Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

M. L. Bendall, K. Luong, K. M. Wetmore, M. Blow, J. Korlach, A. Deutschbauer, R. R. Malmstrom
2013 Journal of Bacteriology  
15 We performed whole genome analyses of DNA methylation in Shewanella 16 oneidensis MR-1 to examine its possible role in regulating gene expression and 17 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 18 revealed extensive methylation of adenine (N6mA) throughout the 19 genome. These methylated bases were located in five sequence motifs, 20 including three novel targets for Type I restriction/modification enzymes. The 21 sequence motifs targeted by putative
more » ... ases were determined via 22 SMRT sequencing of gene knockout mutants. In addition, we found S. 23 oneidensis MR-1 cultures grown under various culture conditions displayed 24 different DNA methylation patterns. However, the small number of differentially 25 methylated sites could not be directly linked to the much larger number of 26 differentially expressed genes in these conditions, suggesting DNA methylation is 27 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 28 of methylated GATC motifs in the origin of replication indicate DNA methylation 29 may regulate genome replication in a manner similar to that seen in Escherichia 30 coli. Furthermore, comparative analyses suggest that many 31 Gammaproteobacteria, including all members of the Shewanellaceae family, may 32 also utilize DNA methylation to regulate genome replication. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 JB Accepts, published online ahead of print on 30 August 2013 J. Bacteriol. on July 13, 2017 by guest http://jb.asm.org/ Downloaded from 47 DNA methylation plays a variety of functional roles in bacteria [1-3]. For 48 example, restriction-modification (R-M) systems use methylation patterns to 49 identify and destroy foreign DNA during viral infections [4, 5]. Bacteria also use 50 DNA methylation to regulate genome replication [6], DNA mismatch repair [7], 51 and gene expression [8-12]. Methylation can even serve as an epigenetic 52 modifier, influencing the expression patterns of daughter cells based on 53 environmental conditions [13, 14]. Because of these varied regulatory roles, 54 DNA methylation should be incorporated into our emerging systems-level view of 55 model microorganisms. 56 Despite the functional significance of DNA methylation, our understanding 57 of its role in bacterial genetics and physiology remains incomplete due to 58 methodological limitations. For example, bisulfite conversion can identify 5-59 methylcytosine modifications [15, 16], but there is no corresponding conversion 60 assay for other common modifications in bacteria such as N6-methyladenine or 61 4-methylcytosine [17]. Methyl-sensitive restriction enzymes have been used to 62 identify the methylation state of specific sequence motifs [18-20], but complete 63 methylome analyses are not possible without 1) prior knowledge of the entire set 64 of methyltransferases and their sequence targets within a genome, and 2) 65 access to methyl-sensitive restriction enzymes targeting these motifs. Single-66 Molecule Real Time (SMRT) sequencing overcomes these limitations and 67 enables genome-wide analysis of DNA methylation with single base resolution 68 [21]. In this approach, modifications in the native state DNA are revealed by 69 on July 13, 2017 by guest http://jb.asm.org/ Downloaded from 3 deviations in the polymerase kinetics observed during sequencing. The specific 70 type of DNA methylation can often be determined from the polymerase kinetics, 71 e.g. N6-methyladenine or 4-methylcytosine. With SMRT sequencing it is now 72 possible to identify the complete set of methylated sequence motifs within a 73 microbial genome as well as the methylation state for each instance of a motif 74 [22-24]. This represents a powerful tool for characterizing the functional roles of 75 DNA methylation in a wide variety of bacteria. 76 Shewanella oneidensis MR-1 is a bacterial isolate belonging to the 77 Shewanellacea, a family distinguished by the wide variety of electron acceptors 78 they can utilize (e.g. iron, manganese, uranium, chromium, and plutonium) [25-79 28]. Because of their flexible respiratory pathways, Shewanella sp. are 80 recognized as potential agents for bioremediation at sites contaminated with 81 heavy metals and radionuclides [29]. To better exploit its metabolic potential, S. 82 oneidensis MR-1 has been characterized extensively, including analysis of gene 83 expression [30, 31], identification of regulatory regions [32], and the 84 determination of fitness levels for thousands of gene knockout mutants 85 [33]. However, the developing systems-level view of Shewanella does not yet 86 incorporate DNA methylation and its potential regulatory roles. Genomic 87 analyses reveal multiple putative methyltransferases in S. oneidensis MR-1 [34, 88 35], including several apparent 'orphans' that lack corresponding restriction 89 enzymes. It remains unclear what role these orphan methyltransferases might 90 play. 91 on July 13, 2017 by guest http://jb.asm.org/ Downloaded from Here we use SMRT sequencing to provide the first look at DNA 92 methylation in S. oneidensis MR-1. We identify methylated sites throughout the 93 genome as well as the sequence motifs targeted by predicted 94 methyltransferases. To determine if DNA methylation regulates gene 95 expression, we examine whether changes in expression level correspond with 96 changes in DNA methylation state when cultures are transferred from one set of 97 growth conditions to another. Finally, we examine the finished genomes of all 98 Gammaproteobacteria, including the Shewanellacea, to determine which groups 99 appear to use DNA methylation for regulating genome replication and DNA 100 mismatch repair. 101 102 RESULTS AND DISCUSSION 103 Methylation profile of S. oneidensis MR-1 104 To identify methylated sites within the genome of S. oneidensis MR-1, we 105 performed SMRT sequencing on DNA extracted from triplicate exponential-phase 106 cultures grown aerobically on minimal media. Our analysis revealed 42,965 107 nucleotides that exhibited significant variations in polymerase kinetics that were 108 diagnostic of DNA modification [21]. Of those modified nucleotides, 41,853 were 109 identified as N6-methyladenine (N6mA) based on their distinct kinetic fingerprint. 110
doi:10.1128/jb.00935-13 pmid:23995632 pmcid:PMC3807482 fatcat:bmqhis3mvjd5xgotax52n7n2gy