligonucleotides (Supplementary Table S2).
AmtR was able to bind and shift the DNA fragments ( Figure 3). Increasing amounts of AmtR and 100 ng DNA fragments were used. As the amount of AmtR increased, the binding of AmtR with DNA fragments gradually increased. AmtR is strongly bound with PcreT-1, PcreT-2, PhyuB-1, and PhyuB-2, and weakly bound with PhyuB-3. The positive control was the protomer region of amtA previously reported, and the negative control was the protomer region of hyuB that did not contain the AmtR binding sequence (Figure 3).

Utilization of Creatine as an Alternative Nitrogen Source in C. glutamicum ATCC 14067
A reciprocal BLASTP search of CreT, CrnA, CshA, and HyuB was done against the genome database in NCBI to identify homologs. CrnA shares 34% sequence identity with creatininase from Pseudomonas putida (Beuth et al., 2003), CshA shares 60% sequence identity with N-carbamoylsarcosine amidase from Arthrobacter sp. (Roma o et al., 1992), and HyuB shares 44% sequence identity with hydantoinase from Pseudomonas sp. NS671. Hydantoinase in Pseudomonas sp. NS671 is encoded 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.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org February 2022 | Volume 10 | Article 816628 by two distinctly different subunits, HyuA and HyuB (Watabe et al., 1992;Ishikawa et al., 1997). No homologous functional protein for CreT has been reported. The model for the location of the trans-membrane helices in CreT was predicted by TMHMM v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM) (Supplementary Figure S3). Hydropathy analysis shows that CreT is an integral membrane protein with 11 putative membrane-spanning domains. Interestingly, CreT has 50% similarities with the MFS transporter encoded by the gene located on upstream of creatinase in P. putida NBRC 14164 (PP4_RS10800). Since clustered genes usually encode proteins that physically interact, or different enzymes in the same metabolic pathway Dandekar et al., 1998), it is possible that creT encodes a protein related to creatine transport. Creatininase catalyzes both the conversion of creatine and creatinine; N-carbamoylsarcosine amidase catalyzes the hydrolysis of N-carbamoylsarcosine to sarcosine with the liberation of carbon dioxide and ammonia; hydantoinase catalyzes the hydrolysis of hydantoin to N-carbamylamino acid.
We hypothesized that CreT, CrnA, CshA, and HyuB were related to creatine and creatinine degradation.
To verify whether C. glutamicum ATCC 14067 can degrade creatine and creatinine, we conducted growth assays of C. glutamicum ATCC 14067 and C. glutamicum ATCC13032 in CGXII medium with creatine or creatinine as a nitrogen source. C. glutamicum ATCC 14067 can grow on creatine or creatinine at a concentration of 10 mM ( Figure 4A). While C. glutamicum ATCC 13032 is able to utilize creatinine but not creatine as a nitrogen source ( Figure 4C), consistent with previous reports. To verify the functions of CreT, CrnA, CshA, and HyuB, the transcripts of creT, crnA, cshA, and hyuB in CGXII medium with creatine or ammonium and urea as nitrogen sources were analyzed by RT-qPCR. In CGXII medium with 10 mM creatine as a sole nitrogen source, the levels of the creT, crnA, cshA, and hyuB transcripts were higher than those growing on ammonium and urea ( Figure 5A). To further validate the results above, PcreT-sfGFP, PcshA-sfGFP, and PhyuB-sfGFP reporter 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). plasmids were transformed into the wild-type strain, and promoter activities of PcreT, PcshA, and PhyuB in these strains were observed with 10 mM creatine or ammonium and urea. The GFP fluorescence of PcreT, PcshA, and PhyuB was significantly higher when the strain was grown with creatine than with ammonium and urea ( Figure 5B). In addition, we tested the growth of C. glutamicum ATCC 14067 and C. glutamicum ATCC 13032 in CGXII medium with 10 mM creatine, 10 mM creatinine, or 10 mM glucose as sole carbon source. CGXII medium without carbon source was used as a negative control. After culturing for 48 h at 30°C, the OD of cultures at 600 nm was measured. C. glutamicum ATCC 14067 and C. glutamicum ATCC 13032 could not grow in CGXII medium with creatine or creatinine as the carbon source (Supplementary Figure S4).
