Published: Vol 6, Iss 24, Dec 20, 2016 DOI: 10.21769/BioProtoc.2071 Views: 10426
Reviewed by: Maria SinetovaElizabeth LibbyLaura Molina-García
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Abstract
This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with the biosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone of cyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivity of the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or its construction would render the mutant strain more sensitive to the respective antibiotic. The method has proven to be useful for phenotyping a mutant of Synechocystis sp. PCC6803 lacking all three genes encoding Deg proteases. Deletion of these ATP-independent serine proteases was shown to have impact on the outer cell layers of Synechocystis cells (Cheregi et al., 2015).
Keywords: CyanobacteriaBackground
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
Materials and Reagents
Equipment
Procedure
Note: it is recommended that handling of cyanobacterial cultures is done under aseptic conditions using a sterile laminar hood.
Data analysis
The average diameters of the areas of inhibition (in cm) and standard deviations were calculated from four biological replicates. Each biological replicate was represented by 3 technical replicates; the technical replicates presented areas of inhibition that were identical. These data are presented in Table 3 and Figure 3 of Cheregi et al. (2015).
Notes
This protocol was adapted from the previously published study of Trautner and Vermaas (2013) and it was performed as in Cheregi et al. (2015). The efficiency of the antibiotic is decreasing after expiration date.
Recipes
Acknowledgments
This work was supported by grants from the Swedish Energy Agency (to C. Funk).
References
Article Information
Copyright
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Cheregi, O. and Funk, C. (2016). Antibiotic Disc Assay for Synechocystis sp. PCC6803. Bio-protocol 6(24): e2071. DOI: 10.21769/BioProtoc.2071.
Category
Microbiology > Antimicrobial assay > Antibacterial assay
Microbiology > Microbial metabolism > Other compound
Cell Biology > Cell metabolism > Other compound
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