ScmR, a Global Regulator of Gene Expression, Quorum Sensing, pH Homeostasis, and Virulence in Burkholderia thailandensis

Abstract

The nonpathogenic soil saprophyte Burkholderia thailandensis is a member of the Burkholderia pseudomallei/B. thailandensis/B. mallei group, which also comprises the closely related human pathogens B. pseudomallei and Burkholderia mallei responsible for the melioidosis and glanders diseases, respectively. ScmR, a recently identified LysR-type transcriptional regulator in B. thailandensis, acts as a global transcriptional regulator throughout the stationary phase and modulates the production of a wide range of secondary metabolites, including N-acyl-l-homoserine lactones and 4-hydroxy-3-methyl-2-alkylquinolines and virulence in the Caenorhabditis elegans nematode worm host model, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in B. thailandensis strain E264 during the exponential phase. We used RNA sequencing transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR using quantitative reverse transcription-PCR or mini-CTX-lux transcriptional reporters, including the oxalate biosynthetic gene obc1 required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, and the bsa (Burkholderia secretion apparatus) type III secretion system genes essential for both B. pseudomallei and B. mallei pathogenicity, as well as the scmR gene itself. We confirmed that the transcription of scmR is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrated that ScmR influences virulence using the fruit fly model host Drosophila melanogaster. We conclude that ScmR represents a central component of the B. thailandensis QS regulatory network. IMPORTANCE Coordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the LysR-type transcriptional regulator, ScmR, which is highly conserved and involved in the control of virulence/survival factors in the Burkholderia genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in B. thailandensis, as well as QS independently. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.

Quorum Sensing Controls Both Rhamnolipid and Polyhydroxyalkanoate Production in Burkholderia thailandensis Through ScmR Regulation

“…Extraction of total RNA from cultures of B. thailandensis E264 and btai123, btaI1, btaI2, btaI3 mutants at 24, 72, and 96 h, cDNA synthesis and amplification were done as before (Le Guillouzer et al, 2020). The scmR gene was amplified using primers SLG_qRThmqR_F…”

“…This gene displayed stable expression in the various genetic contexts (Supplementary Figure S1). The primers used were the same as in our previous studies SLG_qRT_ndh_F (5 -ACCAGGGCGAATTGATCTC-3 ) and SLG_qRT_ndh_R (5 -GATGACGAGCGTGTCGTATT-3 ) (Le Guillouzer et al, 2017Guillouzer et al, , 2018Guillouzer et al, , 2020. Differences in gene expression between B. thailandensis E264 strains were calculated using the 2 − CT formula (Livak and Schmittgen, 2001).…”

“…TnSeq analysis of M63 generated one unique sequence ( Supplementary Table S1); this mutant has a transposon inserted in the BTH_I1403 locus, which encodes the LysRtype transcriptional regulator ScmR. Interestingly, this regulator influences the production of several secondary metabolites in B. thailandensis E264 (Mao et al, 2017;Le Guillouzer et al, 2020). To investigate the impact of this mutation further, we performed cultures of a previously characterized scmR-mutant (ED1023, Table 1; Le Guillouzer et al, 2020) in baffled flasks containing NB medium with 2% glycerol and samples were collected daily.…”

“…We demonstrated that ScmR is involved in both rhamnolipid and PHA biosynthesis. QS activates the transcription of the scmR gene in cultures of B. thailandensis grown in TSB (Le Guillouzer et al, 2020). Also, concentrations of all three AHLs are reduced in a scmR-mutant compared to the WT (Mao et al, 2017) when B. thailandensis is cultured in LB medium.…”

“…In doing so, we identified ScmR as a modulator of the biosynthesis of these metabolites in B. thailandensis. Since this regulator was also reported to be QS-controlled and to impact the production of AHL signals (Majerczyk et al, 2014a,b;Mao et al, 2017;Le Guillouzer et al, 2020), we further investigated the comodulation of the biosynthesis of PHA and rhamnolipids by QS and ScmR in B. thailandensis.…”