Correspondence: Spontaneous secondary mutations confound analysis of the essential two-component system WalKR in Staphylococcus aureus.

Ji et al.1 recently described the structure of the extracytoplasmic Per-Arnt-Sim (PAS) domain of WalK (WalKEC-PAS), the sensor kinase of the essential two-component system WalKR in Staphylococcus aureus. The authors made two independent walK mutants in S. aureus, each with a single amino acid alteration in WalKEC-PAS, inferring from the structure that these residues might be important for signal transduction. We have also been exploring the function of WalKR and were surprised by the striking phenotypic impacts of these single amino acid substitutions.

The authors showed that their WalKEC-PAS mutants (WalKD119A and WalKV149A) caused reduced susceptibility to lysostaphin, loss of sheep blood haemolysis, reduced biofilm formation and reduced virulence compared with parental methicillin-sensitive S. aureus strain Newman2. RNA-seq comparisons of the two mutants to wild type identified substantial transcriptional changes. Structure-based virtual screening was used to predict that 2,4-dihydroxybenzophenone (DHBP) would interact with WalKEC-PAS. As DHBP appeared to stimulate lysostaphin-induced lysis and biofilm formation in strain Newman, the authors postulated that it was activating WalKR. They then measured transcriptional responses of Newman after no treatment, DHBP exposure and in their D119A mutant and reported that there were 41 genes that inversely expressed the WalKD119A mutant compared with DHBP-treated cells, concluding that this supported a role for DHBP in activating WalKR. No direct biochemical evidence of WalKR activation was presented1.

To investigate further, we were provided with S. aureus Newman wild-type, WalKD119A and WalKV149A by the senior author, Chuan He, University of Chicago (UoC)1. We sequenced the genomes of University of Melbourne (UoM) and UoC Newman strains and compared their sequences with the reference2, with the two strains differing by only one synonymous mutation in sbnF (NWMN_0065). Using allelic exchange, we recreated the WalKD119A mutation in the Newman wild type and the USA300 lineage strain NRS3843. We confirmed by genome sequencing that only the AT→CG substitution in WalKEC-PAS was introduced in both strains (NC_009641 chromosome position 25994). We then tested UoM Newman WalKD119A and NRS384 WalKD119A for the key phenotype changes observed by Ji et al.1. However, in contrast to Ji et al.,1 we observed that UoM WalKD119A and NRS384 WalKD119A were fully haemolytic (Fig. 1a) and exhibited identical growth curve kinetics as the parental strains (Fig. 1b). Concurrent screening of UoC Newman WalKD119A confirmed that it was non-hemolytic (Fig. 1a). The mutant also grew to an increased OD600, as reported (Fig. 1b)1, although CFU were identical to wild type, suggesting that the increase in OD was not due to increased growth. Additionally, UoC WalKD119A consistently exhibited larger colonies than Newman or UoM WalKD119A. We next measured the sensitivity of the strains to lysostaphin by cell viability (Fig. 1c). We observed that the UoC WalKD119A mutant was significantly more sensitive (not resistant) to lysostaphin than wild type (3-log10 reduction versus Newman), whereas the UoM WalKD119A mutant showed no change. Interestingly, the WalKD119A mutation in NRS384 caused an increase in lysostaphin sensitivity, suggesting that the mutation contributes to WalKR activation rather than repression, as proposed by Ji et al.14.

Figure 1

Phenotypic screening of walKEC-PAS mutants.

(a) No impact on haemolysis was observed for the WalKD119A mutation in either Newman or NRS384 on sheep blood agar. (b) Growth kinetics were identical for the newly created WalKD119A mutants in TSB at 37 °C with aeration (200 r.p.m.) when compared with the parental strain. Optical density (○) and colony-forming units (□) were enumerated. (c) In a lysostaphin growth sensitivity assay, the UoC WalKD119A exhibited a loss of viability, while the parent or UoM WalKD119A did not. The WalKD119A mutation in the NRS384 background enhanced sensitivity to lysostaphin when compared with the parental strain. Error bars depict the s.d. of the mean from three independent experiments. (d) P1 sae promoter activity with a DsRED reporter. Left: no promoter. Right: P1 sae driving RFP expression; (1) Newman (2) UoM WalKD119A (3) UoC WalKD119A. No expression of sae was observed in UoC WalKD119A. (e) By allelic exchange, the UoC saeS mutation was introduced into Newman or UoM WalKD119A (saeSSTOP) with haemolysis abolished, while introduction of the wild-type saeS gene into UoC WalKD119A (saeSFIX) restored haemolysis.

