Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.     

Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.     

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.     

Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system

F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.     

The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.     

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.