Transposon-mediated directed mutation controlled by DNA binding proteins in Escherichia coli

INTRODUCTION It is a basic principle of genetics that the likelihood of a particular mutation occurring is independent of its phenotypic consequences. The concept of directed mutation, defined as genetic change that is specifically induced by the stress conditions that the mutation relieves (Cairns et al., 1988), challenges this principle (Foster, 1999; Rosenberg, 2001; Wright, 2004). The topic of directed mutation is controversial, and its existence, even its potential existence, as defined above, has been altogether questioned (Roth et al., 2006). Part of the justifiable skepticism concerning directed mutation resulted from experiments that were purported to demonstrate this phenomenon, but were subsequently shown to be explainable by classical genetics (Roth et al., 2006). Mutation rates vary with environmental conditions (e.g., growth state) and genetic background (e. g., mutator genes), a phenomenon known as “adaptive” mutation (Wright, 2004; Foster, 2005), but this does not render the mutation “directed.” To establish the principle of directed mutation, it is necessary to show that the adaptive mutation is “directed” to a specific site, characterize the mechanism responsible, identify the proteins involved, and provide the evolutionary basis for its appearance. One frequently encountered type of mutation results from the hopping of transposable genetic elements, transposons, which can activate or inactivate critical genes (Mahillon and Chandler, 1998; Chandler and Mahillon, 2002). For example, activation of the normally cryptic β-glucoside (bgl) catabolic operon in E. coli can be accomplished by insertion of either of two insertion sequences, IS1 or IS5, upstream of the bgl promoter (Schnetz and Rak, 1992). The E. coli glycerol (glp) regulon consists of five operons, two of which (glpFK and glpD) are required for aerobic growth on glycerol (Lin, 1976). Both operons are subject to negative control by the DNAbinding glp regulon repressor, GlpR (Zeng et al., 1996), which also binds glycerol-3phosphate, the inducer of the glp regulon. The glpFK operon is additionally subject to positive regulation by the cyclic AMP receptor protein (CRP) complexed with cyclic adenosine monophosphate (cAMP; Freedberg and Lin, 1973; Campos et al., 2013), although glpD is not appreciably subject to regulation by this mediator of catabolite repression (Weissenborn et al., 1992). The glpFK regulatory region contains four GlpR binding sites, O1–O4, and two CRP binding sites, CrpI and CrpII, which overlap O2 and O3, respectively, (Figure 1A). The strong CRP dependency of glpFK transcription is reflected by the fact that crp and cya (adenylate cyclase) mutant cells are unable to utilize glycerol (Zhang and Saier, 2009a). We have found that binding of GlpR and the cAMPCRP complex to the glpFK upstream control region negatively influences IS5 hopping specifically into the single site that strongly activates glpFK promoter activity (Zhang and Saier, 2009a, unpublished observations).