To further verify the function of CreT in creatine degradation in C. glutamicum ATCC 14067, creT was deleted, and the inframe deletion mutant △creT was obtained. The ΔcreT strain could grow as the C. glutamicum ATCC 14067 wild-type strain in CGXII medium with ammonium and urea or creatinine as a nitrogen source, but the ΔcreT strain lost the ability to grow on creatine. These results demonstrate that creT encodes a protein related to creatine transport in C. glutamicum ATCC 14067. 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.

AmtR Represses Target Gene Transcription and Expression
To investigate the contribution of AmtR to creT, crnA, cshA, and hyuB, the ΔamtR strain was constructed using homologous recombination. The C. glutamicum ATCC 14067 wild-type and ΔamtR strains were cultured in CGXII medium with ammonium and urea or ammonium, urea, and 10 mM creatine as nitrogen sources for 8 h. When ammonium and urea were used as nitrogen sources, compared with the wildtype strain, the creT, crnA, cshA, and hyuB transcript levels in the ΔamtR strain were increased significantly. The levels of creT, crnA, cshA, and hyuB increased by 2,427-, 998-, 61-, and 229-fold, respectively ( Figure 5A). When the wild-type strain used ammonium, urea, and 10 mM creatine as nitrogen sources, the creT, crnA, cshA, and hyuB transcript levels were higher than those with ammonium and urea as nitrogen sources, but only increased by 1.5-, 1.9-, 2.5-, and 3.0-fold, respectively. The results indicate that creatine could not or very weakly induce the transcription of creT, crnA, cshA, and hyuB when ammonium and urea are abundant. The corresponding sfGFP assays confirmed this result. The PcreT-sfGFP, PcshA-sfGFP, and PhyuB-sfGFP reporter plasmids were transformed into the C. glutamicum ATCC 14067 wild-type and △amtR strains. The resulting strains were cultured in CGXII medium for 8 h, the sfGFP activities were measured ( Figure 5B). Combining RT-qPCR analysis and sfGFP assays, we identified that AmtR represses the transcription and expression of the target genes.

Proteins Related to Creatine or Creatinine Degradation in the Genus Corynebacterium and Related Bacteria
CrnT and CodA have been reported related to creatinine transport and degradation, and SoxA was predicted as a sarcosine oxidase in C. glutamicum ATCC 13032 (Kalinowski et al., 2003). To identify proteins related to creatine or creatinine degradation in bacteria, a reciprocal BLASTP search of CrnT, CodA, CreT, CrnA, SoxA, CshA, and HyuB against the genome database in NCBI was carried out. These proteins were found to be present in some Corynebacterium species. In C. glutamicum, all proteins related to creatine and creatinine degradation in ATCC 14067 are also present in ATCC 15168, BE, and YI strains. CrnT, CodA, CreT, CrnA, and SoxA are present in 17 strains, including ATCC 21831, ATCC 13869, ZL-1, and so on. CrnT, CodA, and SoxA are present in the other strains. In addition, Corynebacterium callunae DSM 20147, Corynebacterium vitaeruminis DSM 20294, and Corynebacterium deserti GIMN1.010 have different proteins related to creatine and creatinine degradation ( Figure 6). Analysis of the promoters' sequence of genes that encode proteins related to creatine and creatinine degradation revealed that the promoter regions have similar sequences to the AmtR binding site (Supplementary Figure S5).
In Corynebacterium terpenotabidum Y-11, Corynebacterium falsenii DSM 44353, and Corynebacterium kroppenstedtii DSM 44385, A606_RS03870, BI88_RS02350, and CKROP_RS06015 encode a putative creatinase that hydrolyzes creatine to sarcosine and urea. According to the sequence alignment using Clustal Omega, those proteins share 59, 58, and 59% sequence identity with creatinase from Arthrobacter sp. TE 1826, respectively (Nishiya et al., 1998). The promoter regions of those genes encoding creatine, and creatinine degradation proteins do not have sequences similar to the AmtR binding site. Further analysis found that these strains do not contain a homolog of AmtR.
