Limits...
Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus.

Burda WN, Miller HK, Krute CN, Leighton SL, Carroll RK, Shaw LN - BMC Microbiol. (2014)

Bottom Line: Through this we identified CymR as a negative effector of sigS expression.Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription.Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

View Article: PubMed Central - PubMed

ABSTRACT

Background: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression.

Results: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS.

Conclusions: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

Show MeSH

Related in: MedlinePlus

Identification of positive regulators ofsigSexpression. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4265319&req=5

Fig3: Identification of positive regulators ofsigSexpression. 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.

Mentions: 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


Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus.

Burda WN, Miller HK, Krute CN, Leighton SL, Carroll RK, Shaw LN - BMC Microbiol. (2014)

Identification of positive regulators ofsigSexpression. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265319&req=5

Fig3: Identification of positive regulators ofsigSexpression. 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.
Mentions: 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

Bottom Line: Through this we identified CymR as a negative effector of sigS expression.Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription.Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

View Article: PubMed Central - PubMed

ABSTRACT

Background: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression.

Results: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS.

Conclusions: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

Show MeSH
Related in: MedlinePlus