The Yersinia enterocolitica pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C

Abstract

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (≤30°C) and virulence plasmid-encoded Yop secretion (37°C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37°C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the positive activator of virulence genes. These bends are detectable at 30°C but melt at 37°C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.

RovA is autoregulated and antagonizes H‐NS‐mediated silencing of invasin and rovA expression in Yersinia pseudotuberculosis

“…It has been shown that a transition from 25∞C to 37∞C induces structural alterations, e.g. relaxation and melting of DNA bends, and these changes can reduce the interaction of H-NS, which no longer prevents the formation of a productive transcriptional complex and allows the expression of virulence genes at higher temperatures (Falconi et al, 1998;Rohde et al, 1999;Madrid et al, 2002;Prosseda et al, 2004). The inv and rovA promoters both contain long and flexible AT-rich sequences, and temperature-dependent changes in the DNA flexibility might trigger a structural transition that modulates the accessibility to H-NS in reverse to the usual situation in other virulence systems.…”

The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo

“…In contrast to yspP, yopH appears to have similar expression levels in both spent DMEM and during HeLa cell infection, which would appear to contradict the results shown in Figure 2. It should be noted, however, that expression of the Ysc T3SS has been shown to be induced at 37°C, due to changes in the secondary structure of the virulence plasmid (Rohde et al 1999) and the ribosome-binding site of the transcriptional activator LcrF (Bohme et al 2012) at that temperature.…”

Thermoregulation of the Escherichia coli O157:H7 pO157 ecf operon and lipid A myristoyl transferase activity involves intrinsically curved DNA

“…DNA with a bent trajectory in its helical axis that is induced solely by innate nucleotide sequences, such as phased homopolymeric adenine:thymine tracts (AT‐tracts), is called intrinsically curved DNA (Ohyama, 2001). We showed previously that multiple intrinsically curved DNAs are present in the virulence plasmids of Yersinia enterocolitica , enteropathogenic E. coli and enterohaemorrhagic E. coli (Rohde et al ., 1999).…”