Detection of N6-methyladenine in GATC sequences ofSelenomonas ruminantium

Abstract

The presence of N6-methyladenine in GATC sequences in DNA of Selenomonas ruminantium was investigated using sensitive methylation discriminating isochizomeric restriction enzymes analysis. Methylated adenine was detected in 8 out of 18 tested strains belonging to the subsp. lactilytica of S. ruminantium. No corresponding restriction activity was detected in three tested strains. No GATC methylation was detected in 3 analysed S. ruminantium subsp. ruminantium strains. Sustainable progress was achieved in the molecular biology of ruminal microorganisms in the last decade. Many different genes acting in the cell wall degradation were cloned and characterized. As practically all cloning experiments were done in Escherichia coli cells, there is a lack of data about regulation of gene(s) expression in the natural hosts. However, much better understanding of molecular genetics of ruminal bacteria is required for improving rumen functions by genetic modifications of rumen bacteria. DNA methylation is main mechanism of the control of gene expression in eukaryotes. In prokaryotes, DNA methylation influences wide variety of important cellular functions as accessibility of DNA to digestion by restriction endonucleases, control of replication initiation, transposition, phage DNA packaging, including positive and negative regulation of gene expression. Most of the DNA methyltransferases; enzymes which facilitate methylation; identified in prokaryotes are part of restriction modification systems (WILSON and MURRAY 1991. Another class of methyltransferase are independent methylases like Dam and Dcm in Escherichia coli. Dam methylase recognizes the sequence GATC and methylates adenine at N6 position (BARRAS and MARINUS 1989). The methylation is the only documented case of prokaryotic methylation involved in the regulation of cellular process (NOYER-WIEDNER and TRAUTNER 1993). Screening of large number of bacteria have detected the presence of Dam methylation in cyanobacteria as well as in the group of related families of Enterobacteriaceae (which includes E. coli), Parvobacteriaceae and Vibrionaceae. Methylated GATC sequences have been found in several other bacterial species (NOYER-WIEDNER and TRAUTNER 1993), however, it could be assumed that some of these methylations are due to the presence of restriction modification systems. There is no data about the presence of Dam methylation neither in Selenomonas ruminantium nor in any other ruminal bacteria. Bacteria of S. ruminantium species are known to contain frequently restriction modification systems. Several restriction endonucleases were characterized from these bacteria (VANAT et al., 1993, PRISTAS et al. 1994, 1995). During characterization of modification activities associated with these endonucleases we have detected the modification of adenine in GATC sequences of DNA of this species.