Extension of bacterial rDNA sequencing to concurrent epigenetic analysis and its application to 16S meta-epigenetics

Although polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA region has been used in a wide range of scientific fields, it does not provide DNA methylation information. We describe a simple add-on method to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical samples or flora. Single-stranded bacterial DNA after bisulfite conversion was preferentially amplified with multiple displacement amplification (MDA) at pH neutral, and the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing. 16S rDNA bisulfite sequencing can provide clinically important bacterial DNA methylation status concurrently with intact 16S rDNA sequence information. We used this approach to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii. Next, we analyzed bacterial flora from clinical specimens of small amount and identified different methylation motifs among Enterococcus faecalis strains. The method developed here, referred to as "add-on" to the conventional 16S rDNA analysis, is the most clinically used bacterial identification genetic test, which provides additional information that could not be obtained with the conventional method. Since the relationship between drug resistance in bacteria and DNA methylation status has been reported, bacterial epigenetic information would be useful in clinical testing as well. Our analysis suggests that M. MmnI has a promotive effect on erythromycin resistance. 16S rDNA bisulfite PCR and sequencing coupled with MDA at pH neutral is a useful add-on tool for analyzing 16S meta-epigenetics.