OPINION ARTICLE published: 01 August 2014 doi: 10.3389/fmicb.2014.00390 Transposon-mediated directed mutation controlled by DNA binding proteins in Escherichia coli

Milton H. Saier Jr* and Zhongge Zhang

Division of Biological Sciences, Department of Molecular Biology, University of California at San Diego, La Jolla, CA, USA *Correspondence: [email protected]

Edited by: Anton G. Kutikhin, Research Institute for Complex Issues of Cardiovascular Diseases under the Siberian Branch of the Russian Academy of Medical Sciences, Russia Reviewed by: Robert Heinzen, NIH/NIAID-RML, USA Claudio Palmieri, Polytechnic University of Marche, Italy Anton G. Kutikhin, Research Institute for Complex Issues of Cardiovascular Diseases under the Siberian Branch of the Russian Academy of Medical Sciences, Russia

Keywords: directed mutation, transposable genetic element, transposon, insertion sequence, gene activation, glpFK operon

INTRODUCTION transposons, which can activate or hopping specifically into the single site that It is a basic principle of genetics that inactivate critical genes (Mahillon and strongly activates glpFK promoter activity the likelihood of a particular mutation Chandler, 1998; Chandler and Mahillon, (Zhang and Saier, 2009a, unpublished occurring is independent of its phenotypic 2002). For example, activation of the nor- observations). consequences. The concept of directed mally cryptic β-glucoside (bgl) catabolic mutation, defined as genetic change that operon in E. coli can be accomplished Glp+ MUTATIONS IN A crp GENETIC is specifically induced by the stress con- by insertion of either of two insertion BACKGROUND ditions that the mutation relieves (Cairns sequences, IS1 or IS5, upstream of the bgl When crp cells were incubated on solid et al., 1988), challenges this principle promoter (Schnetz and Rak, 1992). glycerol minimal medium, Glp+ colonies (Foster, 1999; Rosenberg, 2001; Wright, The E. coli glycerol (glp) regulon con- appeared. We tested the growth of a crp 2004). The topic of directed mutation is sists of five operons, two of which (glpFK Glp+ strain on glycerol in defined liquid controversial, and its existence, even its and glpD) are required for aerobic growth medium. The growth rate was greater than potential existence,asdefinedabove,has on glycerol (Lin, 1976). Both operons are that of wild type (wt) E. coli (Zhang and been altogether questioned (Roth et al., subject to negative control by the DNA- Saier, 2009b). 2006). binding glp regulon repressor, GlpR (Zeng The relative rates of Glp+ mutation Part of the justifiable skepticism con- et al., 1996), which also binds glycerol-3- were determined in minimal and complex cerning directed mutation resulted from phosphate, the inducer of the glp regulon. media. On glycerol plates, colonies first experiments that were purported to The glpFK operon is additionally subject appeared after about 3 days (Figure 1B) demonstrate this phenomenon, but were to positive regulation by the cyclic AMP although wild type (wt)andcrp Glp+ subsequently shown to be explainable receptor protein (CRP) complexed with E. coli cells formed visible colonies in <2 by classical genetics (Roth et al., 2006). cyclic adenosine monophosphate (cAMP; days (Figure 1C). When the same cells Mutation rates vary with environmental Freedberg and Lin, 1973; Campos et al., were plated as before, but small numbers conditions (e.g., growth state) and genetic 2013), although glpD is not appreciably of crp Glp+ mutant cells were included background (e. g., mutator genes), a phe- subject to regulation by this mediator of with the crp cellsbeforeplating,colonies nomenon known as “adaptive” mutation catabolite repression (Weissenborn et al., appeared from the crp Glp+ cells within (Wright, 2004; Foster, 2005), but this 1992). The glpFK regulatory region con- 2 days, and new Glp+ mutants arose at does not render the mutation “directed.” tains four GlpR binding sites, O1–O4,and the same rate as before (Figure 1C). This To establish the principle of directed two CRP binding sites, CrpI and CrpII, experiment showed that the mutants that mutation, it is necessary to show that which overlap O2 and O3,respectively, arose on the plates had not existed in the the adaptive mutation is “directed” to a (Figure 1A). The strong CRP dependency cell population when initially plated. Thus, specific site, characterize the mechanism of glpFK transcription is reflected by the the Glp+ mutants arising from crp cells responsible, identify the proteins involved, fact that crp and cya (adenylate cyclase) on these plates arose during incubation and provide the evolutionary basis for its mutant cells are unable to utilize glyc- on the plate, and no growth inhibitor was appearance. erol (Zhang and Saier, 2009a). We have present. The rate of mutation on glycerol One frequently encountered type found that binding of GlpR and the cAMP- plates proved to be 10 times higher than in of mutation results from the hop- CRP complex to the glpFK upstream minimal sorbitol or complex LB medium, ping of transposable genetic elements, control region negatively influences IS5 and it was over 100 times higher than in

