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

Abstract

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca 2؉ 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 Ca 2؉ was not chelated with EGTA; however, it was released after being heated at 100°C for 1 min, and it rebound to the Ca 2؉ -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 Ca 2؉ , 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 Ca 2؉ . Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca 2؉ -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.

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

“…We detected Sll1951, a large protein that resembles hemolysin and that has been characterized before (22,23), in the growth medium of the ⌬sll1213 mutant strain of Synechocystis (data not shown); this mutant is unable to synthesize fucose and appears to lack the S-layer (24), thus laying the foundation for the present study.…”

“…Changes in composition of the lipopolysaccharide layer or (if present) the S-layer glycan frequently lead to a loss of the glycocalyx (41)(42)(43). The predominant protein in supernatants of ⌬sll1213 cultures was identified as Sll1951, a hemolysinlike protein that has been characterized by Sakiyama et al (22,23). However, these studies did not provide data to demonstrate or suggest that Sll1951 would be the S-layer protein.…”

Ecological Genomics of Marine Picocyanobacteria

“…Alternatively, rather than acting as a protective shield, some of these ORFs may encode exotoxins (some at least encode the nonapeptide repeat shared by the RTX toxin family [319]), though there is evidence in freshwater cyanobacteria that they are nontoxic either because the proteins lack a palmitoylation site or because the bacterium lacks the gene encoding the acyltransferase performing this function (247). Nonetheless, the presence of putative polyketide synthases in Prochlorococcus and Synechococcus genomes (MIT 9303_10861 and CC9311_0156, respectively), which may produce secondary metabolites that confer antimicrobial, antifungal, or antiparasitic activities, might suggest that these isolates have adopted a different strategy against predatory pressure, by producing compounds that provide weaponry against competitors for the same environmental niche.…”

Production and secretion of fatty acids in genetically engineered cyanobacteria

“…2), which encodes the monomer protein of the 6803 S layer (27). In the fourth-generation strain SD243, Ch fatB2 was synthesized in an artificial operon à P psbA2 Ch fatB2 and inserted to knock out slr2001 and slr2002 ( Fig.…”