To resolve the above discrepancies, we subjected UoC WalKD119A and UoC WalKV149 to whole-genome sequencing. Relative to Newman, and in addition to their expected walKEC-PAS changes, both UoC mutants D119A and V149A had acquired four additional mutations (Table 1). Most notably, two independent loss-of-function mutations in saeRS, a major two-component regulator that controls the expression of many genes involved in virulence and biofilm formation5678. The UoC WalKD119A had a TT insertion at position 757519 that introduced a frameshift to saeS. The UoC WalKV149A had a G→T substitution at position 757889 that introduced a premature stop codon to saeR. It is these secondary mutations in saeRS, rather than the targeted mutations in walKEC-PAS, that likely explain the phenotypes observed by Ji et al.1 (reduced biofilm, loss of haemolysis, reduced virulence). We also mapped the authors' RNA-seq reads for WalKD119A and WalKV149A (GSE75731) and readily detected the same saeRS mutations. To confirm the predicted functional consequences of the saeRS mutations, we used a P1 Sae red fluorescence reporter plasmid5. No fluorescence activity was detected in the UoC WalKD119A strain containing the P1 Sae reporter, consistent with the predicted truncation in the histidine kinase preventing phosphorylation of SaeR, whereas high-level expression of P1 Sae from Newman and UoM WalKD119A was observed leading to red colonies (Fig. 1d). We then recreated the mutated saeS allele from UoC WalKD119A in both wild-type Newman and UoM WalKD119A (Fig. 1e). The mutation abolished haemolysis on sheep blood agar. We then repaired the saeS mutation in UoC WalKD119A and observed restoration of wild-type haemolysis (Fig. 1e). These results show that the UoC WalKD119A strain is an sae mutant with the majority of the phenotypic changes reported in this strain (including the reported RNA-seq changes) likely associated with this mutation rather than WalKD119A. The unintended secondary mutations in a major S. aureus regulatory locus preclude analysis of the role of WalKEC-PAS domain in WalKR signal transduction.

Table 1

Whole-genome sequencing identified the introduction of unintended polymorphisms within the S. aureus Newman WalK mutants.

Position of S. aureus Newman (NC_009641)	UoC Newman (Acc: ERR1450026)	UoC WalkD119A (Acc: ERR1450027)	UoC WalkV149A (Acc: ERR1450028)	Locus_tag (NWMN_)	Gene	Comment (product, predicted consequence of mutation) c.=codon; p.=amino acid position
25994	A	C	A	0018	walK	Sensor kinase: missense_variant c.356A>C p.Asp119Ala
26084	T	T	C	0018	walK	Sensor kinase: missense_variant c.446T>C p.Val149Ala
87710	A	A	A	0065	sbnF	Siderophore biosynthesis: synomous c. 425G>A
757519	—	TT	—	0674	saeS	Sensor kinase: frameshift variant c.152_153ins AA p.Thr52fs
757889	C	C	A	0675	saeR	DNA-binding response regulator stop_gained c.469G>T p.Glu157*
820314	C	T	T	0728	hprK	HPr kinase/phosphorylase: missense_variant c.383C >T p.Ala128Val
897870	—	TG	TG	0810		Truncated hypothetical. Created full-length gene. c.98_99ins TG p. 50–119
1914999	T	T	C			Intragenic between NWMN_1716/1717
2370632	C	G	C	2142	rplN	50S ribosomal protein L14: missense_variant c.86G>C p.Gly29Ala

There is precedence for this specific phenomenon. Sun et al.5 showed that elevated temperature and antibiotic selection used during the S. aureus mutagenesis process can aid in the selection of saeRS mutations. How Ji et al.1 managed to complement the mutations (D119A and V149A) by phage integrase plasmid expression (pCL55) of wild-type walKR remains to be explained. We have been unable thus far to obtain the complemented mutants for analysis.

We also observed that UoC WalKD119A and UoC WalKV149A exhibited larger colonies compared with wild type, a phenotypic difference not discussed by Ji et al.1. This change in both mutants might be explained by the C→T substitution observed at 820314, leading to an A128V change in HprK, a serine kinase known to be involved in catabolite repression and associated with a spreading colony phenotype9.

Ji et al.1 used RNA-seq and the inverse expression profiles of the WalKD119A mutant and DHBP treatment of the wild type to ‘prove' that DHBP is signaling through WalKEC-PAS, but this conclusion is confounded by the saeRS mutations. In addition, the authors failed to apply any filter for false discovery rate to their RNA-seq analysis. This analysis without statistical significance thresholds is not meaningful10. We also repeated the lysostaphin assay with and without the addition of 75 μM DHBP. We failed to observe the reported loss of turbidity in Newman pretreated with DHBP upon lysostaphin treatment1 (Supplementary Fig. 1.).

