Background

The cyanobacterium Synechocystis sp. PCC6803 (hereafter, Synechocystis 6803) is a model organism for studying the process of photosynthesis. While its genome was sequenced already in 1996, still more than 50% of its genes encode proteins with hypothetical or unknown function. The three genes slr1204 (htrA), sll1679 (hhoA) and sll1427 (hhoB) encode serine proteases of the Deg (degradation of periplasmic proteins) family; despite detailed analyses (see Cheregi et al., 2016 and references therein) their exact subcellular localization and substrates still are enigmatic. Previous proteomic and metabolomic characterizations of single and triple deg deletion mutants performed in our lab have shown altered expression of proteins with functions in or on the outer cell layers of Synechocystis 6803 (Miranda et al., 2013; Tam et al., 2015; Cheregi et al., 2015).

The antibiotics carbenicillin, colistin and polymyxin inhibit or disrupt the bacterial cell wall and therefore can be used to test the integrity of this cellular component in mutants: polymyxin acts on the outermost lipopolysaccharide layer surrounding the cyanobacterial S-layer, carbenicillin interferes with the peptidoglycan layer and colistin acts on the plasma membrane of gram-negative bacteria (Table 1). An agar diffusion test has been developed (Bauer et al., 1966) in which filter paper discs impregnated with specified concentrations of antibiotics are placed on agar plates inoculated with bacteria. The antibiotics will diffuse from the disc into the agar and inhibit cyanobacterial growth around it. The size of this inhibition zone then reflects the sensitivity of the strain to the antibiotic. The antibiotic disc assay method was used to characterize a triple deg protease mutant, and could be used for the characterization of any cyanobacterial mutant. However, the reader should be aware of the limitations of this assay. Despite the Sll1951 protein being the main component of the outermost cell layer of cyanobacteria, called S-layer, the antibiotic disc assay only had limited effect on a sll1951 deletion mutant (Trautner and Vermaas, 2013). Though the S-layer is compromised in the sll1951 deletion mutant, the underlying layers are still intact, preventing Carbenicillin and Polymyxin B, due to their relatively high molecular masses, to penetrate into the cell.

Table 1 describes the molecular weight, the mode of action and the ordering information for the above mentioned antibiotic discs.

Table 1. Antibiotic discs used in this protocol