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Expression of copA and cusA in Shewanella during copper stress

Maria H. Daleke

Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands

Correspondence
Gerard Muyzer
g.muyzer{at}tudelft.nl

Copper homeostasis is tightly regulated in all living cells as a result of the necessity and toxicity of this metal in free cationic form. In Gram-negative bacteria CPx-type ATPases (e.g. CopA in Escherichia coli) and heavy-metal efflux RND proteins (e.g. CusA in E. coli) play an important role in transport of copper across the cytoplasmic and outer membrane. We investigated the expression of CusA- and CopA-like proteins in Shewanella oneidensis MR1 and Shewanella strain MB4, a Mn(IV)-reducing isolate from a metal-polluted harbour sediment. Q-PCR analysis of total mRNA extracted from cultures grown under aerobic conditions with 25 µM copper showed significantly increased expression of cusA (Student's t-test: MR1, P<0.0001; MB4, P=0.0006). This gene was also induced in the presence of 100 µM copper and 10 or 25 µM cadmium in both tested strains. In the absence of oxygen, with fumarate as final electron acceptor and 100 µM copper, a prolonged lag phase (5 h) was observed and general fitness decreased as evidenced by twofold lower copy numbers of 16S rRNA compared to aerobic conditions. cusA expression in cells grown under these conditions remained at comparable levels (MR1) or was slightly decreased (MB4), compared to aerobic copper challenges. A gene homologous to the copA gene of S. oneidensis was not detected in strain MB4. Although low copA copy numbers were observed in strain MR1 under conditions with 25 and 100 µM copper, copA was not detected in mRNA from cultures grown in the presence of 10 µM cadmium, or in the absence of added heavy metals. However, copA was highly induced under anaerobic conditions with 100 µM copper (P=0.0011). These results suggest essentially different roles for the two proteins CopA and CusA in the copper response in S. oneidensis MR1, similar to findings in more metal-resistant bacteria such as Escherichia coli and Cupriavidus metallidurans.

Present adress: Department of Medical Microbiology and Infection Control, VU Medical Centre, Amsterdam, The Netherlands.