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FIGURE 1 | The appearance of Glp+ mutations in a crp genetic glucose (), or 1% sorbitol (). crp cells from an overnight culture (from a background. (A) The glpFK promoter region. The transcriptional initiation single colony) were applied onto agar plates, and the plates were site (+1), the −10 and −35 hexamers of the promoter, the ribosome incubated at 30◦C. On glycerol minimal plates, mutations were determined binding site (RBS) and the start codon for glpF are shaded. The GlpR by the presence of colonies on the plate. On sorbitol and glucose plates, binding sites (O1–O4; lines above the sequence) and CRP binding sites mutations were determined by washing all cells off the plates and (CrpI and CrpII; lines below the sequence) are also shown. The location of measuring colony formation both on LB plates (total cells) and on minimal the IS5 element upstream of the promoter is indicated by the vertical glycerol plates (Glp+ cells) after 36–48 h of growth. (C) The same minimal white arrow below the horizontal black arrow representing IS5. The two glycerol agar plates, where the crp mutant cells were plated together with 4-nucleotide direct repeats (ctaa), caused by IS5 insertion, are shaded dark various numbers of crp Glp+ cells. Numbers of crp Glp+ cells were: (, and light gray, respectively. (B) Glp+ mutations in a crp genetic background 72; ♦, 38; ,19; ,10;,5;and, 0). These cells were mixed with crp in various media: solid M9 minimal media + 1% glycerol (), 0.01% cells and then applied onto M9 glycerol agar plates. glucose-containing medium (Zhang and DEPENDENCY OF THE Glp+ MUTATION double mutant than in the crp mutant Saier, 2009a). RATE ON GlpR when glycerol was absent. In the pres- Mutation proved to be due to IS5 Glycerol is phosphorylated by glycerol ence of glycerol, the loss of GlpR was hopping to a discrete site, between base kinase (GlpK) to glycerol-3-phosphate without effect. Thus, deletion of glpR is pairs 126 and 127 upstream of the tran- which binds to and releases GlpR from its equivalent to the inclusion of excess glyc- scriptional start site, and always in the operators (Lin, 1976). When GlpR disso- erol in the growth medium. High level same orientation. The increase in muta- ciates from its operators, due to glycerol- overexpression of glpR decreased muta- tion rate was specific to the glpFK operon 3-phosphate binding, a conformational tion rate to background levels (Zhang and did not occur in three other oper- change could be transmitted through the and Saier, 2009a). Clearly, GlpR regulates ons examined (Zhang and Saier, 2009a). DNA, promoting insertion of IS5 at the the increased rate of IS5-mediated inser- Only the downstream 177 bp region of IS5 target CTAA site upstream of the glpFK tional activation of the glpFK promoter by was required for activation of the glpFK promoter. In other words, GlpR binding glycerol. operon, and this proved to be due to the might have two functions: repression of presence of a permanent bend and an over- gene expression and suppression of IS5 GlpR OPERATORS DIFFERENTIALLY lapping IHF binding site, each of which transposition to the upstream activating CONTROL glpFK EXPRESSION AND + wasresponsibleforhalfoftheactivation site. Glp MUTATION RATE (Zhang and Saier, 2009b). This mecha- To test this possibility, the glpR gene There are four GlpR binding sites, O1– nism of activation (but not the actual was deleted, and the rates of appearance O4,intheupstreamglpFK operon reg- transposon hopping event), presumably of Glp+ mutations in the crp glpR dou- ulatory region (see Figure 1A), identified involving DNA looping, could also be ble mutant background were measured by DNA footprinting (Freedberg and Lin, demonstrated for the lactose (lac)operon in the absence and presence of glyc- 1973; Zeng et al., 1996). We mutated the in a crp genetic background of E. coli erol. The numbers of Glp+ cells aris- far upstream site (O1) and the far down- (Zhang and Saier, 2009b). ing was 10-fold higher in the crp glpR stream site (O4) so they no longer could

