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

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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 DNA-binding glp regulon repressor, GlpR (Zeng et al., 1996), which also binds glycerol-3-phosphate, 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 ​(Figure1A).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 cAMP-CRP 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). Figure 1 The appearance of Glp+ mutations in a crp genetic background. (A) The glpFK promoter region. The transcriptional initiation site (+1), the −10 and −35 hexamers of the promoter, the ribosome binding site (RBS) and the start codon for glpF ... Glp+ mutations in a crp genetic background When crp cells were incubated on solid glycerol minimal medium, Glp+ colonies appeared. We tested the growth of a crp Glp+ strain on glycerol in defined liquid medium. The growth rate was greater than that of wild type (wt) E. coli (Zhang and Saier, 2009b). The relative rates of Glp+ mutation were determined in minimal and complex media. On glycerol plates, colonies first appeared after about 3 days (Figure ​(Figure1B)1B) although wild type (wt) and crp Glp+ E. coli cells formed visible colonies in <2 days (Figure ​(Figure1C).1C). When the same cells were plated as before, but small numbers of crp Glp+ mutant cells were included with the crp cells before plating, colonies appeared from the crp Glp+ cells within 2 days, and new Glp+ mutants arose at the same rate as before (Figure ​(Figure1C).1C). This experiment showed that the mutants that arose on the plates had not existed in the cell population when initially plated. Thus, the Glp+ mutants arising from crp cells on these plates arose during incubation on the plate, and no growth inhibitor was present. The rate of mutation on glycerol plates proved to be 10 times higher than in minimal sorbitol or complex LB medium, and it was over 100 times higher than in glucose-containing medium (Zhang and Saier, 2009a). Mutation proved to be due to IS5 hopping to a discrete site, between base pairs 126 and 127 upstream of the transcriptional start site, and always in the same orientation. The increase in mutation rate was specific to the glpFK operon and did not occur in three other operons examined (Zhang and Saier, 2009a). Only the downstream 177 bp region of IS5 was required for activation of the glpFK operon, and this proved to be due to the presence of a permanent bend and an overlapping IHF binding site, each of which was responsible for half of the activation (Zhang and Saier, 2009b). This mechanism of activation (but not the actual transposon hopping event), presumably involving DNA looping, could also be demonstrated for the lactose (lac) operon in a crp genetic background of E. coli (Zhang and Saier, 2009b).