Abstract
Quorum sensing (QS) is a form of communication used by bacteria to initiate population-wide changes in gene expression, enabling modulation of phenotypes that maximise fitness. Synthetic gene circuits, which often employ QS systems to synchronise a population’s gene expression, are designed to approximate electronic circuits which produce predictable outcomes. However, QS systems are vulnerable to stochastic variations in gene expression, termed biological noise, which can cause genetic circuits to behave unpredictably. Few components capable of insulating biological noise are available. The research presented here is part of a larger study that aims to develop noise-isolating components for QS-integrated synthetic biological circuits and to uncover the mechanisms of novel proteins governing integrative and conjugative element (ICE) transfer.
Initiation of transfer of the Mesorhizobium japonicum R7A symbiosis ICE is dependent on the RdfS protein, which induces excision. Expression of the rdfS operon is controlled by a multi-component regulatory circuit, which is overarched by a QS system. Duplication of a QS gene linked the QS system to a novel regulatory circuit that includes two proteins containing the domain of unknown function (DUF) 2285. One of these proteins, FseA, activates expression of the rdfS operon. The other, QseM, is an antiactivator of both FseA and the QS transcriptional activator TraR. In this work, we aimed to determine how FseA interacts with QseM, with the ultimate aims of defining an FseA sequence-tag that can be used to place recombinant proteins under QseM control, and to determine how FseA activates transcription of the rdfS promoter.
An initial investigation of FseA residues critical to rdfS promoter activation suggested FseA was prone to destabilisation. Moreover, past experiments showed recombinant FseA expression produced little soluble protein.
Maltose-binding protein (MBP)-fused FseA and FseA mutants appeared to gain in stability/solubility, leading to the identification of seven FseA residues critical for rdfS promoter activation. To identify FseA residues critical for QseM antiactivation, regions of interest were explored by protein-protein interaction studies. The experiments revealed that QseM binds FseA amino acids in the region 15-55 and specifically to the amino-acid Arg37.
An inverted repeat (IR) separated by ~2 DNA turns was previously identified upstream of the core rdfS promoter. To determine the role of this IR, rdfS promoter mutations were tested in vivo for FseA-dependent activation. The results showed that the IR likely binds specifically to FseA for promoter activation and possibly negatively regulates rdfS expression.
An accurate FseA structure was determined using computational modelling with a curated FseA database. Analysis of the structure revealed that the C-terminus formed a novel helix-turn-helix (HTH) domain, the turn of which was extended and featured an additional α-helix. Furthermore, the FseA structure supported the FseA-QseM interaction results. Purification of MBP-FseA revealed FseA likely forms a dimer and enabled in vitro assays. Additional FseA mutagenesis coupled with in vitro DNA-binding assays demonstrated the HTH domain bound the rdfS promoter IR sequence.
In summary, this study provided insight into the DNA-binding capability of DUF2285-containing transcriptional activators and the mechanism of QseM antiactivation. FseA amino-acids 15-55 were identified as a starting candidate for developing a QseM-targetable FseA sequence tag.