Copper homeostasis-related genes in three separate transcriptional units regulated by CsoR in Corynebacterium glutamicumby Haruhiko Teramoto, Hideaki Yukawa, Masayuki Inui

Appl Microbiol Biotechnol


Applied Microbiology and Biotechnology / Biotechnology


Vanillate Metabolism in Corynebacterium glutamicum

Hedda Merkens, Gabriele Beckers, Astrid Wirtz, Andreas Burkovski

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Putrescine production by engineered Corynebacterium glutamicum

Jens Schneider, Volker F. Wendisch



Copper homeostasis-related genes in three separate transcriptional units regulated by CsoR in Corynebacterium glutamicum

Haruhiko Teramoto & Hideaki Yukawa & Masayuki Inui

Received: 27 November 2014 /Revised: 23 December 2014 /Accepted: 28 December 2014 # Springer-Verlag Berlin Heidelberg 2015

Abstract In Corynebacterium glutamicum R, CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the copZ1-copB-cgR_0126 operon. It is predicted that copA and copB encode P-type ATPases for copper efflux and copZ1 encodes a metallochaperone. Here, a

CsoR-binding motif was found upstream of another copZ-like gene, copZ2, and the in vitro binding of the CsoR protein to its promoter was confirmed. The monocistronic copZ2 transcript was upregulated by excess copper in a CsoR-dependent manner. Among the extended CsoR regulon, deletion of copA, but not of copB, copZ1, or copZ2, resulted in decreased resistance to copper, indicating a major role of the CopA copper exporter in the multilayered systems for copper homeostasis. A redundant role of copZ1 and copZ2 in copper resistance was also indicated by double deletion of these genes. The copper-dependent activation of the copA, copZ1, and copZ2 promoters was confirmed by lacZ reporter assays, consistent with the coordinated derepression of the three transcriptional units. The copZ1 promoter activity showed the highest responsiveness to copper and was also induced by excess zinc and nickel.

Furthermore, zinc-inducible expression observed for the

CsoR-regulated genes was independent of Zur, recently found to uniquely act as a transcriptional repressor of zinc efflux genes. These results implied complicated cross talk between homeostasis of multiple transition metals.

Keywords Corynebacterium . Metal homeostasis .

Metal-dependent gene expression . Transcriptional regulation


Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the class Actinobacteria, has a long history in the industrial production of amino acids such as glutamate and lysine (Hermann 2003; Ikeda and Takeno 2012; Kinoshita et al. 1957). It has been further used in the development of bioprocesses for production of various compounds including isobutanol, expected as one of the next-generation biofuels (Blombach et al. 2011; Inui et al. 2004a; Jojima et al. 2012;

Okino et al. 2008a, b; Smith et al. 2010; Wieschalka et al. 2013; Yamamoto et al. 2013). Essential transition metals, such as iron, manganese, zinc, and copper, are required for various types of metabolic enzymes but have toxic effects on cellular functions when present in excess amounts. Thus, their intracellular levels should be maintained within tight limits. The transcriptional regulatory systems for homeostasis of iron, zinc, and copper in C. glutamicum have been recently identified. Two different types of transcriptional regulators, DtxR and RipA, are hierarchically involved in the global gene expression in response to iron deficiency (Brune et al. 2006;

Wennerhold and Bott 2006; Wennerhold et al. 2005). Zur acts as a transcriptional repressor of not only zinc-repressible genes for zinc uptake but also zinc-inducible genes for zinc efflux in distinct mechanisms, indicating that Zur plays a unique role in zinc homeostasis by its response to both zinc deficiency and excess in this bacterial species (Schröder et al. 2010; Teramoto et al. 2012b). The two-component system

Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6373-z) contains supplementary material, which is available to authorized users.

H. Teramoto :H. Yukawa :M. Inui (*)

Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan e-mail:

Appl Microbiol Biotechnol

DOI 10.1007/s00253-015-6373-z consisting of CopS and CopR is involved in the copperinducible expression of a set of genes for copper resistance (Schelder et al. 2011), while a different set of copper/zincinducible genes are under the control of a member of the copper-responsive transcriptional regulator CsoR family (Teramoto et al. 2012a).

Several types of regulatory systems for copper homeostasis have been identified in bacteria (Ma et al. 2009b; Rademacher and Masepohl 2012; Waldron and Robinson 2009). Among them, CsoR has been characterized as a copper-sensing transcriptional repressor of a copper resistance operon containing a copper-translocating P-type ATPase in close genetic proximity to the csoR gene in various bacteria such as

Mycobacterium tuberculosis (Liu et al. 2007; Ward et al. 2010), Bacillus subtilis (Smaldone and Helmann 2007), and

Listeria monocytogenes (Corbett et al. 2011). Copper inhibits the DNA-binding activity of CsoR, resulting in derepression of the copper resistance systems.

We previously reported that CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the cgR_0124-copB-cgR_0126 operon, which is distant from csoR on the chromosome of C. glutamicum R (Teramoto et al. 2012a). copA and copB code for putative copper-translocating P-type ATPases, while cgR_0124, designated here copZ1, codes for a homolog of the copper chaperone CopZ. It has been recently reported elsewhere that CsoR directly regulates the distantly encoded copper homeostasisrelated genes including copA and copZ in Staphylococcus aureus (Baker et al. 2011; Grossoehme et al. 2011) and

Streptomyces lividans (Dwarakanath et al. 2012). Therefore,

CsoR acts as a transcriptional repressor of multiple operons supposed to be concertedly involved in copper homeostasis in some bacterial species. However, the roles of these respective gene products remain largely unclear. Our previous study also showed that the CsoR-regulated genes inC. glutamicumR are upregulated by excess zinc as well as by excess copper, implying cross talk between the regulatory systems responsive to these essential transition metals (Teramoto et al. 2012a).