The identification of proteins that modulate sigS expression

Previous studies on sigS expression by our group have demonstrated significant variability across different wild-type strains. This has led us to hypothesize in the past that there may be regulatory circuits that exists in S. aureus to control the expression of this gene [8]. As such, in order to identify direct regulators of sigS expression, a pull down assay was performed using a biotinylated sigS promoter fragment immobilized on streptavidin magnetic beads. Protein lysates from strain RN4220 were utilized in these assays, as our group previously demonstrated sigS expression to be highest in this background under standard conditions [8]. Protein lysates from RN4220 grown under standard conditions for 2 h were incubated with the biotinylated sigS promoter fragment. This time point was selected in order to identify modulators of sigS that effect transcript levels as the bacteria transition from minimal sigS expression (2 h) to maximal expression by hour 3 [8]. Proteins that were bound to the promoter region were harvested, and separated by SDS PAGE analysis followed by silver staining (Figure 1A). We observed that while there were no proteins detected in our no DNA control lane, several distinct bands were detected in the sigS test lanes. For our control promoter (nsaXRS), we observed a number of proteins bound to this fragment, which likely results from the fact that this locus is very highly expressed, and subject to complex, multifactorial regulation [30],[31]. The identity of proteins bound to the sigS promoter was then determined using mass-spectrometric analysis, identifying 21 different factors (Additional file 1: Table S2). Whilst a number of proteins were identified with known or predicted DNA-binding roles (including DNA replication and transcription, stress proteins and exonucleases), only one was a known transcriptional regulator: the cysteine biosynthesis pathway transcriptional regulator, CymR. As this did not appear in either of the control samples, we next set out to determine if CymR had a measurable impact on sigS expression using qPCR and mutant strains deficient in the cymR gene. This analysis was performed in strains RN4220 and 8325-4: the former because this is the background where the identification was made and the latter as we have previously shown that this strain is highly sensitive to sigS modulation [8]. For RN4220 and 8325-4, RNA was harvested at 3 and 5 hours, respectively, as this is the time for each strain when sigS is maximally expressed [8]. Upon analysis, we observed a 2.7- and 2.6-fold increase, respectively, in sigS expression in the 8325-4 and RN4220 cymR mutants compared with the wild-type strains (Figure 1B). This would seem to indicate that the CymR protein plays a role in the negative regulation of sigS expression in S. aureus. To determine if this effect is indeed direct, we sought to recapitulate the binding of this protein to the sigS promoter in vitro using electrophoretic mobility shift assays (EMSA) (Figure 1C). The promoter region of mccAB was used as a positive control because Soutourina et al. have previously shown that CymR directly binds to this region [28]. When the mccAB promoter fragment was incubated with increasing amounts of CymR, a clear shift is observed at higher concentrations of protein. Interestingly, parallel experiments with the sigS promoter fragment revealed similar shifts, which would indicate that CymR is a direct regulator of sigS expression. In order to demonstrate that this effect was specific and not a result of promiscuity, we performed EMSAs using a biotinylated DNA fragment that was amplified from the coding region of the gene rseP. In these studies we did not observe any shift of the DNA, validating that the binding observed for sigS is indeed specific, and that CymR directly regulates, and represses, transcription from the sigS promoter.

Figure 1 CymR is a direct repressor of transcription from the sigS promoter. (A) A pull down assay was performed using crude protein lysates harvested from RN4220 at hour 2 and a biotinylated sigS promoter DNA probe. The identity of protein bands was determined by LC/MS analysis. (B) qPCR analysis of sigS expression in a cymR mutant compared to its respective parental strain, in the 8325-4 and RN4220 backgrounds. Error bars are shown as ± SEM, * = p <0.05 using a Student’s t-test. (C) An electrophoretic mobility shift assay was performed using purified CymR, and the promoter region of mccAB (positive control), the promoter region of sigS (test), and an intergenic region from the rseP gene (rseP; negative control). CymR was added at increasing concentrations of 0.01, 0.1 and 1 μM in all panels. Full size image

A global screen to identify genes that negatively regulate sigS expression

To assess whether additional regulators of sigS expression exist in S. aureus beyond CymR, we next used a global genetic approach to identify modulating factors. Therefore, we employed transposon mutagenesis screen in conjunction with an 8325-4 sigS-lacZ reporter fusion strain. This background was chosen because our previous works have shown that 8325-4 is the most sensitive wild-type strain to sigS modulation, and that information gained using this strain is conserved amongst other S. aureus wild-types [8]. As such, we screened >10,000 clones from our transposon library, and identified 123 that had increased sigS expression. A secondary screen was performed on these mutations, by transducing them into a clean 8325-4 sigS-lacZ fusion background. This was to ensure that the increase in expression is due to direct gene disruption by Tn551, rather than the result of SNPs that may have accumulated during construction of our library. We determined that 96 mutants from our secondary screen recapitulated the increase in sigS expression on media containing X-GAL. Upon sequence analysis, we identified 58 unique insertion sites, with 49 found to be in coding regions, and a further 9 found to be intergenic between open reading frames (Additional file 1: Table S3).

