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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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Effect of deleting five “key” transcriptional regulators and sensitivity of thecysRdeletion mutant to carolacton treatment. (A) Inhibition of viability caused by carolacton treatment was tested for the biofilms of gene deletion mutants of 5 key transcriptional regulators identified from network analysis. The inhibition of viability was determined by live/dead staining of 20 h-old static biofilms of mutant, wild type and cysR complementation strains under carolacton treatment and is expressed as inhibition of the green/red fluorescence ratio. The bars show the mean of three independent biological replicates. (B) The effect of carolacton treatment on the number of colony forming units (cfu’s) of biofilms of the S. mutans UA159 wildtype and cysR gene deletion mutant was also investigated. The Cfu experiment was repeated in two biological replicates. The sequences of primers used for generating the deletion mutants are given in Additional file 9.
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Fig7: Effect of deleting five “key” transcriptional regulators and sensitivity of thecysRdeletion mutant to carolacton treatment. (A) Inhibition of viability caused by carolacton treatment was tested for the biofilms of gene deletion mutants of 5 key transcriptional regulators identified from network analysis. The inhibition of viability was determined by live/dead staining of 20 h-old static biofilms of mutant, wild type and cysR complementation strains under carolacton treatment and is expressed as inhibition of the green/red fluorescence ratio. The bars show the mean of three independent biological replicates. (B) The effect of carolacton treatment on the number of colony forming units (cfu’s) of biofilms of the S. mutans UA159 wildtype and cysR gene deletion mutant was also investigated. The Cfu experiment was repeated in two biological replicates. The sequences of primers used for generating the deletion mutants are given in Additional file 9.

Mentions: The susceptibility to carolacton treatment of 20 h-old static biofilms of the corresponding key regulator gene deletion mutants was tested using Live/Dead viability staining. As shown in Figure 7A, carolacton only marginally reduced the viability (~5-10% inhibition) of the biofilms of the cysR gene deletion strain, while complementation of cysR in trans fully restored the carolacton sensitive phenotype of the wildtype (approximately 45-55% inhibition of viability). It was previously shown that carolacton exclusively damages growing biofilms [5]. The observed strong loss in sensitivity of the cysR gene deletion strain to carolacton was not biased due to poor or significantly slower growing mutant biofilms since the final growth yield and doubling time of the cysR gene deletion mutant was only slightly reduced in comparison to the wildtype (data not shown). Strikingly, the spxA gene deletion strain displayed impaired ability to grow under acidic conditions [92], a condition known to be essential for the carolacton induced membrane damage in S. mutans[5]. However, all the tested strains with the fabT or cysR deletion showed similar susceptibilities to carolacton relative to the wildtype (Figure 7A). For the fabT deletion mutant though, no final conclusion regarding its sensitivity to carolacton treatment could be drawn since the mutant strain grew very poorly under the tested conditions and formed very thin biofilms. Since growth is a prerequisite for the membrane damage caused by carolacton treatment [5], the results of the Live/Dead staining for the fabT mutant should be interpreted with caution. However, the membrane integrity of the fabT deletion mutant was highly compromised (data not shown), as determined by live/dead staining. The importance of fabT for maintaining membrane integrity in S. mutans biofilms might also be indicative of its involvement in the membrane damage caused by carolacton treatment. Furthermore, fabT was identified as a top structural hub of the inferred co-expression network, indicating its essential physiological role, and the deletion of which is therefore expected to have a high chance to cause poor growth.Figure 7


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)

Effect of deleting five “key” transcriptional regulators and sensitivity of thecysRdeletion mutant to carolacton treatment. (A) Inhibition of viability caused by carolacton treatment was tested for the biofilms of gene deletion mutants of 5 key transcriptional regulators identified from network analysis. The inhibition of viability was determined by live/dead staining of 20 h-old static biofilms of mutant, wild type and cysR complementation strains under carolacton treatment and is expressed as inhibition of the green/red fluorescence ratio. The bars show the mean of three independent biological replicates. (B) The effect of carolacton treatment on the number of colony forming units (cfu’s) of biofilms of the S. mutans UA159 wildtype and cysR gene deletion mutant was also investigated. The Cfu experiment was repeated in two biological replicates. The sequences of primers used for generating the deletion mutants are given in Additional file 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: Effect of deleting five “key” transcriptional regulators and sensitivity of thecysRdeletion mutant to carolacton treatment. (A) Inhibition of viability caused by carolacton treatment was tested for the biofilms of gene deletion mutants of 5 key transcriptional regulators identified from network analysis. The inhibition of viability was determined by live/dead staining of 20 h-old static biofilms of mutant, wild type and cysR complementation strains under carolacton treatment and is expressed as inhibition of the green/red fluorescence ratio. The bars show the mean of three independent biological replicates. (B) The effect of carolacton treatment on the number of colony forming units (cfu’s) of biofilms of the S. mutans UA159 wildtype and cysR gene deletion mutant was also investigated. The Cfu experiment was repeated in two biological replicates. The sequences of primers used for generating the deletion mutants are given in Additional file 9.
Mentions: The susceptibility to carolacton treatment of 20 h-old static biofilms of the corresponding key regulator gene deletion mutants was tested using Live/Dead viability staining. As shown in Figure 7A, carolacton only marginally reduced the viability (~5-10% inhibition) of the biofilms of the cysR gene deletion strain, while complementation of cysR in trans fully restored the carolacton sensitive phenotype of the wildtype (approximately 45-55% inhibition of viability). It was previously shown that carolacton exclusively damages growing biofilms [5]. The observed strong loss in sensitivity of the cysR gene deletion strain to carolacton was not biased due to poor or significantly slower growing mutant biofilms since the final growth yield and doubling time of the cysR gene deletion mutant was only slightly reduced in comparison to the wildtype (data not shown). Strikingly, the spxA gene deletion strain displayed impaired ability to grow under acidic conditions [92], a condition known to be essential for the carolacton induced membrane damage in S. mutans[5]. However, all the tested strains with the fabT or cysR deletion showed similar susceptibilities to carolacton relative to the wildtype (Figure 7A). For the fabT deletion mutant though, no final conclusion regarding its sensitivity to carolacton treatment could be drawn since the mutant strain grew very poorly under the tested conditions and formed very thin biofilms. Since growth is a prerequisite for the membrane damage caused by carolacton treatment [5], the results of the Live/Dead staining for the fabT mutant should be interpreted with caution. However, the membrane integrity of the fabT deletion mutant was highly compromised (data not shown), as determined by live/dead staining. The importance of fabT for maintaining membrane integrity in S. mutans biofilms might also be indicative of its involvement in the membrane damage caused by carolacton treatment. Furthermore, fabT was identified as a top structural hub of the inferred co-expression network, indicating its essential physiological role, and the deletion of which is therefore expected to have a high chance to cause poor growth.Figure 7

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