Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 25 Nov 2018 00:26:30 Three different DnaK proteins are functionally expressed in the cyanobacterium Synechocystis sp. PCC 6803 Eva Rupprecht, 1,2 Sven Gathmann, 1,2 Eva Fuhrmann 1,2 and Dirk Schneider 1 Correspondence Dirk Schneider Dirk.Schneider@biochemie. uni-freiburg.de 1 Institut fu ¨ r Biochemie und Molekularbiologie, Zentrum fu ¨ r Biochemie und Molekulare Zellforschung, Albert-Ludwigs-Universita ¨ t Freiburg, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany 2 Fakulta ¨t fu ¨ r Biologie, Albert-Ludwigs-Universita ¨ t, 79104 Freiburg, Germany Received 9 January 2007 Revised 6 February 2007 Accepted 7 February 2007 Multiple dnaK genes appear to be common in cyanobacteria; the function of the encoded proteins is, however, still elusive. To characterize the dnaK gene family from the cyanobacterium Synechocystis sp. PCC 6803 in detail, genetic analyses were combined with analyses of the expression and localization patterns of the three encoded proteins. While significant expression of all three genes was found, the results obtained clearly indicate physiological differences of the three proteins in vivo, and DnaK2 seems to have a key function in Synechocystis. Expression of DnaK3 appears also to be as essential as expression of DnaK2, whereas the dnaK1 gene was deleted without resulting in any distorted phenotype. In line with a suggested privileged function, expression of DnaK2 altered most significantly after heat shock. INTRODUCTION Proteins of the 70 kDa heat-shock protein family (Hsp70) are ubiquitously distributed in all kingdoms of life, and in eukaryotes members of this family can be found in several organelles as well as in the cytosol (Bukau & Horwich, 1998; Bukau et al., 2006). The main functions of the proteins are to ensure proper folding of polypeptide chains, to keep proteins in a translocation-competent state after synthesis, and to prevent misfolding and aggregation of proteins caused by e.g. cellular stress. Although some Hsp70 proteins are involved in the cellular heat-shock response, the proteins are usually constitutively expressed and functional, and therefore members of the Hsp70 family are better described as chaperones rather than as heat- shock proteins (Rospert & Chacinska, 2006). Besides the structure of an almost complete bovine Hsc70 protein (Jiang et al., 2005), the structure of the ~45 kDa N- terminal ATPase domain has been resolved recently (Flaherty et al., 1990; Harrison et al., 1997; Zhang & Zuiderweg, 2004) as well as the structure of the 25 kDa peptide-binding domain (Cupp-Vickery et al., 2004; Wang et al., 1998; Zhu et al., 1996), which is linked to the ATPase domain via a short flexible linker. The C-terminal part of Hsp70 proteins is not well conserved, and the physiological function of this domain is less characterized. In recent years, the Hsp70 protein from Escherichia coli (DnaK) has become a paradigm for studying the activity and function of Hsp70s, and the general mechanism of DnaK activity has been mainly investigated in E. coli. Interestingly, the dnaK gene can be easily deleted in E. coli, although the deletion strain displayed defects in cell growth at low and high temperatures (Bukau & Walker, 1989a, b; Paek & Walker, 1987). The ATPase activity of E. coli DnaK is regulated by the two co-chaperones DnaJ and GrpE. DnaJ enhances the substrate-binding activity of Hsp70 by stimulating its ATPase activity, and GrpE is a nucleotide exchange factor. When ATP is bound to the ATPase domain of Hsp70s the protein possesses weak chaperone activity, whereas when ADP is bound the protein binds tightly to substrates (Harrison, 2003; Liberek et al., 1991). While in eukaryotes several different Hsp70 proteins exist in the various cellular compartments, in bacteria usually only a single DnaK protein is present. Nevertheless, in a few bacteria more than one DnaK homologous protein exists, and in E. coli, for example, two DnaK-homologous proteins can be found besides the canonical DnaK (Lelivelt & Kawula, 1995; Seaton & Vickery, 1994; Yoshimune et al., 1998). While this is rather uncommon in other bacteria, all cyanobacteria appear to encode several DnaK proteins as deduced from the available genomic sequence information. A dnaK multigene family was originally identified in the cyanobacterium Synechococcus sp. PCC 7942 (Nimura et al., 1994a, b, 1996, 2001), but later on it became evident that also in the genomes of other completely sequenced cyanobacteria dnaK gene families exist, the functions of which are still largely unknown. As in Synechococcus, in the genome of the mesophilic Microbiology (2007), 153, 1828–1841 DOI 10.1099/mic.0.2007/005876-0 1828 2007/005876 G 2007 SGM Printed in Great Britain