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Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

Sudhakar P, Reck M, Wang W, He FQ, Wagner-Döbler I, Dobler IW, Zeng AP - BMC Genomics (2014)

Bottom Line: To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed.These sub-networks were significantly enriched with genes sharing common functions.Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21073 Hamburg, Germany. iwd@helmholtz-hzi.de.

ABSTRACT

Background: Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.

Results: The co-expression response network constructed from transcriptomic data using the reverse engineering algorithm called the Trend Correlation method consisted of 8284 gene pairs. The regulatory response network inferred by superimposing transcription factor binding site information into the co-expression network comprised 329 putative transcriptional regulatory interactions and could be classified into 27 sub-networks each co-regulated by a transcription factor. These sub-networks were significantly enriched with genes sharing common functions. The regulatory response network displayed global hierarchy and network motifs as observed in model organisms. The sub-networks modulated by the pyrimidine biosynthesis regulator PyrR, the glutamine synthetase repressor GlnR, the cysteine metabolism regulator CysR, global regulators CcpA and CodY and the two component system response regulators VicR and MbrC among others could putatively be related to the physiological effect of carolacton. The predicted interactions from the regulatory network between MbrC, known to be involved in cell envelope stress response, and the murMN-SMU_718c genes encoding peptidoglycan biosynthetic enzymes were experimentally confirmed using Electro Mobility Shift Assays. Furthermore, gene deletion mutants of five predicted key regulators from the response networks were constructed and their sensitivities towards carolacton were investigated. Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

Conclusion: The network approach used in this study revealed important regulators and interactions as part of the response mechanisms of S. mutans biofilm cells to carolacton. It also opens a door for further studies into novel drug targets against streptococci.

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Experimental verification of the predicted transcriptional regulation of themurMN-SMU_718c operon by the response regulator MbrC. The predicted transcriptional regulatory relationship was based on a well correlated (A) expression profile between mbrC and the murMN-SMU_718 operon as well as the presence of a (B) putative MbrC binding site (TTACAA-AT-TTCTAC) in the upstream regulatory regions of the murMN-SMU_718 operon. The alignment among the MbrC binding sites in other experimentally verified targets (black) reported by Ouyang et al. [50] and the putative site upstream of the predicted target (red) murMN-SMU_718 operon is shown. The signature repeats of the MbrC binding motif are italicized, underlined and shown in bold. (C) Binding of MbrC to the promoter region of the gene SMU_1006 (positive control) was verified using Electro Mobility Shift Assays (EMSA), as already reported by Ouyang et al.[50]. (D) EMSA also provided the verification of the in-vitro binding of the MbrC protein to the promoter region of the predicted target murMN-SMU_718c operon via the putative binding site thus confirming that the latter is a transcriptional regulatory target of MbrC. The triangles indicate increasing concentrations of MbrC in the binding reactions. Black triangles followed by IR indicate target DNA fragments lacking the MbrC binding site.
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Fig6: Experimental verification of the predicted transcriptional regulation of themurMN-SMU_718c operon by the response regulator MbrC. The predicted transcriptional regulatory relationship was based on a well correlated (A) expression profile between mbrC and the murMN-SMU_718 operon as well as the presence of a (B) putative MbrC binding site (TTACAA-AT-TTCTAC) in the upstream regulatory regions of the murMN-SMU_718 operon. The alignment among the MbrC binding sites in other experimentally verified targets (black) reported by Ouyang et al. [50] and the putative site upstream of the predicted target (red) murMN-SMU_718 operon is shown. The signature repeats of the MbrC binding motif are italicized, underlined and shown in bold. (C) Binding of MbrC to the promoter region of the gene SMU_1006 (positive control) was verified using Electro Mobility Shift Assays (EMSA), as already reported by Ouyang et al.[50]. (D) EMSA also provided the verification of the in-vitro binding of the MbrC protein to the promoter region of the predicted target murMN-SMU_718c operon via the putative binding site thus confirming that the latter is a transcriptional regulatory target of MbrC. The triangles indicate increasing concentrations of MbrC in the binding reactions. Black triangles followed by IR indicate target DNA fragments lacking the MbrC binding site.

Mentions: Among our predicted MbrC target genes, SMU_718c codes for a hypothetical protein with a haloacid dehalogenase-like domain, and SMU_716 and SMU_717 encode two different enzymes, MurN and MurM respectively. These enzymes catalyze the last steps of the peptidoglycan biosynthesis pathway and also play an important role in imparting resistance to cell wall-acting antibiotics [83–86]. Figures 6A and B illustrate the coexpression of mbrC with the murMN-SMU_718c operon genes and the presence of the potential MbrC binding site (TTACAA-AT-TTCTAC) upstream of the putative target murMN-SMU_718c operon respectively. This potential binding site differs from the motif consensus identified by Ouyang et al. [50] by the presence of two substitutions in the inverted repeat and is located upstream (between −33 and −20) of the transcriptional start site of the murMN-SMU_718c operon.Figure 6


Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

Sudhakar P, Reck M, Wang W, He FQ, Wagner-Döbler I, Dobler IW, Zeng AP - BMC Genomics (2014)

Experimental verification of the predicted transcriptional regulation of themurMN-SMU_718c operon by the response regulator MbrC. The predicted transcriptional regulatory relationship was based on a well correlated (A) expression profile between mbrC and the murMN-SMU_718 operon as well as the presence of a (B) putative MbrC binding site (TTACAA-AT-TTCTAC) in the upstream regulatory regions of the murMN-SMU_718 operon. The alignment among the MbrC binding sites in other experimentally verified targets (black) reported by Ouyang et al. [50] and the putative site upstream of the predicted target (red) murMN-SMU_718 operon is shown. The signature repeats of the MbrC binding motif are italicized, underlined and shown in bold. (C) Binding of MbrC to the promoter region of the gene SMU_1006 (positive control) was verified using Electro Mobility Shift Assays (EMSA), as already reported by Ouyang et al.[50]. (D) EMSA also provided the verification of the in-vitro binding of the MbrC protein to the promoter region of the predicted target murMN-SMU_718c operon via the putative binding site thus confirming that the latter is a transcriptional regulatory target of MbrC. The triangles indicate increasing concentrations of MbrC in the binding reactions. Black triangles followed by IR indicate target DNA fragments lacking the MbrC binding site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Experimental verification of the predicted transcriptional regulation of themurMN-SMU_718c operon by the response regulator MbrC. The predicted transcriptional regulatory relationship was based on a well correlated (A) expression profile between mbrC and the murMN-SMU_718 operon as well as the presence of a (B) putative MbrC binding site (TTACAA-AT-TTCTAC) in the upstream regulatory regions of the murMN-SMU_718 operon. The alignment among the MbrC binding sites in other experimentally verified targets (black) reported by Ouyang et al. [50] and the putative site upstream of the predicted target (red) murMN-SMU_718 operon is shown. The signature repeats of the MbrC binding motif are italicized, underlined and shown in bold. (C) Binding of MbrC to the promoter region of the gene SMU_1006 (positive control) was verified using Electro Mobility Shift Assays (EMSA), as already reported by Ouyang et al.[50]. (D) EMSA also provided the verification of the in-vitro binding of the MbrC protein to the promoter region of the predicted target murMN-SMU_718c operon via the putative binding site thus confirming that the latter is a transcriptional regulatory target of MbrC. The triangles indicate increasing concentrations of MbrC in the binding reactions. Black triangles followed by IR indicate target DNA fragments lacking the MbrC binding site.
Mentions: Among our predicted MbrC target genes, SMU_718c codes for a hypothetical protein with a haloacid dehalogenase-like domain, and SMU_716 and SMU_717 encode two different enzymes, MurN and MurM respectively. These enzymes catalyze the last steps of the peptidoglycan biosynthesis pathway and also play an important role in imparting resistance to cell wall-acting antibiotics [83–86]. Figures 6A and B illustrate the coexpression of mbrC with the murMN-SMU_718c operon genes and the presence of the potential MbrC binding site (TTACAA-AT-TTCTAC) upstream of the putative target murMN-SMU_718c operon respectively. This potential binding site differs from the motif consensus identified by Ouyang et al. [50] by the presence of two substitutions in the inverted repeat and is located upstream (between −33 and −20) of the transcriptional start site of the murMN-SMU_718c operon.Figure 6

Bottom Line: To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed.These sub-networks were significantly enriched with genes sharing common functions.Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21073 Hamburg, Germany. iwd@helmholtz-hzi.de.

ABSTRACT

Background: Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.

Results: The co-expression response network constructed from transcriptomic data using the reverse engineering algorithm called the Trend Correlation method consisted of 8284 gene pairs. The regulatory response network inferred by superimposing transcription factor binding site information into the co-expression network comprised 329 putative transcriptional regulatory interactions and could be classified into 27 sub-networks each co-regulated by a transcription factor. These sub-networks were significantly enriched with genes sharing common functions. The regulatory response network displayed global hierarchy and network motifs as observed in model organisms. The sub-networks modulated by the pyrimidine biosynthesis regulator PyrR, the glutamine synthetase repressor GlnR, the cysteine metabolism regulator CysR, global regulators CcpA and CodY and the two component system response regulators VicR and MbrC among others could putatively be related to the physiological effect of carolacton. The predicted interactions from the regulatory network between MbrC, known to be involved in cell envelope stress response, and the murMN-SMU_718c genes encoding peptidoglycan biosynthetic enzymes were experimentally confirmed using Electro Mobility Shift Assays. Furthermore, gene deletion mutants of five predicted key regulators from the response networks were constructed and their sensitivities towards carolacton were investigated. Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

Conclusion: The network approach used in this study revealed important regulators and interactions as part of the response mechanisms of S. mutans biofilm cells to carolacton. It also opens a door for further studies into novel drug targets against streptococci.

Show MeSH
Related in: MedlinePlus