ORIGINAL ARTICLE Copper and cadmium: responses in Pseudomonas putida KT2440 C.D. Miller 1 , B. Pettee 2 , C. Zhang 2 , M. Pabst 3 , J.E. McLean 3 and A.J. Anderson 2 1 Biological and Irrigation Engineering Department, Utah State University, Logan, UT, USA 2 Biology Department, Utah State University, Logan, UT, USA 3 Water Research Laboratory, Utah State University, Logan, UT, USA Introduction Cells respond to heavy metals with protective mechanisms including efflux systems and sequestration (Silver and Phung 2005). This paper compares the response of a soil bacterium, Pseudomonas putida strain KT2440, to the toxic metals Cu and Cd that contaminate water and soils from natural and industrial sources. Cu is essential for cellular function because of roles as a cofactor in certain enzymes. There are no essential functions documented for Cd (Nies 1999). We are interested in understanding the risks of heavy metal contamination of water and soils. EPA-established guidelines for Cu include a water hardness factor (USEPA 2007). Currently, there are no guidelines specifically for Cd. Pseudomonas putida KT2440 was studied because its genome is sequenced (Dos Santos et al. 2004) and Cano- vas et al. (2003) published an in silico analysis of heavy metal-responsive genes. For Cu cell homoeostasis may involve a P-type ATPase, encoded by pacS and chelation by Cop and other binding proteins (Canovas et al. 2003). For Cd, there is no specific predicted uptake system, rather it may enter microbial cells by transport systems for Mn (Tynecka et al. 1981) or Zn (Laddaga and Silver 1985). Three loci for the Czc (Cd 2+ , Zn 2+ and Co 2+ ) chemiosmotic efflux systems are present (Hu and Zhao 2007). The toxicity of both metals in part is due to induced oxidative stress (Ferianc et al. 1998; Wang and Crowley 2005; Teitzel et al. 2006) because ion binding to cysteine and histidine in proteins disrupts structure and function (Dameron and Harrison 1998). Cd damages the Escherichia coli respiratory chain (Pacheco et al. 2008) and causes temporary growth stasis (Ferianc et al. 1998). Pseudomonas Keywords genomics, heavy metal responses, Pseudomonas putida KT2440, RNA accumulation. Correspondence Anne J. Anderson, Biology Department, Utah State University, Logan, UT, USA 84322-5305. E-mail: anderson@biology.usu.edu 2009 ⁄ 2124: received 11 December 2008, revised 15 July 2009 and accepted 9 September 2009 doi:10.1111/j.1472-765X.2009.02741.x Abstract Aims: To compare responses of a soil bacterium to Cu and Cd. Methods and Results: In minimal medium, Cd caused a dose-dependent growth stasis of logarithmic phase cells of Pseudomonas putida, strain KT2440, whereas Cu did not compromise growth up to 10 mg l )1 . Proteomics showed changes in accumulation of both membrane and soluble proteins by 6 h of treatment; increased Krebs cycle enzymes were apparent. Transcript analysis showed Cd- and Cu-induced different genes. Cd-induced genes encoding the transcriptional regulator CzrR2; an outer membrane protein associated with lipopolysaccharide stability, H1; two oxidative stress protective proteins and the P-type ATPase, CadA2, associated with Cd 2+ efflux. The genes most responsive to Cu encoded the regulator CopR1 and the outer membrane resistance protein regulated by CopR1, CopB1; a putative porin, PorD and the Cu-binding protein, PacZ or CopZ, and CopA2. Conclusions: These findings support that a soil pseudomonad restricts internal- ization of the metals by using different sets of binding proteins and efflux pumps. Activation of mechanisms to protect against oxidative stress also was evident especially with Cd exposure. Significance and Impact of the Study: The differential cellular responses to Cd and Cu suggest that risk assessment for Cd and Cu should be different. Letters in Applied Microbiology ISSN 0266-8254 ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 775–783 775