Genetic and functional integration of SrcA with type III secretion 
The genes encoding the srcA chaperone and the effector cargos (pipB2 and sseL) are found in all serotypes of Salmonella enterica that contain the SPI-2-encoded T3SS.
Conversely, these genes are absent from S. bongori, which lacks the SPI-2-encoded T3SS.
The expression of srcA is coordinated with T3SS transcriptional activity via the SsrA-SsrB two-component regulatory system encoded in SPI-2.
The direct binding of SsrB to the promoter region upstream of srcA, along with SsrB-regulation of both sseL [19],[20] and pipB2 [32] is indicative of multiple cis-regulatory mutation events that have allowed for functional coordination of the distributed secretion apparatus, chaperone and effector cargos.
We recently described this type of regulatory evolution for pathogenic adaptation of Salmonella to its host [21] and srcA is consistent with regulatory evolution of chaperone-effector gene pairs that are not co-transcribed in operons.
Due to low G+C base content compared to the genome average of 52%, it's likely that srcA (32% G+C) and an adjacent gene, STM2137 (37% G+C), were acquired as a foreign islet that was retained in organisms containing the SPI-2 T3SS due to the selective advantage afforded by the new protein interactions so created.
Interestingly, STM2137 (also known as SseK2) is a likely paralog of SseK1, an effector translocated by the SPI-2 T3SS [33].
SseK2 is also regulated by the SsrA-SsrB two-component system but compared to SseK1, it is translocated in much less abundance into host cells [33].
Using the methods described here, we were not able to detect SseK2 secretion or a physical interaction with SrcA, however it remains possible that SrcA also chaperones SseK2 for low-level translocation.
SrcA is unique among other multi-effector chaperones most closely related to it in that it is unlinked from the T3SS genomic island.
For example, InvB and SicP (Salmonella SPI-1), CesT (enteropathogenic E. coli locus of enterocyte effacement) and Spa15 (Shigella mxi/spa virulence plasmid region) chaperones are all encoded within the T3SS structural operons, implying they have co-evolved as a single genetic entity from a common ancestor.
Given its genetic neighborhood, srcA appears to be a genetic acquisition separate from SPI-2 that functionally links some effectors to the T3SS apparatus via the ATPase.
The role of horizontal gene transfer and regulatory evolution in allowing for uncoupling of chaperones, effectors and the T3SS has many possible implications for T3SS function, including plasticity in chaperone-effector interaction networks, expansion of effector repertoires, and alterations to the kinetics and hierarchical delivery of effectors to a host cell.
These events may improve host adaptability or even expand the host range of bacteria that acquire and integrate new functional secretion chaperones.
