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

Hao Zhang,Nannan Zhao,Shuangyan Han,Zhilin Ouyang,Suiping Zheng

doi:10.3389/fbioe.2022.816628

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.

Highlights

Corynebacterium glutamicum is a Gram-positive and generally regarded as safe (GRAS) microorganism with less endotoxicity
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)

Summary

INTRODUCTION

Corynebacterium glutamicum is a Gram-positive and generally regarded as safe (GRAS) microorganism with less endotoxicity. Most C. glutamicum strains cannot use L-arabinose as a carbon source, the gene cluster for L-arabinose utilization and its regulation have been reported in C. glutamicum ATCC 31831 (Kuge et al, 2015). 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). ChIP-seq was performed to identify the regulon of AmtR in the non-type strain C. glutamicum ATCC 14067. 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. Real-time quantitative PCR (RTqPCR) 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

MATERIALS AND METHODS

RESULT

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DATA AVAILABILITY STATEMENT