Marjan W. van der Woude

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.

Phase variation: how to create and coordinate population diversity

Phase variation yields phenotypic heterogeneity in a clonal population as the result of one of a limited number of known molecular mechanisms. These include slipped strand mispairing, site-specific recombination and epigenetic regulation mediated by DNA methylation. Recently new regulatory variants utilizing these mechanisms have been identified, which is facilitating the identification of additional phase variation events solely from genome sequence analysis. Furthermore, it is becoming increasingly clear that in many cases phase variation control is integrated with regulatory networks and with cellular processes of a growing cell. This review focuses specifically on these recent advances in the understanding of the regulation of phase variation.

Regulation and Function of Ag43 (Flu)

Antigen 43 (Ag43) is an abundant outer membrane protein in Escherichia coli belonging to the autotransporter family. Structure-function relationships of Ag43 proposed on the basis of experimental work and in silico analysis are discussed in context of insights derived from molecular modeling. New sequence analysis sheds light on the phylogeny of the allelic variants of the Ag43-encoding gene and identifies two distinct families that appear to be distributed between specific pathogenic and commensal isolates. The molecular mechanism that controls expression by phase variation to create population heterogeneity is discussed. Proposed roles of Ag43 expression for E. coli are summarized and the studies are put into perspective regarding the role of allelic variants, genetic background of the bacterial strain, and control of expression by phase variation. We conclude that future studies need to take into account these variables to obtain a complete understanding of the contribution of Ag43 expression to E. coli biology.

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.

Slipped-Strand Mispairing Can Function as a Phase Variation Mechanism in Escherichia coli

Slipped-strand mispairing (SSM) has not been identified as a mechanism of phase variation in Escherichia coli. Using a reporter gene, we show that sequences that cause phase variation by SSM in Haemophilus influenzae also lead to phase variation when introduced onto the chromosome of E. coli, and the frequencies of switching are in the biologically relevant range. Thus, the absence of SSM-mediated phase variation in E. coli does not appear to be due to a mechanistic constraint.

Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR

OxyR is a DNA binding protein that differentially regulates a cells response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of agn43 from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of agn43. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses agn43 transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of agn43. OxyR(ox) repressed in vitro transcription but only from an unmethylated agn43 template. The -10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the agn43 regulatory region DNA with an affinity similar to that for the regulatory regions of katG and oxyS, which are activated by OxyR(ox), indicating that binding at agn43 can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at agn43 is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.

The essential role of the promoter‐proximal subunit of CAP in pap phase variation: Lrp‐ and helical phase‐dependent activation of papBA transcription by CAP from −215

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP–CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP‐dependent activation of papBA requires binding of the leucine‐responsive regulatory protein (Lrp) at binding sites that extend from −180 to −149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP‐dependent promoters. Also, like class I promoters, the C‐terminal domain of the α‐subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an ‘oriented heterodimer’ approach with wild‐type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter‐distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H‐NS. The results presented here show that AR1 of the promoter‐proximal CAP subunit was required for papBA transcription even in the absence of the histone‐like protein H‐NS. These results show that the promoter‐proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C‐terminal domain of the α‐subunit of RNA polymerase.

Dam-dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant

In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 gene is mediated by DNA methylation and the global regu‐lator OxyR. Transcription of agn43 occurs (ON phase) when three Dam target sequences in the agn43 regulatory region are methylated, which prevents the repressor OxyR from binding. Conversely, transcription is repressed (OFF) when these Dam target sequences are unmethylated and OxyR binds. A change in expression phase requires a concomitant change in the DNA methylation state of these Dam target sequences. To gain insight into the process of inheritance of the expression phase and the DNA methylation state, protein–DNA interactions at agn43 were examined. Binding of OxyR at agn43 was sufficient to protect the three GATC sequences contained within its binding site from Dam‐dependent methylation in vitro, suggesting that no other factors are required to maintain the unmethylated state and OFF phase. To maintain the methylated state of the ON phase, however, Dam must access the hemimethylated agn43 region after DNA replication, and OxyR binding must not occur. OxyR bound hemimethylated agn43 DNA, but the affinity was severalfold lower than for unmethylated DNA. This presumably contributes to the maintenance of the methylated state but, at the same time, may allow for infrequent OxyR binding and a switch to the OFF phase. Hemimethylated agn43 DNA was also a binding substrate for the sequestration protein SeqA. Thus, SeqA, OxyR and Dam may compete for the same hemimethylated agn43 DNA that is formed after DNA replication in an ON phase cell. In isolates with a mutant seqA allele, agn43 phase variation rates were altered and resulted in a bias to the OFF phase. In part, this can be attributed to the observed decrease in the level of DNA methylation in the seqA mutant.

CdiA promotes receptor-independent intercellular adhesion

CdiB/CdiA proteins mediate inter‐bacterial competition in a process termed contact‐dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI+ cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto‐aggregation and biofilm formation in several species, but the contribution of CdiA–receptor interactions to these multi‐cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA–BamA mediated cell–cell adhesion. However, CdiA also promotes auto‐aggregation in a BamA‐independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA‐independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA–CdiA interactions. The BamA‐dependent and BamA‐independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth‐inhibition activity. This adhesion should enable ‘greenbeard’ discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind‐selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer.

Horizontally Acquired Glycosyltransferase Operons Drive Salmonellae Lipopolysaccharide Diversity

The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.