The Influence of Deletion in the Non-Catalytic Domain of GdpP Mediated by Genome Editing with CRISPR/Cas9 on the Phenotype of <i>Staphylococcus aureus</i>

Abstract

The high cellular level of cyclic di-adenosine monophosphate (c-di-AMP), which in turn results in resistance to cell-wall targeted antibiotics in Staphylococcus aureus. An increase in intracellular molecules of c-di-AMP is due to mutations in the DHH/DHHA1 domain of the GdpP protein. The influence of mutations in the other domains of GdpP has not been fully studied. The aim of this study was to obtain a targeted non-frameshifting deletion in the GdpP protein’s linker region between the GGDEF and DHH/DHHA1 domains. Restriction-modification deficient strain S. aureus RN4220 was used for genome editing. Two vectors with thermosensitive origins of replication were used. The first pCN-EF2132tet vector contained the Enterococcus faecalis EF2132 recombinase gene; the second pCAS9counter vector contained the Streptococcus pyogenes RNA-directed Cas9 nuclease gene. The S. aureus RN4220 strain was transformed with the pCN-EF2132tet vector to obtain recombining competent cells, and then a donor oligonucleotide was introduced simultaneously with the counterselection vector. A recombinant strain with a target deletion (90 bp) in GdpP (amino acids 308–337) was obtained after two sequential transformations. The mutant strain showed no changes in the phenotype: lag phase, growth rate, doubling time, and colony morphology did not differ from the progenitor strain. Susceptibility to cell-wall targeted antibiotics was the same as in the progenitor strain. Thus, mutations in the linker region between the GGDEF and DHH/DHHA1 domains of the GdpP do not affect susceptibility to antibiotics in S. aureus.