Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5′-GATC-3′ appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential.
展开▼