Analysis of transposon insertion sites revealed many were clustered in one particular region of the genome (SACOL1411 to SACOL1490). We have previously identified this region as a hot-spot for Tn551 insertion [17], and therefore sought to validate our findings further to ensure they specifically related to sigS expression. To achieve this, we made use of the Nebraska Transposon Mutant Library (NTML), a collection of bursa aurealis transposon mutants in almost all non-essential S. aureus gene [32]. Of the 49 identified insertions from our screen that were in coding regions, 40 had NTML mutants available (Additional file 1: Table S3). Therefore, each of the NTML insertions for these 40 genes were separately introduced into our 8325-4 reporter strain. In order to determine if the NTML mutants recapitulated our sigS expression findings from the Tn551 screen, all mutants were first analyzed using a plate based assay. Upon analysis, 20 of the transduced NTML mutants were blue when plated on media containing X-GAL (Table 2), whilst the remaining 20 did not reproduce the findings of their counterpart Tn551 insertions. β-galactosidase assays were then performed on the NTML mutants that tested positive, to specifically quantify changes in sigS expression. As such, NTML reporter fusion strains were grown for 5 h in TSB, as we have previously shown this to be the window of peak sigS expression in strain 8325-4 [8]. Interestingly, only three mutants were found to have increased sigS expression at this time point (Figure 2A): insertions in ald1 (an alanine dehydrogenase, 2.6-fold), lacR (the lactose phosphotransferase system repressor, 31-fold) and sucB (dihydroipoamide succinyltransferase, 4.2-fold). For the 17 mutants that did not demonstrate increased sigS expression at this time point, we next performed transcriptional profiling every hour over a 10 hour time course. In doing so, we observed increased sigS expression for an additional 2 mutants (Figure 2B and C): insertions in arlR (a DNA-binding response regulator), and sucA (2-oxoglutarate dehydrogenase E1 component). For arlR we observed an increase in sigS expression at all hours assayed (maximal change =6.1 fold at 9 h) except at 5 h; whilst for sucA, a gene transcribed upstream of sucB, we observed a 2.2 fold increase at hour 4. With regards to the remaining 15 transposon mutants, we did not see an increase in sigS expression over the 10 hour time course. However, we did observe a blue coloration when plated on media containing X-GAL, which does not occur with wild-type 8325-4 sigS-lacZ fusions. As such, the increase in sigS expression observed for these strains either takes place deeper into stationary phase than assessed herein; or as a result of growth on a solid surface compared with liquid culture.

Table 2 Transposon insertions resulting in increased expression of sigS Full size table

Figure 2 Transcriptional profiling of sigS in transposon mutants found to negatively effect expression. (A, B, and C) Mutant strains bearing a sigS-lacZ fusion were grown in TSB at 37°C and samples withdrawn at the times specified. β-Galactosidase activity was measured using 4-MUG as a substrate to determine sigS expression levels. Assays were performed on duplicate samples and the values averaged. The results presented are from three independent experiments. Error bars are shown as ± SEM. Significance was determined by a Student t test; *indicates a p value of <0.05. Full size image

Transposon mutagenesis to identify positive activators of sigS expression

The advantage of using the 8325-4 sigS-lacZ fusion for transposon mutagenesis is that it can be performed in reverse, meaning that we can use a chemical that induces expression of sigS alongside X-GAL, and identify positive regulators by a lack of blue coloration upon transposon insertion. As such, media was prepared using 0.25 mM MMS and X-GAL, and our Tn551 mutant library was again subject to screening. In total, we assessed >10,000 clones and identified 349 that had no observable sigS expression, determined by a lack of blue coloration of colonies. A secondary screen was performed with these strains, yielding 86 that recapitulated the phenotype. This relatively low number of clones that retained phenotype, compared to our original screen, is most likely due to point mutations caused through the use of the DNA damaging agent MMS. Upon sequencing, we identified 35 unique insertion sites (Additional file 1: Table S4), 24 of which occurred within genes, and 11 that were found to be intergenic. Interestingly, 12 insertions occurred within the known hotspot region, whilst a further 5 were identified in both screens (one of which failed to validate in our previous screen, and the other four failed to validate in this screen, see below). In an effort to define which of these elements legitimately influence sigS expression in a positive manner, we again made use of the NTML collection. Of the 24 insertions within ORFs, 21 mutants were available in the Nebraska Transposon Mutant Library. As such, each of these mutations was again transduced into a clean 8325-4 sigS-lacZ strain, and their effects on sigS expression validated by plating on media containing 0.25 mM MMS and X-GAL. Of the 21 mutations assayed, 10 of them were blue when plated on media containing MMS and X-GAL (Table 3), including the remaining 4 that were identified in both screens (thus excluding them from further study). The remaining 11 mutants had abrogated sigS expression, as expect, and were thus subject to transcription profiling in liquid culture in the presence of 0.25 mM MMS for validation (Figure 3). This time, all mutants tested resulted in decreased sigS expression after 5 h of growth, ranging from 2.2-fold (SACOL2143) to 12.2-fold (SACOL1412). Of the elements identified in this screen to positively influence sigS expression, there were insertions in genes whose products are involved in regulation, transport, protein synthesis and modification, amino acid biosynthesis, and cell envelope biosynthesis.

Table 3 Transposon insertions resulting in decreased expression of sigS Full size table

Figure 3 Identification of positive regulators of sigS expression. Mutant strains bearing a sigS-lacZ fusion were grown in TSB at 37°C and sampled after 5 h of growth. β-Galactosidase activity was measured using 4-MUG as a substrate to determine sigS expression levels. Assays were performed on duplicate samples and the values averaged. The results presented are from three independent experiments. Error bars are shown as ± SEM. Significance was determined using a Student t test; *indicates a p value of <0.05. Full size image

Inducing sigS expression in strain SH1000

S. aureus strain SH1000 is identical to 8325-4, apart from an 11 bp deletion in the σB controlling phosphatase, rsbU. Despite this similarity, SH1000 does not demonstrate detectable sigS expression during regular growth; an effect that appears to be more complex than mere σB mediated control [7],[8]. Therefore, to explore whether sigS upregulation could be achieved in this strain, we next exposed our SH1000 sigS-lacZ fusion strain to the DNA mutagen, methyl nitro-nitrosoguanidine (MNNG). MNNG acts by adding alkyl groups to O6 of guanine and O4 of thymidine, which can lead to transition mutations. This has an advantage over transposon mediated technologies as it can assess both loss- and gain-of function, rather than only the former. Upon analysis, we identified 76 strains that had an increase in expression of sigS as determine by blue coloration when plated on media containing X-GAL. To quantitate the increased sigS expression observed on a plate, we selected two strains, HKM15 and HKM16, and subjected them to continuous growth analysis in liquid media (Figure 4A). We determined that both HKM15 and HKM16 resulted in a 75-fold increases in sigS expression after 5 h of growth, compared to the wild-type. To identify the specific mutations that result in these outcomes, we next subjected these two strains to whole genome sequencing, alongside the SH1000 parent, with resulting data analyzed using the CLC Genomic Workbench software. When one compares these two datasets, we found 9 mutations that were common to both strains (Table 4). Importantly, within this list are two known transcriptional regulators, LacR, which we have already identified in this study as influencing sigS expression, and KdpD, a membrane sensor histidine kinase. In order to evaluate the effect these mutations have on sigS expression, the relevant NTML mutations were transduced into SH1000, and qPCR profiling was performed (Figure 4B). We observed a 2.0-fold increase in sigS expression in the SH1000 lacR mutant, while we observed a 4.0-fold increase in sigS expression in SH1000 kdpD. This would indicate that lack of lacR and kdpD also have a role in regulating sigS expression.

Figure 4 Nitrosoguanidine mutagenesis identifies additional regulators of sigS expression. (A) The SH1000 sigS-lacZ fusion strain was subjected to MNNG mutagenesis. Two resulting clones were selected for further analysis during growth in TSB at 37°C. HKM15 and HKM16 were grown along side the parent strain, SH1000, with samples collected at 5 hours. β-Galactosidase activity was measured using 4-MUG as a substrate to determine sigS expression levels. Assays were performed on duplicate samples and the values averaged. The results presented are from three independent experiments. Error bars are shown as ± SEM. (B) qPCR for sigS expression was performed on SH1000 strains containing a mutation in either lacR or kdpD. Error bars are shown as ± SEM. Significance was determined using a Student t test; *indicates a p value of <0.05. Full size image

Table 4 SNPs common to both SH1000 sigS-lacZ NTG generated mutants HKM15 and HKM16 Full size table

In an effort to determine if the effect on sigS expression is direct or indirect we performed EMSAs using purified LacR and KdpE. KdpE was used because it is the partner response regulator, phosphorylated by KdpD. With regards to LacR, the promoter region of the lac operon was used as positive control, as it has previously shown that LacR directly binds to this region [27]. As such, we incubated the lac promoter fragment with increasing amounts of LacR, and, as expected, observed a shift for the lac promoter fragment at higher protein concentrations (Figure 5). However, when the sigS promoter fragment was used, no such shift was observed. This suggests that, despite LacR clearly influencing sigS expression (it was identified in two of our screens), these effects appear to be indirect.

Figure 5 LacR does not directly regulate sigS expression. Electrophoretic mobility shift mobility assays were performed using purified LacR, the lac promoter region (positive control), and the promoter region of sigS (test). LacR was added at increasing concentrations of 0.01, 0.1 and 1 μM in all panels. Full size image

With regards to KdpE, we first used the promoter region of kdpFABC as a positive control, because it has previously been shown that KdpE directly binds this region [29]. As such, we incubated the kdpFABC promoter fragment with increasing amounts of KdpE, and detected a shift for this fragment at higher protein concentrations (Figure 6). Importantly, we also detected a shift for the sigS promoter fragment at similar concentrations, which would indicate that KdpE is a direct regulator of sigS expression. To determine if this is specific effect, and not the result of promiscuous binding by KdpE, we again used our internal fragment from the coding region of rseP as a negative control. In these studies we did not observe any shift of the DNA, validating that the binding observed for sigS is indeed specific, and that KdpDE directly regulates, and represses, transcription from the sigS promoter.