One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus

Abstract

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress‐associated sigma B factor (σB). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σB activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs.

RNA thermoswitches modulateStaphylococcus aureusadaptation to ambient temperatures

“…The resulting vector was digested using SphI and AscI and the fragments of interest were inserted into the pCN47 plasmid ( 44 ). Plasmids p5′UTR cspB - gfp and p5′UTR cspC - gfp were generated by amplifying the 5′UTR sequences from the pCspB 3xF and pCspC 3xF plasmids ( 36 ) with oligonucleotide pairs 5UTR_CspB_FW_EcoRI/5UTR_CspB_RV_SpeI and 5′UTR_CspC_FW_EcoRI/5′UTR_CspC_RV_SpeI ( Supplementary Table S3 ), respectively. The amplified PCR products were then digested using the EcoRI and SpeI enzymes and ligated into pHRG.…”

“…The csp mutant strains were obtained by marker-less homologous recombination, using the pMAD plasmid system ( 45 ) as previously described ( 36 ). Briefly, the cspB 3xF , cspC 3xF , cspB 3xF Δ cspA , cspC 3xF Δ cspA , Δ cspBC , Δ 24cspB , Δ 24cspC , Δ 24cspB 3xF , Δ 24cspC 3xF and Δ 24cspBC strains were generated by a two-step procedure that inserts or replaces a gene of interest by a mutant allele contained within the pMAD plasmids ( Supplementary Table S2 ) ( 46 ).…”

“…Western blot analyses were performed as previously described ( 36 ). The chromosomally 3xFLAG-tagged CSPs were developed with anti-FLAG antibodies (Sigma) and the GFP samples with monoclonal anti-GFP antibodies 1:5000 (Living Colors, Clontech) and peroxidase-conjugated goat anti-mouse immunoglobulin G and M antibodies 1:2500 (Pierce-Thermo Scientific).…”

“…The S. aureus genome encodes three CSP paralogues (CspA, CspB and CspC) with a protein identity >70% between them ( 36 ). It has been shown that the cspB mRNA levels are induced after cold shock ( 25 , 26 ), while the cspA mRNA levels remain unaltered by shifts in the temperature ( 37 ).…”

Cold-Shock Domains—Abundance, Structure, Properties, and Nucleic-Acid Binding

“…In Staphylococcus aureus , for example, only Sa CspA but no other CSP can stimulate the biosynthesis of the pigment staphyloxanthin (STX). However, a single amino-acid mutation (E58P) enables the paralog Sa CspC to restore STX production in a cspA deletion strain [ 39 ]. Sa CspA post-transcriptionally modulates target-gene expression by binding to sites in the 3′-untranslated regions (3′UTRs) of their mRNAs [ 40 ].…”