There are many bacterial species distinct from Corynebacterium that can degrade creatine or creatinine as nitrogen sources. For example, Pseudomonas (Yamada et al., 1985), Arthrobacter (Nishiya et al., 1998), and Roseobacter (Wawrik et al., 2017) strains ( Figure 6B). Interestingly, the homologous transporters are ubiquitous and usually adjacent to creatinase or creatininase in these genomes ( Figure 6). This supports the speculation that CreT is related to creatine transport. In addition, R. denitrificans Och114 can degrade creatine as a

DISCUSSION
In bacteria, the PII signal transduction proteins act in conjunction with various transcription factors to control nitrogen metabolism, such as nitrate assimilation through the global nitrogen control factor NtcA in all cyanobacteria characterized to date (Llácer et al., 2010). Nitrogen limitation in enteric bacteria is regulated by NtrBC (Caballero et al., 2005), which activates the expression of over 100 genes; nitrogen metabolism in Bacillus subtilis under conditions of nitrogen limitation is regulated by TnrA (Wray et al., 1996); the GlnR controls nitrogen metabolism in Streptomyces coelicolor and regulates at least 50 nitrogen response genes (Tiffert et al., 2008). The AmtR is the central nitrogen regulator in C. glutamicum, and it has been reported that AmtR in conjunction with the PII signal transduction protein GlnK triggers the dissociation of AmtR from DNA. The conservative sequence of the AmtR binding site contains two conserved 4-bp sequences forming an inverted repeat separated by a random 6-bp spacer, which is CTAT-N 6 -ATAG. In addition, in Streptomyces avermitilis, Rhodococcus jostii RHA1, Arthrobacter aurescens, Mycobacterium smegmatis, and Nocardia farcinica, both glnR and amtR-homologous genes are present in the genome. The GlnR is the central nitrogen regulator in those strains, and AmtR has a 22 bp consensus TAtCTGTCa-n2-cGACAGATAT sequence (Chen et al., 2013), similar to the AmtR binding site sequence in C. glutamicum. In different species, AmtR and its homologous protein have similar binding sequences. The binding site of AmtR in the C. glutamicum ATCC 14067 genome was identified by ChIP-seq in BHI medium. Ten peaks 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.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org February 2022 | Volume 10 | Article 816628 were obtained in the C. glutamicum ATCC 14067 genome including two new peaks related to three operons. ChIP-seq detected a total of 30 genes include four new target genes, fewer than the 35 genes previously reported (Hasselt et al., 2011). There may be some genes not strictly regulated by AmtR. In a nitrogen-rich medium, the transcription and expression of gdh and gln, which encode L-glutamate dehydrogenase and L-glutamine synthetase, are only loosely controlled by AmtR (Hasselt et al., 2011). To identify the transcription and expression of creT, crnA, cshA, and hyuB regulated by AmtR, we used 50 bp doublestranded DNA containing the potential AmtR binding sites annealed using two complementary single-stranded oligonucleotides. EMSA showed that PcreT-1, PcreT-2, PhyuB-1, and PhyuB-2 have a robust affinity when binding with AmtR, and PhyuB-3 has a very weak affinity with AmtR. PhyuB-1, PhyuB-2, PhyuB-3 are located in the intergenic region of cshA and hyuB, cshA is transcribed divergently from hyuB. RT-qPCR and other assays were employed to investigate the effect of the △amtR strain on the promoter activity of cshA and hyuB. The result of the analyses showed that the transcription and expression of cshA and hyuB in the ΔamtR strain were significantly higher than in the wild-type strain. In previous studies, urea acted as an alternative nitrogen source that could diffuse across the cytoplasmic membrane present in high concentrations in the medium; the transporter of urea is strictly and fully repressed by AmtR in C. glutamicum (Siewe et al., 1998). Creatinine acts as a membrane-impermeable nitrogen source, and the genes that code transport and degrade creatinine are limited to nitrogen starvation and strictly regulated by AmtR (Bendt et al., 2004). The ΔcreT strain cannot use creatine as a nitrogen source, which indicates that creatine cannot diffuse across the cytoplasmic membrane. The operons creT-crnA, cshA, and hyuB may be limited to nitrogen starvation and be strictly regulated by AmtR.
