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Journal of Bacteriology, May 2006, p. 3535-3542, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3535-3542.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Hemolysin-Like Protein, Sll1951, a Nontoxic Member of the RTX Protein Family from the Cyanobacterium Synechocystis sp. Strain PCC 6803

Tetsushi Sakiyama, Hironori Ueno, Hideya Homma, Osamu Numata, and Tomohiko Kuwabara^*

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan

Received 28 December 2005/ Accepted 9 March 2006

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca²⁺ binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca²⁺ was not chelated with EGTA; however, it was released after being heated at 100°C for 1 min, and it rebound to the Ca²⁺-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca²⁺, respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca²⁺. Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca²⁺-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.

Present address: Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8562, Japan.