Novel Methyltransferase Recognition Motif Identified in Chania multitudinisentens RB-25T gen. nov., sp. nov.

Abstract

DNA methylation, defined by the addition of a methyl group to adenine or cytosine bases in DNA catalyzed by DNA methyltransferases (MTases), is one of the most studied post-replicative DNA modification mechanism in bacteria (Roberts et al., 2003b). The three forms of nucleotide methylation identified to date are: N6-methyladenine(m6A), N4-methylcytosine (m4C), and 5-methylcytosine (m5C) (Gromova and Khoroshaev, 2003). Generally, MTases can be classified into two main groups: as part of a restriction modification (RM) system in which the MTase is associated with a cognate restriction endonuclease (REase) or as a solitary MTase, which as the name suggests, serves the role of an independent MTase (Murphy et al., 2013; Roberts et al., 2015). The RM systems, which are found to occur exclusively in unicellular organisms, can be further classified into four groups based on their subunit composition, recognition sequence specificity, substrate specificity, cofactor requirements, and DNA cleavage positions (Wilson and Murray, 1991; Casadesus and Low, 2006). In brief, Type I RM systems comprise three polypeptides which form a hetero-oligomeric protein complex, namely the R (restriction), M (modification), and S (specificity) subunits, that recognizes asymmetric, and bipartite recognition sequences (Murray, 2000). Type II systems, the most ubiquitous, and simplest systems, are composed of two functionally-independent R and M genes, which are responsible for restriction and methylation activity respectively. The recognition sequences of the Type II systems are most often symmetrical, but can also be asymmetric (Wilson and Murray, 1991). Type III system also consist of two subunits, named res, and mod. The mod subunit can function independently as a MTase, but only methylates one strand of the DNA. The res subunit must form a complex with the mod subunit to express its DNA restriction activity, because the recognition specificity is encoded in the mod subunit (Wilson and Murray, 1991). The recognition sequences of the known Type III systems are asymmetric and four to six bases in length. Furthermore, when known, the res enzyme requires the presence of two unmethylated recognition sites for efficient DNA cleavage (Rao et al., 2013). Lastly, the Type IV systems are restriction enzymes that recognize and cleave only methylated DNA (Vasu and Nagaraja, 2013). The sequence specificities of the Type IV systems are not well studied. MTases can be further sub-classified according to the order of their conserved amino acid motifs, which represent the DNA binding domain, the target recognition domain (TRD) and the catalytic domain (Malone et al., 1995; Jeltsch, 2002). RM systems are often considered as the most primitive defense mechanism of prokaryotes against the invasion of extraneous DNA elements (Wilson and Murray, 1991; Bickle, 2004). However, more recent studies have offered new insights into their additional biological roles, including genomic island stabilization, species identity maintenance, generating, and enhancing genomic diversity for host fitness and adaptability as well as the regulation of gene expression (Vasu and Nagaraja, 2013). The development of Single Molecule Real-Time (SMRT) sequencing technology which enables the simultaneous genome-wide detection of MTase activity during genome sequencing has permitted the rapid detection of novel MTase recognition motifs in various prokaryotes including Helicobacter pylori, Salmonella enterica, Escherichia coli, and Campylobacter coli (Krebes et al., 2014; Forde et al., 2015; Lee et al., 2015; Pirone-Davies et al., 2015; Zautner et al., 2015). These data are important not only for the identification of novel types of MTases, but they also lay the groundwork for the discovery of new biological roles for DNA methylation. Furthermore, these new data on previously characterized methylomes can further extend our understanding of these RM systems. Chania multitudinisentens RB-25T gen. nov., sp. nov. is a fully characterized newly proposed novel genus in the family of Enterobacteriaceae. Isolated in a soil sample collected from a former municipal landfill site, C. multitudinisentens RB-25T was initially misidentified as a member of the Serratia genus (Ee et al., 2014a). Further in-depth investigation later reclassified C. multitudinisentens RB-25T as a novel genus (Ee et al., 2016). To date, C. multitudinisentens RB-25T is only characterized for its quorum sensing properties (the production of C4-HSL, C6-HSL, and 3-oxo-C6-HSL) and its potential chitinolytic activity (Ee et al., 2014a; Lim et al., 2015b). In this study, we report a novel methyltransferase recognition motif in C. multitudinisentens RB-25T, which will serve as the foundation for future investigation of the role of methylation in the genus Chania.