Crucial Role ofSalmonellaGenomic Island 1 Master Activator in Parasitism of IncC plasmids

Abstract

IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1’s lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters in both SGI1 and IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential for the excision and replication of SGI1, and the destabilisation of the helper IncC plasmid.

Salmonella Genomic Island 1 requires a self‐encoded small RNA for mobilization

“…SGI1‐family elements are sophisticated parasites of IncA and IncC plasmids as they do not simply utilize the plasmid‐encoded T4SS but exploit the control mechanisms of the helper plasmid for timing of their excision and plasmid‐like replication (Kiss et al., 2015; Szabó et al., 2021). They modify the T4SS for their own benefit (Carraro et al., 2017) and destabilize the helper (Durand et al, 2021b; Harmer et al., 2016; Szabó et al., 2021), possibly in order to ensure the stable vertical transfer. These interventions increase the efficiency of SGI1 transfer and the stability of the island in the host bacteria when coexisting with the helper plasmid.…”

“…SGI1 encodes an FlhDC‐family activator and three T4SS components that are related to and can substitute their helper‐encoded homologues. These SGI1 derived proteins have important roles in the parasitism of SGI1 on the helper plasmid (Carraro et al., 2017; Durand et al, 2021b).…”

“…These broad host range plasmids are prevalent in Gram‐negatives and seem to be key players in the distribution of many AR genes including novel metallo‐β‐lactamase genes among clinical isolates of Enterobacteriaceae (Wu et al., 2019; Zhang et al., 2020; Zheng et al., 2020). The conjugative system of IncA and IncC plasmids is classified into the MOB H12 group (Garcillán‐Barcia et al., 2009) and controlled by the FlhDC‐family master activator, AcaCD, which activates 18 promoters in the IncC backbone, including promoters of the entire conjugative apparatus and several genes of unknown functions (Carraro et al., 2014; Durand et al, 2021b). SGI1 exploits this conjugative system and its control mechanisms in multiple ways.…”

“…Furthermore, SGI1 also encodes an FlhDC‐family activator, FlhDC SGI1 (also known as SgaDC) (Kiss et al., 2015), which acts on the AcaCD‐responsive promoters of SGI1 and the IncC plasmids (Murányi et al., 2016). FlhDC SGI1 encroaches on the regulatory circuits of IncC transfer and appears to have a key role in the parasitism by SGI1 on IncC plasmids (Durand et al, 2021a, 2021b; Kiss et al., 2015; Szabó et al., 2021).…”

Conjugative IncC plasmid entry triggers the SOS response and promotes effective transfer of the integrative antibiotic resistance element SGI1

“…Moreover, IncC plasmids and SGI1 share a complex transcriptional regulatory network, each element having homologous master activators, i.e. AcaDC and SgaDC, respectively, which have been shown to activate the same regulons in both elements (15, 18, 19, 21). Durand et al recently demonstrated that SgaDC and AcaDC activate the expression of AcaB, an IncC transcriptional regulator which in turn in cooperation with AcaDC have been shown to trigger the expression of the conjugative IncC machinery through a positive feedback loop of mutual expression (18, 24).…”