Dataset Information

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression.

ABSTRACT: DNA adenine methyltransferase (Dam) is widespread and conserved among the ?-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). Taken together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.

SUBMITTER: Horton JR

PROVIDER: S-EPMC4417163 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression.

Horton John R JR Zhang Xing X Blumenthal Robert M RM Cheng Xiaodong X

Nucleic acids research 20150406 8

DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam ...[more]

PMID: 25845600

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Project description:DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In Escherichia coli K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focused on the motifs associated with methylated adenine (meA), the frequency of meA across the genome, and temporal dynamics during early periods of incubation, here we conduct the first study on the temporal dynamics of adenine methylation in E. coli by Dam throughout all five phases of the bacterial life cycle in the laboratory. Using single-molecule real-time sequencing, we show that virtually all GATC sites are significantly methylated over time; nearly complete methylation of the chromosome was confirmed by mass spectroscopy analysis. However, we also detect 66 sites whose methylation patterns change significantly over time within a population, including three sites associated with sialic acid transport and catabolism, suggesting a potential role for Dam regulation of these genes; differential expression of this subset of genes was confirmed by quantitative real-time PCR. Further, we show significant growth defects of the dam mutant during long-term stationary phase (LTSP). Together these data suggest that the cell places a high premium on fully methylating the chromosome and that alterations in methylation patterns may have significant impact on patterns of transcription, maintenance of genetic fidelity, and cell survival. IMPORTANCE While it has been shown that methylation remains relatively constant into early stationary phase of E. coli, this study goes further through death phase and long-term stationary phase, a unique time in the bacterial life cycle due to nutrient limitation and strong selection for mutants with increased fitness. The absence of methylation at GATC sites can influence the mutation frequency within a population due to aberrant mismatch repair. Therefore, it is important to investigate the methylation status of GATC sites in an environment where cells may not prioritize methylation of the chromosome. This study demonstrates that chromosome methylation remains a priority even under conditions of nutrient limitation, indicating that continuous methylation at GATC sites could be under positive selection.

Project description:Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS induction to result in increased mutability. Phenotypic characteristics of parental strains were restored in dam complement strains. Dam's vital role in DNA methylation and gene expression in local UPEC isolates was confirmed. Similarly to dam-deficient Enterohemorrhagic E. coli (EHEC), these findings suggest unsuccessful therapeutic use of Dam inhibitors against UPEC or dam-deficient UPEC strains as attenuated live vaccines. However, further investigations are necessary to determine the post-transcriptional influence of dam on the regulatory network of virulence genes central to pathogenesis.

Dataset Information

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression.

Publications

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression.

Similar Datasets

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