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

Abstract

Summary 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.

Epigenetic Gene Regulation in the Bacterial World

“…OxyR affinity for fully methylated agn43 regulatory DNA is too low to be measured by electrophoretic mobility shift, but the K d of binding to nonmethylated agn43 is about 2 nM. Binding of OxyR to hemimethylated agn43 methylated on the top or bottom strand is similar, with at least a sixfold reduction in affinity compared to nonmethylated DNA (60). The intermediate affinity of OxyR for hemimethylated agn43 provides a switch transition mechanism: immediately following DNA replication OxyR presumably dissociates from (Fig.…”

“…2A). Exceptions are the DNA replication origin oriC, the dnaA promoter, and possibly additional GATC sites in the chromosome which bind SeqA (60). SeqA preferentially binds to clusters of two or more hemimethylated GATC sites spaced one to two helical turns apart (Fig.…”

“…The mechanism(s) by which agn43 expression switches between the OFF and ON states is not known, though it likely requires DNA replication to generate a hemimethylated DNA intermediate (60). OxyR affinity for fully methylated agn43 regulatory DNA is too low to be measured by electrophoretic mobility shift, but the K d of binding to nonmethylated agn43 is about 2 nM.…”

“…6D) It is not clear if environmental or cellular factors directly regulate Ag43 switching, but it is possible that SeqA may play a role. SeqA binds to agn43 regulatory DNA containing hemimethylated GATC sites but does not bind to fully methylated or nonmethylated DNAs (60). The OFF-to ON-phase rate was reduced in a seqA mutant, but much of this effect could be accounted for by a reduction in the Dam/DNA ratio caused by increased asynchronous initiation of DNA replication that occurs in the absence of SeqA, which normally sequesters oriC and plays a critical role in timing of DNA replication (36).…”

Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

“…Dam methylation is a postreplicative process (28), and consequently the newly synthesized daughter strand is unmethylated for a short time after passage of the replication fork. This transient hemimethylated state following DNA replication plays a crucial role in processes such as the regulation of gene expression (11,26,45), DNA mismatch repair (3,45,55), and the timing of chromosome replication initiation (2,24,42,52). By altering the recognition sequences of transcriptional regulators and RNA polymerases, Dam methylation may affect the ability of proteins to bind the upstream regions of genes and in such a way may serve to regulate gene expression.…”