A slipped‐mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate δ‐ and α‐haemolysins

Abstract

Summaryagr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the nonhaemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3 ′ ′ ′ ′ end of agrA ( agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the Cterminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate α α α α -and δ δ δ δ -haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate α α α α -and δ δ δ δ -haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.

Various checkpoints prevent the synthesis ofStaphylococcus aureuspeptidoglycan hydrolase LytM in the stationary growth phase

“…The same experiment was performed on RN4220 (strain EL101), which is capable of accepting foreign DNA, 39,40 and which produces low levels of RNAIII. 41 First, we have verified by Northern blot analysis the levels of lytM mRNA in backgrounds HG001 and RN4220 ( Fig. 2A).…”

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola

“…Most RRs interact with DNA through a recognition alpha helix located within a DNA binding domain. Studies of the S. aureus AgrA (accessory gene regulator A), a LytTR RR, have provided insight into the LytTR-DNA interaction (Koenig et al 2004;Nicod et al 2014;Rode et al 2007;Sidote et al 2008;Traber and Novick 2006). AgrA is a cytoplasmic protein that possesses an N-terminal REC domain of 124 amino acids and a C-terminal LytTR domain of 98 residues.…”

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Rapid Detection and Quantification of agr Functionality in Clinical Staphylococcus aureus Isolates

“…However, S. aureus agr dysfunction has also been associated with longer duration of bacteremia and attenuated vancomycin bactericidal activity, as well as increased mortality among bacteremic patients (4,13). A number of reports have indicated that the emergence of vancomycin-intermediate S. aureus (VISA) was accompanied by agr dysfunction, mainly due to loss-offunction mutations within the agr operon (11,12,18).…”

“…AgrA also activates the agr P3 promoter, which drives the synthesis of RNAIII, the effector of target gene regulation. RNAIII also encodes ␦-hemolysin, the expression of which serves as a surrogate for agr functionality (5,18,19).…”