Construction and verification of the transcriptional regulatory response network of Streptococcus mutansupon treatment with the biofilm inhibitor carolacton

Abstract

BackgroundCarolacton 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.ResultsThe 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-362) contains supplementary material, which is available to authorized users.

Stress Physiology of Lactic Acid Bacteria

“…A later study identified four additional genes that belong to the MbrABRS regulon, including those for the cell surface antigen SpaP and for MurM and MurN, which are alanine-adding enzymes involved in the branched-peptide PG biosynthetic pathway. These observations suggest that MbrABRS plays an important role in the cell envelope stress response of S. mutans (388).…”

Streptococcus mutans copes with heat stress by multiple transcriptional regulons modulating virulence and energy metabolism

“…VicR is the response regulator of the vicKR TCS system, which is essential for the survival of S. mutans and plays a major role in the stress response, competence development, sugar metabolism and biofilm formation 27 . A previous study found that vicR was an essential response regulator of S. mutans upon treatment with carolacton (a biofilm inhibitor) by regulating the expression of gtfB , gtfD and gbpB 28 . In this work, we observed a set of genes, including gene encoding glucosyltransferase-SI ( gtfC ), transcriptional regulators ( malR and smu_439 ), gene encoding endolysin ( smu_707c ) and UDP-N-acetylglucosamine 2-epimerase ( epsC ), were regulated by vicR in S. mutans under heat stress ( Fig.…”

“…The microarray data were confirmed using q RT-PCR with the expression of eight transcripts using a previously published method and the primers in Table S6 52 55 . By integrating the transcript units information 56 , promoter binding motifs 28 , and experimental validation, we summarized heat stress-induced regulons of S. mutans (see Table S5 for details).…”

Collaboration in Natural Product Total Synthesis: Carolacton – A Decade of Discovery

“…8 Further evidence for transcriptional regulation interference was found in 2014 when Sudhakar et al published that CodY, PyR, GlnR, CysR, CcpA, and Mbr were manipulated by carolacton, and reconfirmed VicKR's connection. 9 Then, in 2017, Muller and co-workers identified folate dehydrogenase (FolD) as the target of carolacton in an efflux deficient E. coli strain and confirmed the target in Streptococcus pneumoniae planktonic inhibition assays. 10 However, the connection between FolD and the anti-biofilm activity has yet to be explained.…”