RsaM: a transcriptional regulator of Burkholderia spp. with novel fold

Abstract

Burkholderia cepacia complex (Bcc) is a set of closely related bacterial species that are notorious pathogens of cystic fibrosis patients, responsible for life-threatening lung infections. Expression of several virulence factors of Bcc is controlled by a mechanism known as quorum sensing (QS). QS is a means of bacterial communication used to coordinate gene expression in a cell-density–dependent manner. The system involves the production of diffusible signaling molecules (N-acyl-L-homoserine lactones, AHLs), that bind to cognate transcriptional regulators and influence their ability to regulate gene expression. One such system that is highly conserved in Bcc consists of CepI and CepR. CepI is AHL synthase, while CepR is an AHL-dependent transcription factor. In most members of the Bcc group, the cepI and cepR genes are divergently transcribed and separated by additional genes. One of them, bcam1869, encodes the BcRsaM protein, which was recently postulated to modulate the abundance or activity of CepI or CepR. Here we show the crystal structure of BcRsaM from B. cenocepacia J2315. It is a single-domain protein with unique topology and presents a novel fold. The protein is a dimer in the crystal and in solution. This regulator has no known DNA binding motifs and direct binding of BcRsaM to the cepI promoter could not be detected in in vitro assays. Therefore, we propose that the modulatory action of RsaM might result from interactions with other components of the QS machinery rather than from direct association with the DNA promoter.

TworsaMhomologues encode central regulatory elements modulating quorum sensing expression inBurkholderia thailandensis

“…The btaI 1 ( BTH_II1512 ) and btaR 1 ( BTH_II1510 ) genes, respectively encoding the BtaI1 AHL synthase and the BtaR1 transcriptional regulator of the B. thailandensis E264 QS-1 system, are separated by the BTH_I1511 gene that codes for an hypothetical protein conserved in the Burkholderia genus (7, 10, 11, 21-23). This hypothetical protein of 147 amino acids is similar to RsaM-like proteins and displays 35.8% identity with the negative AHL biosynthesis modulatory protein RsaM of the phytopathogen P. fuscovaginae UPB0736 ( Fig.…”

“…Additionally, these AHL biosynthesis regulatory proteins were shown to be an integral part of the QS modulatory circuitry, contributing to the temporal expression of the multiple AHL-based QS circuits of B. thailandensis E264. The precise underlying molecular mechanism of action of RsaM-like proteins remains currently unknown and has to be further investigated in the future given their importance in the regulation of QS-controlled genes in the Burkholderia genus and other Proteobacteria (7-11, 21-23).…”

“…A gene conserved in the Burkholderia genus within the QS-1 system (7, 10, 11, 21-23) is divergently transcribed from btaR 1 and oriented in the same direction as btaI 1 (Fig. 8 and Fig.…”

“…This gene encodes an hypothetical protein, homologous to the negative AHL biosynthesis modulatory protein RsaM originally identified in the plant pathogen P. fuscovaginae UBP0736 (8, 9), hence designated RsaM1. In B. cenocepacia H111, the homologue Bc RsaM was described as an important repressor of the CepI/CepR QS system, and proposed to inhibit the production of C 8 -HSL by regulating the activity and/or stability of the LuxI-type synthase CepI and the LuxR-type transcriptional regulator CepR, as well as the orphan LuxR-type transcriptional regulator CepR2 (10, 11). While the transcription of the QS cepI , cepR , and cepR 2 genes were shown to be lowered in the rsaM -mutant of B. cenocepacia H111 (10), btaI 1 and btaR 1 expressions were both increased in our RsaM1 of B. thailandensis E264, correlating with the accumulation of C 8 -HSL observed in this background.…”

“…The function of RsaM-like proteins has been investigated in the Burkholderia genus, and could be important for balancing and fine-tuning of QS-dependant regulation (10). RsaM-like proteins do not possess any sequence similarity with biochemically or structurally characterized proteins, such as DNA-binding motifs, and constitute single-domain proteins with unique topology presenting a novel fold (11). Their precise underlying regulatory mechanism is currently unknown.…”