Replication of the Salmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss

Abstract

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis . SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC + cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 ( rep ) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.

IncC helper dependent plasmid-like replication of Salmonella Genomic Island 1

“…This arrangement of oriV is characteristic for the iteron-containing replicons ( 50 ). Similar conclusions have been reported on the SGI1-derived replicon in a very recent study published after the submission of this work ( 51 ). Both repA and the cis elements of oriV are well conserved in the IncN2/N3 group of plasmids having closely related replicon to that of SGI1 ( Supplementary Figure S2 ).…”

“…In contrast, we have documented that SGI1 exists as a circular extrachromosomal plasmid-like element in a host also carrying an IncC plasmid and that SGI1 replicates transiently in a helper-induced manner. In this situation, SGI1 is maintained in 4–8 extrachromosomal copies due to the AcaCD-induced expression of RepA as has also been reported recently ( 51 ). Therefore, the copy number of SGI1 significantly exceeds that of the IncC helpers, which are known to be very low or even single-copy plasmids ( 55 ).…”

“…The only exception is the chromosome of the S. enterica serovar Kentucky strain VNSEC0 (CP039439), where an SGI1-like element showing less than 40% coverage to the SGI1 backbone also contains the homolog of SGI1 rep region. A search for more divergent SGI1-related elements revealed two SGI1-like GIs sharing a similar structure to SGI1, but having a different rep gene ( 51 ). These elements, GI Vch O27–1 and GI Sen -26, identified in V .…”

“…enterica serovar Muenster 26 strains, respectively, are also located at the 3′ end of trmE and show 94% identity to each other but have much lower sequence similarity to SGI1. Their repA is unrelated to that of the IncN2/N3 group and encodes a Rep-Primase family protein, whose homologs occur also in several SGI1-related elements found in P. mirabilis , S. enterica serovars and some other species ( 51 ). The repA of GI Vch O27–1 and GI Sen -26, like in SGI1, is preceded by an S004 homolog whose putative protein product shares only 33% identity to S004 of SGI1.…”

Antibiotic Resistance in Vibrio cholerae: Mechanistic Insights from IncC Plasmid-Mediated Dissemination of a Novel Family of Genomic Islands Inserted at trmE

“…In SGI1, AcaCD binding sites have been identified upstream of xis, S004-rep, traN S , traHG S , and S018 and corresponding promoters shown to respond to AcaCD activation (9,17,(45)(46)(47). In MGIVchHai6, putative AcaCD binding sites were predicted at the 5= end of 84 and upstream of an operon-like gene cluster containing xis, which encodes a putative protein sharing only 37% identity with the RDF Xis of SGI1 (13).…”