Bruce A. Braaten

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

Abstract We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC 1028 and GATC 1130 , were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC 1028 blocks formation of the ON state by inhibiting the binding of Lrp and Papl regulatory proteins to this site. Conversely, methylation of GATC 1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional small immunity protein (CdiI) protects CDI+ cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.

Epigenetic phase variation of the pap operon in Escherichia coli

Expression of the pyelonephritis-associated pilus (pap) operon in Escherichia coli is regulated by a complex epigenetic phase-variation mechanism involving the formation of differential DNA-methylation patterns. This review discusses how DNA-methylation patterns are formed by protein-DNA interactions and how methylation patterns, in turn, control pap gene expression.

Self-perpetuating epigenetic pili switches in bacteria

Bacteria have developed an epigenetic phase variation mechanism to control cell surface pili–adhesin complexes between heritable expression (phase ON) and nonexpression (phase OFF) states. In the pyelonephritis-associated pili (pap) system, global regulators [catabolite gene activator protein (CAP), leucine-responsive regulatory protein (Lrp), DNA adenine methylase (Dam)] and local regulators (PapI and PapB) control phase switching. Lrp binds cooperatively to three pap DNA binding sites, sites 1–3, proximal to the papBA pilin promoter in phase OFF cells, whereas Lrp is bound to sites 4–6 distal to papBA in phase ON cells. Two Dam methylation targets, GATCprox and GATCdist, are located in Lrp binding sites 2 and 5, respectively. In phase OFF cells, binding of Lrp at sites 1–3 inhibits methylation of GATCprox, forming the phase OFF DNA methylation pattern (GATCdist methylated, GATCprox nonmethylated). Binding of Lrp at sites 1–3 blocks pap pili transcription and reduces the affinity of Lrp for sites 4–6. Together with methylation of GATCdist, which inhibits Lrp binding at sites 4–6, the phase OFF state is maintained. We hypothesize that transition to the phase ON state requires DNA replication to dissociate Lrp and generate a hemimethyated GATCdist site. PapI and methylation of GATCprox act together to increase the affinity of Lrp for sites 4–6. Binding of Lrp at the distal sites protects GATCdist from methylation, forming the phase ON methylation pattern (GATCdist nonmethyated, GATCprox methylated). Lrp binding at sites 4–6 together with cAMP-CAP binding 215.5 bp upstream of the papBA transcription start, is required for activation of pilin transcription. The first gene product of the papBA transcript, PapB, helps maintain the switch in the ON state by activating papI transcription, which in turn maintains Lrp binding at sites 4–6.

Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.

Contact-dependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB

Contact‐dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell‐to‐cell contact via the CdiA/CdiB two‐partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi‐component machines. The Bam machine assembles outer membrane β‐barrel proteins into the outer membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI+ inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, α‐BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor–target cells binding and CDI. A second class of CDI‐resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner membrane component of a multidrug efflux pump that normally forms a cell envelope‐spanning complex with the membrane fusion protein AcrA and the outer membrane protein TolC. Strikingly, the requirement for the BamA and AcrB proteins in CDI is independent of their multi‐component machines, and thus their role in the CDI pathway may reflect novel, import‐related functions.

Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap.

Pyelonephritis‐associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non‐methylated pap regulatory DNA region containing the sequence ‘GATC’ and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved ‘GATC‐box’ domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.

Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli.

Pyelonephritis‐associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine‐responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC‐I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns. Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter. A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the −35 and −10 RNA polymerase binding region of papBAp. The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription. The pap GATC‐I and GATC‐II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.

Regulation of pyelonephritis-associated pili phase-variation in Escherichia coli : binding of the Papl and the Lrp regulatory proteins is controlled by DNA methylation

Expression of pyelonephritis‐associated pili (Pap) in Escherichia coli is under a phase‐variation control mechanism in which individual cells alternate between pili+ (ON) and pili− (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC1028 site of phase ON cells is non‐methylated and the GATC1130 site is fully methylated. Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non‐methylated. Two transcriptional activators, Papl and Lrp (leucine‐responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non‐methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas Papl does not appear to bind to pap regulatory DNA. However, addition of Lrp and Papl together results in an additional DNasel footprint around the GATC1028 site. Moreover, Dam methylation inhibits binding of Lrp/Papl near the GATC1028 site and alters binding of Lrp at the GATC1130 site. Our results support a model in which Dam and Lrp/Papl compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.

Thermoregulation of Escherichia coli pap transcription: H‐NS is a temperature‐dependent DNA methylation blocking factor

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26°C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23°C. The histone‐like nucleoid structuring protein H‐NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H‐NS or a factor regulated by H‐NS bound within the pap regulatory region at 23°C but not at 37°C, as evidenced by H‐NS‐dependent inhibition of methylation of the pap GATC sites designated GATC‐I and GATC‐II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H‐NS bound specifically to a pap DNA fragment containing the GATC‐I and GATC‐II sites. Moreover, H‐NS blocked methylation of these pap GATC sites in vitro : H‐NS blocked pap GATC methylation at 1.4 μM but was unable to do so at higher concentrations at which non‐specific binding occurred. Thus, non‐specific binding of H‐NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H‐NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H‐NS with pap regulatory DNA. We hypothesize that a function of H‐NS such as oligomerization was altered at 23°C, which enabled H‐NS to repress pap gene expression through the formation of a specific nucleoprotein complex.