title: Dam-dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant.
abstract: In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 gene is mediated by DNA methylation and the global regulator 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.
authors.affiliation: 202A Johnson Pavilion, Department of Microbiology, School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, Philadelphia 19104-6076, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Correnti
authors.forename: Vincent
authors.initials: V
authors.lastname: Munster
authors.forename: Teresa
authors.initials: T
authors.lastname: Chan
authors.forename: Marjan van der
authors.initials: Mv
authors.lastname: Woude
mesh_terms: D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors; C118376:antigen 43, E coli; C499299:oxyR protein, E coli; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli