A plasmid-encoded H-NS protein selectively binds its own plasmid

Abstract

H-NS is an abundant nucleoid-associated protein found in Enterobacterales species. Some conjugative plasmids encode H-NS homologues, which are thought to facilitate plasmid maintenance and reduce the fitness costs associated with plasmid carriage. Here, we characterize HppXCROD2, an H-NS homologue encoded by the IncX4 plasmid pCROD2 of Citrobacter rodentium. Our data indicate that HppXCROD2 has a strong preference for binding pCROD2 over the chromosome or other plasmids. By contrast, chromosomally encoded H-NS displays no preference for plasmid sequence. When expressed from a heterologous plasmid in Escherichia coli, HppXCROD2 showed similar DNA-sequence preference to chromosomally encoded H-NS. Moreover, HppXCROD2 binding to a sequence from pCROD2 was much lower when that sequence was cloned in a laboratory plasmid. Thus, HppXCROD2 preferentially binds DNA in the context of the plasmid where it is encoded, a phenomenon we term "cognate plasmid specificity". We propose that cognate plasmid specificity occurs through recognition of plasmid-specific DNA topology generated by plasmid-encoded topoisomerases. Cognate plasmid specificity may insulate regulation of plasmid genes from the effects of host DNA, while minimizing disruption of host chromosome regulation due to plasmid carriage. IMPORTANCEMany bacteria carry conjugative plasmids, mobile DNA molecules that spread traits such as antibiotic resistance. Some conjugative plasmids encode proteins related to the bacterial DNA-binding protein H-NS. We show that an H-NS-like protein from the IncX4 plasmid pCROD2 binds almost exclusively to the plasmid from which it originates, while largely ignoring the host chromosome. Our findings reveal a previously unrecognized mechanism that allows plasmids to regulate their own genes with high specificity while minimizing interference with host gene expression.