Growth phase‐dependent control of R27 conjugation is mediated by the interplay between the plasmid‐encoded regulatory circuit TrhR/TrhY‐HtdA and the cAMP regulon

Abstract

Plasmids of the incompatibility group HI1 (IncHI1) have been isolated from several Gram-negative pathogens and are associated with the spread of multidrug resistance. Their conjugation is tightly regulated and it is inhibited at temperatures higher than 30°C, indicating that conjugation occurs outside warm-blooded hosts. Using R27, the prototype of IncHI1 plasmids, we report that plasmid transfer efficiency in E. coli strongly depends on the physiological state of the donor cells. Conjugation frequency is high when cells are actively growing, dropping sharply when cells enter the stationary phase of growth. Accordingly, our transcriptomic assays show significant downregulation of numerous R27 genes during the stationary phase, including several tra (transfer) genes. Growth phase-dependent regulation of tra genes transcription is independent of H-NS, a silencer of horizontal gene transfer, and ppGpp and RpoS, regulators of the stationary phase, but highly dependent on the plasmid-encoded regulatory circuit TrhR/TrhY-HtdA. The metabolic sensor cAMP, whose synthesis is chromosomally encoded, is also involved in the growth phase regulation of R27 conjugation by modulating htdA expression. Our data suggest that the involvement of regulators encoded by both chromosome and plasmid are required for efficient physiological control of IncHI1 plasmid conjugation.

Growth‐stage‐dependent regulation of conjugation

“…Surprisingly, 5 mM cAMP added throughout the growth did not significantly change the conjugation efficiency in Δcya background and did not restore the growth‐dependent regulation (Figure S2). Such observation disagrees with some prior studies showed that Δcya ‐dependent regulation deficiency in mating of IncF pRK100 and IncH R27 plasmids was overcome by adding extracellular cAMP . Nevertheless, a nonmonotonic response of F‐conjugation to cAMP was recorded before and may reflect a complex nature of the cAMP regulation …”

“…We found that the transcripts for the essential transfer components were elevated in the stationary state donor cells (Figure a). Similar observations correlating mRNA levels for essential components and conjugation efficiency were previously reported for prototypic F plasmids and R27 IncH system . Therefore, we concluded that levels of the transfer proteins indeed correlate with the observed levels of the transcripts for the growth‐dependent conjugative systems.…”

“…. In plasmids pRK100 and R27, growth‐stage dependence was attributed to intricate transcription regulation via plasmid‐encoded factors (TraJ and TrhR/TrhY‐HtdA, respectively) and chromosomally encoded metabolism . For plasmids F, pRK100, and R27 it was shown previously that cyclic AMP (cAMP) regulation is important for growth‐stage dependence .…”

“…Interestingly, for pSLT plasmid no conjugative repression in the stationary phase was observed . Another Enterobacteriaceae plasmid R27 was found to be strongly downregulated in conjugative transfer during stationary phase . In addition, indirect data suggested growth‐stage‐dependent regulation of plasmid pRK100, for which the central transfer region transcription activator TraJ was found to be upregulated in glucose‐deficient and high cAMP donor states .…”

Crosstalk between bacterial conjugation and motility is mediated by plasmid‐borne regulators

“…Its complete nucleotide sequence is available, and the replication and conjugation determinants are well characterized (Taylor et al, ; Couturier et al, ; Maher et al, ; Gabant et al, ; Newnham and Taylor, ; Sherburne et al, ; Lawley et al, , ; Alonso et al, ). The interplay between the R27 plasmid and the bacterial chromosome has been studied, and functional interactions between regulators from both genetic elements have been characterized (Doyle et al, ; Baños et al, ; Dillon and Dorman, ; Gibert et al, , ). Moreover, the impact of the presence of R27 on the global expression profile of Salmonella under some environmental conditions has been described (Paytubi et al, ).…”