Targets of the QseM Antiactivator in Mesorhizobium loti

Abstract

Quorum Sensing (QS) is a system used by bacteria to coordinate gene expression in response to population density using secreted diffusible signalling molecules, known as autoinducers. Many QS systems are similar to the model LuxR/I system originally discovered in Vibrio fischeri, where constitutive expression of the autoinducer synthase luxI produces acyl homoserine lactone molecules (AHLs) known as autoinducers at low levels. Once the population density reaches a threshold level, the regulator LuxR recognises and responds to the AHLs, activating downstream gene expression. These systems may also involve an antiactivator, that acts on the LuxR protein to prevent premature activation of the system by low AHL levels. Mesorhizobium loti strain R7A contains a mobile 502-kb symbiosis island known as ICEMlSymR7A which can transfer to nonsymbiotic mesorhizobia in both the laboratory and the environment. The excision and transfer of ICEMlSymR7A is directly controlled through QS via the actions of the the regulator TraR that acts in conjunction with AHL made by the autoinducer synthase TraI1. TraR activity in turn is controlled by the antiactivator QseM, through direct interaction with the TraR+AHL signalling molecule complex to block promoter activation. In this work, RT-qPCR was used to demonstrate that QseM had an effect on downstream TraR-regulated gene expression. Strong expression of the ICEMlSymR7A excisionase gene rdfS or the TrbC protease gene traF is known to have an inhibitory effect on cell growth. These genes are regulated by QS through the intermediacy of the msi172-msi171 gene product which is a single protein, FseA, that is produced by frame-shifting. A conjugation-based growth-inhibition assay involving introduction of a potentially lethal plasmid overexpressing target proteins into cells either overexpressing or not expressing QseM was developed to detect targets of QseM. The assay confirmed that TraR was a target of QseM and further suggested that FseA was a further target. RdfS and TraF were eliminated as targets. Bacterial two-hybrid analyses confirmed FseA as a target and narrowed the interacting portion down to the Msi172 portion of the frame-shifted protein. Furthermore β-galactosidase assays showed that FseA was unable to activate the rdfS promoter in the presence of QseM. Overall, this work confirmed the role of QseM as an antiactivator within the ICEMlSymR7A transfer system regulatory network and revealed it has more than one target. A 6-His tag was attached to QseM and a high concentration of protein was purified. Attempts at determining QseM interacants through Mass Spectrometry from a R7AΔqseM lysate proved difficult despite distinct bands being seen. QseM was subjected to circular dichroism that inferred that QseM is composed solely of α-helices, as is TraM, an antiactivator that targets TraR from the Agrobacterium tumefaciens QS system.