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

202A Johnson Pavilion, Department of Microbiology, School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, Philadelphia 19104-6076, USA.
Molecular Microbiology (Impact Factor: 4.42). 05/2002; 44(2):521-32. DOI: 10.1046/j.1365-2958.2002.02918.x
Source: PubMed


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.

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    • "The repression of agn43 transcription by OxyR is independent of its oxidation state (Wallecha et al., 2003). Comparable to the other switch mechanisms described above OxyR binding prevents Dam methylation (Correnti et al., 2002). "
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    ABSTRACT: Epigenetic modification plays a dual role in bacterial infection. On the one hand, the expression of cer- tain genes in the bacterial organism is regulated by epigenetic mechanisms. For example, the expression of components of the type IV secretion system in Gram-negative bacteria like enteroaggregative Esche- richia coli (EAggEC), which is responsible for the secretion of proteins involved in host cell invasion and bacterial killing, is regulated by DNA methylation (Brunet et al., 2011). Differences in the digestion-pattern of endonucleases recognizing methylated adenine at 5’- GATC-3’-sites in the genomes of several Gram-negative bacterial species like Campylobacter coli (Wright et al., 2010) and Campylobacter jejuni (Gu et al., 2009) suggest that further genes are regulated by epigenetic modifications as well. Bioinformatic investigations demonstrated epigenetic regulation of type III secretion system (TTSS) associated toxicity in the non-fermentative Gram-negative facultative pathogen Pseudomonas aeruginosa (Filopon et al., 2006). A generalized logical analysis indicated epigenetic control of Pseudo- monas aeruginosa’s mucoidy (Guespin-Michel & Kaufman, 2001). There are even hints for epigenetic regulation of multistationary of bacterial growth (Guespin-Michel & Kaufman, 2001). On the other hand, bacterial infections induce epigenetic changes in the cells of the infected organ- ism. It is a well described phenomenon that viral proteins affect the activity of cellular promoters via epi- genetic mechanisms. In the last years similar effects were demonstrated for bacterial infections. Promotor silencing by hypermethylation at CpG dinucleotides was described for Campylobacter rectus in placenta tissue (Bobetsis et al., 2007) and for Helicobacter pylori in gastric mucosa (Chan et al., 2003). This epi- genetic reprogramming might play a critical role in fetal growth and ontogenetic programming but also in carcinogenesis. In contrast, methylation of bacterial 16S rRNA is a resistance mechanism providing high-level re- sistance to aminoglycosides in actinomycetes, Enterobacteriaceae and Gram-negative, non-fementing, rod-shaped bacteria like Pseudomonas aeruginosa (Yokoyama et al., 2003; Doi et al., 2004; Périchon et al., 2008). However, this phenomenon is no epigenetic mechanism as gene expression is not affected. Infection with Gram-negativ bacteria induces epigenetic modifications not only in the invading bacterial cells, but also in the host tissue. Epigenetic consequences of bacterial infection are important for the understanding of the pathomechanisms and may have principal therapeutic implications.
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    • "Subsequent investigations revealed that binding of OxyR to the promoter acted as a transcriptional 'road block' preventing RNA polymerase from transcribing the gene and that this repression could only be relieved by DNA replication and subsequent Dam-mediated methylation of GATC nucleotide motifs within the OxyR-binding site (Haagmans & van der Woude, 2000; Waldron et al., 2002; Wallecha et al., 2002, 2003). Recent investigations have indicated that other factors play a role in the switching events which lead to Ag43 expression (Phase ON) or Ag43 repression (Phase OFF) (Correnti et al., 2002; Lim & van Oudenaarden, 2007). Many years ago Diderichsen (1980) described a phase variable phenomenon characterized by the ability of one population of E. coli to settle in static liquid culture and an isogenic population which would remain in suspension. "
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    • "Thus, like NagC at GATC NagC , an additional cofactor could be required for NanR to prevent Dam methylation of GATC NanR in vivo. To our knowledge, this is the fourth time that methylation protection has been demonstrated for a bacterial regulatory system in vitro, with the interactions of Lrp with the papA-papB intergenic region, OxyR with ag43 and GutR with its operator sequence being the first three (van der Woude et al., 1998; Correnti et al., 2002). However, unlike Lrp and OxyR binding to their operator sequences at pap and ag43 respectively (Nou et al., 1993; Haagmans and van der Woude, 2000), methylation does not seem to affect the binding of NagC to O NC1. "
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