Self-Perpetuating Epigenetic Pili Switches in Bacteria
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Colloquium Self-perpetuating epigenetic pili switches in bacteria Aaron Hernday*, Margareta Krabbe†, Bruce Braaten*, and David Low*‡ *Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93117; and †Karolinska Institutet, SE-171 77 Stockholm, Sweden Bacteria have developed an epigenetic phase variation mechanism three helical turns apart which control transcription at the papBA to control cell surface pili–adhesin complexes between heritable promoter. Two DNA GATC sites designated GATCprox and expression (phase ON) and nonexpression (phase OFF) states. In GATCdist (proximal and distal with respect to the papBA the pyelonephritis-associated pili (pap) system, global regulators promoter) are located within Lrp binding sites 2 and 5, respec- [catabolite gene activator protein (CAP), leucine-responsive regu- tively. DNA GATC sites are target sites for Dam, which methy- latory protein (Lrp), DNA adenine methylase (Dam)] and local lates the adenosine of the GATC sequence. regulators (PapI and PapB) control phase switching. Lrp binds Mutational analyses showed that disruption of Lrp binding cooperatively to three pap DNA binding sites, sites 1–3, proximal sites 2 or 3 resulted in a higher OFF to ON switch rate or a phase to the papBA pilin promoter in phase OFF cells, whereas Lrp is locked ON phenotype, respectively. In contrast, disruption of bound to sites 4–6 distal to papBA in phase ON cells. Two Dam Lrp sites 4 or 5 resulted in a phase locked OFF phenotype (Fig. methylation targets, GATCprox and GATCdist, are located in Lrp 1) (8). These results suggested that binding of Lrp proximal to binding sites 2 and 5, respectively. In phase OFF cells, binding of Lrp at sites 1–3 inhibits methylation of GATCprox, forming the phase the papBAp promoter inhibits transcription whereas binding of OFF DNA methylation pattern (GATCdist methylated, GATCprox Lrp at the distal site activates transcription. In vitro DNA nonmethylated). Binding of Lrp at sites 1–3 blocks pap pili tran- footprint analyses indicated that Lrp binds with highest affinity scription and reduces the affinity of Lrp for sites 4–6. Together to Lrp sites 1–3, and with lower affinity to sites 4–6 (8–10). with methylation of GATCdist, which inhibits Lrp binding at sites Insertion or deletion of a single base pair between Lrp binding 4–6, the phase OFF state is maintained. We hypothesize that sites 1 and 2 locks transcription in the ON phase, consistent with transition to the phase ON state requires DNA replication to the observation that proper spacing between Lrp binding sites is dissociate Lrp and generate a hemimethyated GATCdist site. PapI necessary for the high cooperativity in binding (N. Kozak and and methylation of GATCprox act together to increase the affinity D.L., unpublished results). Examination of the pap DNA meth- of Lrp for sites 4–6. Binding of Lrp at the distal sites protects ylation patterns showed that, in phase OFF cells, GATCprox is GATCdist from methylation, forming the phase ON methylation nonmethylated and GATCdist is methylated, whereas the con- pattern (GATCdist nonmethyated, GATCprox methylated). Lrp bind- verse pattern exists in phase ON cells (GATCdist nonmethylated, ing at sites 4–6 together with cAMP-CAP binding 215.5 bp up- GATCprox methylated) (Fig. 2). These methylation patterns stream of the papBA transcription start, is required for activation depend on Lrp (9, 11, 12). Moreover, addition of Lrp to pap of pilin transcription. The first gene product of the papBA tran- DNA in vitro blocks methylation of the pap regulatory GATC script, PapB, helps maintain the switch in the ON state by activating sequences (12). Together, these data indicate that in phase OFF papI transcription, which in turn maintains Lrp binding at sites 4–6. cells Lrp is bound at sites 1–3, blocking methylation of GATCprox within site 2. In contrast, Lrp bound to sites 4–6 in phase ON Cis- and Trans-Acting Pap Switch Components cells blocks methylation of GATCdist within site 5 (Fig. 2). acteria have developed phase variation mechanisms to con- Translocation of Lrp from sites 1–3 to sites 4–6 requires PapI. Btrol cell surface pili–adhesin complexes between expression Lrp thus plays dual roles in regulating pap transcription. Lrp acts (phase ON) and nonexpression (phase OFF) states. Pili phase as a repressor when bound proximal to the pap BA pilin variation can occur by site specific (1, 2) and homologous promoter. Lrp activates pilin transcription when, together with recombination (3) mechanisms. In addition, a large group of pili PapI, it is bound distal to the pilin promoter (13). Activation of operons including pyelonephritis-associated pili (pap) are regu- pap transcription also requires binding of cAMP-CAP at a site lated by an epigenetic switch directly controlled by DNA meth- 60 bp upstream of Lrp binding site 4 (14–16) and binding of the ylation pattern formation (4, 5). The focus of this paper is on the papI mechanisms by which the phase OFF and phase ON states are PapB regulatory protein at a site proximal to the promoter perpetuated, the transition between phase OFF and phase ON (17, 18) (Fig. 1). states, and the external inputs that control phase switching. Pap Phase Variation Model The expression of pap is positively controlled by the local regulators PapI (8 kDa) and PapB (12 kDa) in concert with the A model for Pap phase variation is shown in Fig. 3. Specific global regulators leucine-responsive regulatory protein (Lrp) details and supporting data for the model are discussed below. and catabolite gene activator protein (CAP). DNA adenine methylase (Dam) is also required for pap transcription (Table 1). The Self-Perpetuating Phase OFF State. Only the distal GATC site Knockout mutations in each of the genes encoding these regu- of the pap regulatory region is methylated in phase OFF cells latory proteins inhibit the pap phase OFF to phase ON switch (Table 1). In addition, histone-like nucleoid structuring protein This paper results from the Arthur M. Sackler Colloquium of the National Academy of (H-NS) modulates pap phase switching because hns mutants Sciences, ‘‘Self-Perpetuating Structural States in Biology, Disease, and Genetics,’’ held show a decreased OFF to ON switch rate (6, 7). March 22–24, 2002, at the National Academy of Sciences in Washington, DC. The pap regulatory region encompasses the divergently tran- Abbreviations: Lrp, leucine-responsive regulatory protein; Pap, pyelonephritis-associated scribed papI and papB genes together with the 416-bp intergenic pili; Dam, DNA adenine methylase; CAP, catabolite gene activator protein; H-NS, histone- region (Fig. 1). There are six pap DNA Lrp binding sites, like nucleoid structuring protein. designated sites 1–6, within the pap regulatory region spaced ‡To whom reprint requests should be addressed. E-mail: [email protected]. 16470–16476 ͉ PNAS ͉ December 10, 2002 ͉ vol. 99 ͉ suppl. 4 www.pnas.org͞cgi͞doi͞10.1073͞pnas.182427199 Downloaded by guest on September 27, 2021 Table 1. Trans-acting factors that regulate Pap phase variation Genotype Description Switch rates (OFF to ON)* Wild type 7 ϫ 10Ϫ4 per cell per generation lrpϪ Leucine-responsive regulatory protein Locked OFF crpϪ Catabolite gene activator protein Locked OFF damϪ DNA adenine methylase Locked OFF papIϪ Local regulatory protein Locked OFF papBϪ Local regulatory protein Locked OFF hnsϪ Histone-like nucleoid structuring protein 2 ϫ 10Ϫ4 per cell per generation *Switch rates measured in M9 glycerol minimal medium. Data are from refs. 6, 7, 9–11, 13, and 22. (GATCdist methylated, GATCprox nonmethylated). Initial anal- mutual binding exclusion effect is reduced from 10-fold to only ysis indicated that methylation of GATCdist was required for about 2-fold when unsupercoiled DNAs are used (unpublished maintenance of the OFF transcription state because introduc- data), showing a strong dependence on DNA topology. Because tion of an A to C transversion within GATC results in a Lrp is known to form higher oligomers under certain conditions phase-locked ON phenotype (Fig. 1) (10). Although the aden- (19, 20) and bend DNA (21), one possible mechanism is that the osine within GATC is obviously required for methylation of this conformation of pap sites 4–6 might be altered as a result of a site by Dam, the affinities of Lrp for wild-type pap DNA and binding of Lrp at sites 1–3 located 102 bp away (measured from DNA containing the GCTCdist mutation appear similar (10), and site 2 to site 5) (Fig. 1). Because the binding of Lrp to pap DNA dimethyl sulfate footprint analyses indicate that Lrp does not is highly cooperative (8), this could serve as a signal amplifica- closely contact the adenosine of GATC (8). Further studies (10) tion mechanism. As discussed below, when the affinity of Lrp for showed that overproduction of Dam prevented the phase OFF sites 1–3 is reduced by mutation of Lrp binding site 3, mutual to ON transition in cells containing a wild-type pap sequence but exclusion works in reverse, lowering the affinity of Lrp at sites not in cells containing the GCTCdist mutation (Fig. 4). Thus, 1–3 as a result of binding of Lrp at sites 4–6 (Fig. 5B). overmethylation of GATCdist prevents the phase OFF to ON Activation of papBA transcription requires binding of Lrp at transition, consistent with the hypothesis that methylation of this sites 4–6 (8), and thus, binding of Lrp at sites 1–3 indirectly distal GATC site helps maintain cells in the OFF state. inhibits transcription because of mutual exclusion of Lrp binding Another factor that may contribute to maintenance of the at sites 4–6. In addition, Lrp binding at sites 1–3 appears to phase OFF state is a mutual binding exclusion phenomenon.