CtaM Is Required for Menaquinol Oxidase aa3 Function in Staphylococcus aureus

Abstract

Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability of S. aureus to cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (o type, bd type, and aa3 type), the genome contains genes encoding only two respiratory oxygen reductases, cydAB and qoxABCD Previous investigation showed that cydAB and qoxABCD are required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of the cydAB and qoxABCD in virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is an aa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as a bo3-type menaquinol oxidase. However, the bo3 form of QoxABCD restricts the carbon sources that can support the growth of S. aureus In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiring Firmicutes In total, these studies establish the heme A biosynthesis pathway in S. aureus, determine that QoxABCD is a type aa3 menaquinol oxidase, and reveal CtaM as a new protein required for type aa3 menaquinol oxidase function in multiple bacterial genera.

Importance: Staphylococcus aureus relies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of the S. aureus electron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain of S. aureus is comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration.

FIG 1

The cytochrome profiles of terminal oxidase mutants reveal the preferred heme cofactors utilized by each enzyme. The reduced-minus-oxidized spectra of Staphylococcus aureus and isogenic terminal oxidase mutants (ΔcydA and ΔqoxA) are shown. Heme A peaks are located at 430 nm and 609 nm. AU, arbitrary units.

FIG 2

CtaB is predicted to be the heme O synthase in S. aureus. (A) Putative heme cofactor biosynthesis pathway in S. aureus. CtaA is the heme A synthase in S. aureus (21). (B) Genomic locus containing the heme cofactor biosynthesis genes in S. aureus strain Newman. 0982 denotes the locus tag NWMN_0982 (referred to herein as ctaM). Scale bar = 250 bp.

FIG 3

Heme O is synthesized by CtaB and is required for QoxABCD function. (A) Liquid chromatography (LC) analysis of membranes isolated from wild-type (WT) staphylococci. Tandem mass spectroscopy was used to identify heme B in fraction 37. (B) LC analysis of membranes isolated from an isogenic ΔctaB deletion mutant. (C) Overnight cultures of WT S. aureus and isogenic mutants were grown at 37°C in tryptic soy broth (TSB) and streaked for isolated colonies on tryptic soy agar (TSA). The ΔcydAB ΔqoxA and ΔctaB ΔcydA mutants are respiration-arrested small colony variants.

FIG 4

In the absence of heme A synthesis, QoxABCD utilizes heme O. (A) Overnight cultures of WT S. aureus and isogenic mutants were grown at 37°C in TSB and streaked for isolated colonies on TSA. The ΔcydAB ΔqoxA mutant is a respiration-arrested small colony variant. (B) LC analysis of membranes isolated from a ΔcydAB ΔctaA mutant. Tandem mass spectroscopy was used to identify heme B in fraction 37 and heme O in fraction 52. (C) The growth of the WT and isogenic mutants of S. aureus was monitored over time by optical density at 600 nm. The final concentrations of glucose (glc) and Casamino Acids (CAS) added to the growth medium were 25 mM (open symbols) and 0.5% (red symbols), respectively. The average results from three independent experiments are shown. Error bars represent one standard deviation from the mean.

FIG 5

QoxABCD function is supported by CtaM. (A) PCR was performed on cDNA enriched from staphylococci grown at 37°C to early stationary (6 h) or late stationary (12 h) phase. RT, reverse transcriptase. (B) Overnight cultures of WT S. aureus and isogenic mutants were grown at 37°C in TSB and streaked for isolated colonies on TSA. The ΔqoxB ΔcydA and ΔcydAB ΔctaM double mutants are respiration-arrested small colony variants. (C) LC analysis of membranes isolated from the ΔctaM strain.

FIG 6

ctaB, ctaA, and ctaM homologues are conserved among respiring Firmicutes and show a strong tendency to co-occur in the same genome. The phylogenic distribution of ctaM (orange, outer ring) was mapped onto the Tree of Life (29, 45). The distributions of ctaB, the gene that encodes the heme O synthase (purple, outer ring), and of ctaA, the gene that encodes the heme A synthase (teal, outer ring), are also presented.