Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

Abstract

Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δspx strain harbored suppressor mutations that allowed it to grow without spx One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δspx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism.

Importance: The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.

FIG 1

Physical map of the spx genomic region and features used for genetic analysis. Ordered sequence tags for strain NCTC8325 (NCBI database accession no. NC_007795) are shown, together with arrows that depict the direction of transcription. The limits of the original spx disruption in AR738 are shown, together with the Δspx::kanA allele engineered in this study. The position of the erythromycin resistance gene, ermC, placed in the intergenic region near spx (5 kb upstream) and used for bacteriophage-mediated transduction experiments, is also shown.

FIG 2

Physical map depicting the positions of single nucleotide changes detected by deep-sequencing analysis of strain AR738 Δspx. (A) rpoB-P519L. (B) ytxJ promoter transition mutation. (C) P37S in SAOUHSC_01464 and nearby open reading frames in three different reading frames. Conceptual translation reveals that loci SAOUHSC_01464 and SAOUHSC_01463 overlap by four nucleotides. Within the presumptive SAOUHSC_01463 reading frame, a stem-loop (predicted ΔG of −11.7) was detected in close proximity to the gpsB promoter. The orientations of transcription are shown by arrows, together with the positions of kanamycin or erythromycin resistance markers site specifically engineered in the chromosome and used for genetic analysis. Ordered sequence tag numbers for the indicated reading frames are shown as in Fig. 1.

FIG 3

RpoB rifampin resistance mutations that support deletion of spx are located in close spatial proximity. Shown is a spatial projection of amino acids of S. aureus RpoB surrounding the rifampin binding pocket, using S. aureus coordinates as in the alignment shown in Fig. 4 but based upon the X-ray structure determination of the T. aquaticus RNA polymerase core in complex with rifampin (16). The rifampin binding pocket is oriented with the rifampin functional groups as follows: naphthol ring (planar and perpendicular to the page, upper left), methyl-piperazine (below the naphthol ring), and O-acetyl (center right). Amino acids mutated in this study are boxed: the amino acids whose mutations support the viability of Δspx strains (P519L and R484H) are highlighted in dark blue; those whose mutations support Δspx strains less effectively (A477V and D471Y) are shown in light blue; and the amino acid whose mutation is not a Δspx suppressor mutation (S486L) is not highlighted.

FIG 4

The amino acid residues encoded by the rpoB mutations P519L and R484H are located in a highly conserved region. Shown is a sequence alignment of RNAP β subunit regions comprising the discontinuous rifampin binding pocket regions I and II (16) in various bacteria (marked rpoB) or eukaryotic organisms, together with their corresponding amino acid coordinates. The positions of mutations described herein for S. aureus are depicted with colored circles consistent with the highlighting in Fig. 3. Regions that are boxed and highlighted in gray are highly conserved. Sa, S. aureus; Ec, E. coli; Bs, B. subtilis; Mt, Mycobacterium tuberculosis; Ta, T. aquaticus; Hs, Homo sapiens; Dm, Drosophila melanogaster, Sc, Saccharomyces cerevisiae.

FIG 5

Colony-forming assay showing certain concordant phenotypes attributable to the Δspx allele. (A) Phenotypic analysis of rifampin-resistant suppressor strains and their Δspx derivatives. Those marked P519L* still possess the original mapped SNPs. Strains marked +spx are the complemented strains. Spots made with the 10⁻⁴ dilution from serial dilutions of overnight cultures are depicted for all phenotypes except growth with diamide (0.2 mM) and NaCl (2.2 M), for which we show spots made with the 10⁻² dilution. (B) Phenotypic analysis of trx-dependent suppressor strains and their Δspx derivatives.