The new regulons of AmtR in ATCC 14067 are not present in the type strain C. glutamicum ATCC 13032. The sequences of CreT, CrnA, CshA, and HyuB were analyzed in C. glutamicum strains. CreT and CrnA are present in 17 strains. CreT is related to creatine transport, and its homologous protein is ubiquitous in species that can utilize creatine. The marine bacteria Roseobacter denitrificans Och114 can grow on creatine as a nitrogen source, but it has no protein homologous to CreT ( Figure 6). However, there may be other ways to take up creatine into the cell. CrnT and CodA related to creatinine transport and degradation are present in all C. glutamicum strains. All strains of C. glutamicum can use creatinine as a nitrogen source, and only some strains can use creatine as a nitrogen source. It has been reported that creatinine inhibits bacterial replication. C. glutamicum can resist creatinine stress in the environment by degrading creatinine to N-methylhydantoin and ammonia. Ammonia is used in other pathways, and N-methylhydantoin is a dead-end product accumulating in the cells (Bendt et al., 2004). Incomplete degradation of creatinine is detrimental, leading to the arrest of C. glutamicum growth when the concentration of creatinine is high. In C. glutamicum ATCC 14067, CshA and HyuB can further react with the N-methylhydantoin to yield sarcosine, and CrnA can catalyze the conversion of both creatine and creatinine (Beuth et al., 2003). When the creatinine concentration is high, it is converted into creatine that does not inhibit bacteria. If N-methylhydantoin is further degraded to sarcosine or creatinine is converted to creatine, it can improve the survival ability of C. glutamicum ATCC 14067 when the concentration of creatinine is high. Intraspecies variation of C. glutamicum is characterized by different strains with different abilities to degrade creatine and creatinine. Similar characteristics have been seen in Pseudomonas. P. putida NBRC 14164 has the genes related to the degradation of creatine, but these are not present in P. putida KT2440 (Nelson et al., 2002;Ohji et al., 2014). Possibly, these strains gain the ability to degrade creatine via horizontal gene transfer. C. glutamicum ATCC 14067 cannot use creatine or creatinine as the sole carbon source (Supplementary Figure S4). Creatine and creatinine produce ammonia and glycine during the degradation process, but glycine cannot be re-utilized. The glycine cleavage system that degrades glycine is also lacking in C. glutamicum (Hüser Andrea et al., 2005), and thus glycine will accumulate in the cells. In some bacteria, creatine or creatinine can be used as the sole carbon source since creatine or creatinine can be degraded into glycine. Serine hydroxymethyltransferase and 10-formyltetrahydrofolate hydrolase further catalyze glycine into serine and pyruvate (Chlumsky et al., 1995;Meskys et al., 2001;Willsey and Wargo, 2015).
It has been reported that the degradation pathway of creatine and related metabolites in bacteria is regulated by transcription factors. Creatinine transport and degradation are regulated by AmtR in C. glutamicum (Bendt et al., 2004), and sarcosine catabolism is transcriptionally regulated by GbdR and SouR in Pseudomonas aeruginosa (Willsey and Wargo, 2015). The pathway degrading creatine and creatinine into glycine is regulated by AmtR in C. glutamicum ATCC 14067 (Figure 7).

DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the National Center for Biotechnology Information repository, accession number PRJNA798895.

AUTHOR CONTRIBUTIONS
HZ and SZ designed the experiments. HZ, ZO and NZ performed the experiments. HZ wrote the manuscript. HZ, SH and SZ conceived the project. All authors contributed to the manuscript, read and approved the final version.