The discovery of small-molecule inhibitors of WalKR function would represent a major advance in the fight against multidrug-resistant S. aureus. Unfortunately, the presence of unintended saeRS mutations in their walKEC-PAS mutants invalidate their conclusions with respect to role of the WalK extracytoplasmic domain in controlling WalKR function. Using a clean D119A mutant, we observed opposing results: with increased sensitivity to lysostaphin, a phenotype previously linked with enhanced activity of WalKR4. In our own WalKR research, we have observed a propensity for mutations introduced into this locus to yield secondary compensatory events11. These secondary changes can confound analysis of this essential two-component system and highlight the extreme care needed when manipulating this locus and then attributing specific phenotypes to specific mutational changes.

Methods

Bacteria and molecular tools

The S. aureus UoM Newman was obtained from Professor Tim Foster (Trinity College Dublin); NRS384 was obtained from BEI resources (www.beiresources.org). S. aureus was routinely grown in Tryptic Soy Broth (TSB-Oxoid) at 37 °C with aeration at 200 r.p.m. Primers were purchased from IDT (www. idtdna.com) with primer sequences detailed in Supplementary Table 1. Restriction enzymes, Phusion DNA polymerase and T4 DNA ligase were purchased from New England Biolabs. Genomic DNA was isolated from 1 ml of an overnight culture (DNeasy Blood and Tissue Kit—Qiagen) pretreated with 100 μg of lysostaphin (Sigma cat. no. L7386). DHBP was purchased from Sigma (cat. no. 126217; 100 g).

Lysostaphin sensitivity assay

Overnight cultures of S. aureus were diluted 1:100 in fresh, prewarmed TSB in the presence of 0.2 μg ml−1 of lysostaphin with or without 75 μM DHBP (100 mM stock in methanol). Broths were incubated statically at 37 °C. Colony-forming units were determined by spot plate dilution on Brain heart infusion agar (Difco) at 0 and 90 min. Limit of detection for the assay was 103 CFU ml−1.

Construction of pIMC8-RFP and SLiCE cloning

The S. aureus codon optimized DsRED red fluorescent protein and upstream TIR sequence from pRFP-F (ref. 12) was PCR amplified with primers IM314/IM315. The product was digested with KpnI/SacI and cloned into the complementary digested pIMC8 (non-temperature-sensitive version of pIMC5 (ref. 13)), creating pIMC8-RFP. To clone into pIMAY-Z3 and pIMC8-RFP, primers were tailed with 30 nt of complementary sequence to the plasmid. Amplimers were inserted with seamless ligation cloning extract (SLiCE; ref. 14) into the vector (pIMAY-Z: walRKD119A, sae, sae; pIMC8-RFP: P1 sae). Either vector was linearized with KpnI, gel extracted and PCR amplified with primers IM1/IM2 (pIMAY-Z) or IM1/IM385 (pIMC8-RFP). Both amplimers (vector and insert) were combined in a 10 μl reaction containing 1 × T4 ligase buffer, with 1 μl of SLiCE extract. The reaction was incubated at 37 °C for 1 h and then transformed into Escherichia coli strain IM08B3, with selection on Luria agar plates containing chloramphenicol 10 μg ml−1. Plasmids were extracted and directly transformed by electroporation into the target S. aureus strain3.

Production of SLiCE extract

The SLiCE was isolated from DY380 (ref. 14) grown in 50 ml 2xYT (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) at 30 °C after a 1:100 dilution of the overnight culture. Once the culture reached an OD600 of ∼2.5, the cells were moved to 42 °C for 25 min (addition of 50 ml of 42 °C 2xYT). Cells were processed as described by Zhang et al.14, with the pellet lysed in 500 μl of CelLytic B cell lysis reagent (C87040; 10 ml; Sigma).

Whole-genome sequencing and data analysis

Whole-genome sequencing was performed using the Illumina NextSeq (2 × 150 bp chemistry), with library preparation using Nextera XT (Illumina). Resulting reads were mapped to the S. aureus Newman reference (Accession: NC_009641) using Snippy v3.1 (https://github.com/tseemann/snippy). Note that 89 substitutions, 20 deletions and 25 insertions were shared between UoC and UoM Newman strains compared with the NC_009641 reference sequence, representing likely sequencing errors in the 2008 published reference2.

Data availability

All sequencing data used in this study have been deposited in the National Center for Biotechnology Information BioProject database and are accessible through the BioProject accession number PRJEB14381 (https://www.ncbi.nlm.nih.gov/bioproject/325902). An updated S. aureus Newman genome sequence is available (Genbank reference: NZ_LT598688.1).

Additional information

How to cite this article: Monk, I. R. et al. Correspondence: Spontaneous secondary mutations confound analysis of the essential two component system WalKR in Staphylococcus aureus. Nat. Commun. 8, 14403 doi: 10.1038/ncomms14403 (2017).

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