1998

1998. 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 ATN-161 trifluoroacetate salt the cell surface layer. The localization and Ca2+-induced reversible conformational change suggest that HLP is usually 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 ATN-161 trifluoroacetate salt 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. (1). Biochemical and molecular biological studies have best characterized the following RTX proteins: HlyA of (4, 6, 31) and CyaA FTDCR1B of (10, 11, 29). CyaA is usually a natural fusion protein of adenylate cyclase and hemolysin and exhibits toxicity that modifies the host cellular functions by increasing the intracellular concentration of cyclic AMP (20). The binding of Ca2+ ions is essential for these proteins to acquire the toxic conformation (4, 29). HlyA (K564 and K690) and CyaA (K983) are palmitoylated at the lysine residues by the acyltransferases HlyC (31) and CyaC (11), respectively. This palmitoylation is essential for the toxicity of the proteins. The RTX proteins are secreted with a noncleavable C-terminal signal peptide (6, 20) by the type I secretion system that consists of three membersHlyB, HlyD, and TolC for HlyA (35, 37) and CyaB, CyaD, and CyaE for CyaA (10). forms an operon with forms an operon with (10, 37). RTX proteins of sp. strain WH8102 (5) and oscillin of (13), have been shown to be necessary for cell motility. Thus, there may be a functional diversity of RTX proteins in pathogenic bacteria and cyanobacteria. However, the mechanisms underlying the involvement of the cyanobacterial RTX proteins in motility have not been clarified. sp. strain PCC 6803 (referred to as PCC 6803) is usually a unicellular freshwater cyanobacterium; the wild-type strain (WT) of PCC 6803 has type IV pili that mediate motility (2). A glucose-tolerant strain (GT) which is usually capable of photoheterotrophic growth was generated by spontaneous mutation of the WT (38) and has been used to study the photosynthetic genes. In contrast to the WT, the GT is usually immotile on 1.2% agar plates due to an unknown mechanism that developed during the spontaneous mutation (33). The genomic DNA sequences of a single representative clone of the GT have been decided (14, 15). The product of sll1951 (referred to as or for 10 min. The supernatant was filtered through a GF/F filter (average pore size, 0.7 m; Whatman, Maidstone, United Kingdom) and then through a Nuclepore ATN-161 trifluoroacetate salt filter ATN-161 trifluoroacetate salt (pore size, 0.2 m; Whatman). The cell-free supernatant was collected as the filtrate. It was concentrated 50-fold by using a dialysis membrane and polyethylene glycol 20000 (Wako, Osaka, Japan) at 4C overnight. Subsequently, the concentrated supernatant was precipitated at 10% saturation of ammonium sulfate, incubated at 4C for 1 h, and centrifuged at 10,000 and 4C for 10 min. The precipitates were dissolved in 1.5 ml of 2% sodium dodecyl sulfate (SDS) and centrifuged at 10,000 for 10 min. The supernatant was subjected to the first preparative SDS-polyacrylamide gel electrophoresis (PAGE) with a 2-mm-thick gel plate. After electrophoresis, the gel plate was stained, destained, and washed twice with deionized water for 10 min each time. The band (height, 3 mm) of compact HLP (cHLP) at 90 kDa was excised and subjected to protein extraction by using Maxyield-NP (ATTO, Tokyo, Japan) as described for the manufacturer’s protocol, with the exception that the reverse electrophoresis was performed at 100 V for 1 min at the end of the extraction step. The extract was concentrated ATN-161 trifluoroacetate salt 20-fold by using a Centricon YM-10 apparatus (Millipore, Bedford, Mass.), supplemented with SDS buffer, heated at 100C for 1 min, and subjected to the second preparative SDS-PAGE. The gel plate was stained, destained, and washed as described above. The band (height, 2 mm) of extended HLP (eHLP) at 200 kDa was excised, briefly washed with deionized water, and stored at ?80C until use. Polyclonal antibodies were prepared in two rabbits by Shibayagi (Shibukawa, Japan) using a gel slurry with Freund’s.