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bind GlpR, and compared the effects on CONTROL OF IS5-MEDIATED glpFK had actually evolved under the pressures of glpFK expression using a lacZ reporter OPERON ACTIVATION BY THE natural selection. gene fusion construct vs. mutation rate cAMP-CRP COMPLEX We consequently undertook a system- + to Glp during growth in LB medium. As noted above, IS5-mediated activation atic analysis of the cAMP dependency of Mutation of O4 increased glpFK operon of the glpFK promoter was observed in IS5-mediated glpFK activation to under- expression about 5-fold although muta- a crp genetic background. Initial attempts stand why crp mutants but not wt cells tion of O1 was almost without effect in our laboratory and elsewhere to isolate gave rise to IS5-activated mutants. The (Zhang and Saier, 2009a). By contrast, loss such mutants in a wild type genetic back- cya gene, encoding the cAMP biosyn- of O1 yielded a 7-fold increase in muta- ground proved unsuccessful (Ibarra et al., thetic enzyme, adenylate cyclase (Cya), tion rate although loss of O4 had only a 2002; Honisch et al., 2004; Fong et al., was deleted, and as expected, IS5-mediated 2-fold effect on mutation rate. We con- 2005; Zhang and Saier, 2011, unpublished glpFK activation was observed at high fre- firmed that IS5 was always in the same observations; Cheng et al., 2014). Since quency.Whensub-mMconcentrationsof position and orientation (Zhang and Saier, crp mutants are not found in nature, this cAMP were added to the growth medium, 2009a). Thus, O1 primarily controls the brought into question the suggestion that the frequency of these mutations was dras- rate of IS5 insertion into the activating our discovery of directed mutation in a tically reduced; 1 mM exogenous cAMP site, while O4 primarily controls glpFK crp mutant of E. coli wasrelevanttothe essentially prevented the appearance of expression. wild type situation, and hence whether it these mutants. When glpR was deleted in

FIGURE 2 | Schematic diagram illustrating dual GlpR-mediated/cAMP- and IS5 hopping increase about 10×. Binding of GlpR to operator O1 CRP-mediated control of (right) the level of glpFK transcription and (left) preferentially blocks IS5 insertion, while binding of GlpR to operator O4 the rate of IS5 hopping into the activating site upstream of the glpFK preferentially blocks transcription as indicated. Binding of cAMP-CRP to its promoter (directed mutation). With GlpR bound to its operators (O1–O4)(in transcriptional activating sites, CrpI and CrpII, similarly inhibits IS5 hopping the presence of GlpR and the absence of glycerol), transcription and IS5 even though binding of this complex promotes glpFK transcription. Glucose hopping both occur at low rates. When GlpR is not bound to its operators (in the inhibits IS5 insertion by a mechanism independent of glycerol, GlpR, cAMP,and absence of GlpR or in the presence of glycerol), both transcriptional initiation CRP.( , activation; , inhibition or repression).

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the cya genetic background, the frequen- have confirmed and extended their results are synthesized by many organisms and cies of IS5 insertion increased by about (Zhang et al., 2013). Moreover, prelim- therefore are present in nature. This exam- 20-fold (Zhang and Saier, unpublished inary results suggest that the E. coli fuc ple of directed mutation could therefore observations). (fucose; propanediol) operon may also be have been selected for during evolution. These observations led us to experi- subject to IS5-mediated directed muta- It appears to be a genuine example of ment with wt E. coli cells. Glycerol utiliza- tion (Zhang and Saier, 2011; Zhang et al., directed mutation, with mutations aris- tion in these cells is strongly inhibited by 2013). It seems that transposon-mediated ing at a greater rate under conditions that the presence of the non-metabolizable directed mutation will prove to be impor- allow benefit to the organism. The fact glucose analogs, 2-deoxyglucose and tant to microbial evolution, and possibly that mutation rate is influenced by the α-methyl glucoside, which also lower to that in other organisms, partly account- presence of glycerol in a process medi- cytoplasmic cAMP levels by inhibit- ingfortheprevalenceofthesegenetic ated by the glycerol repressor, and by ing adenylate cyclase activity (Saier and elements in virtually all living organisms. cAMP in a process mediated by CRP, Feucht, 1975; Castro et al., 1976; Saier provides mechanistic explanations for et al., 1976; Feucht and Saier, 1980; CONCLUSIONS AND PERSPECTIVE IS5-mediated directed mutational control. Saier, 1989). Wild type cells were there- Directed mutation has been defined as This mechanism, illustrated in Figure 2, fore plated on minimal salts medium genetic change that is specifically induced allows rationalization of the presence of containing 0.2% glycerol and 0.1% 2- by the stress conditions that the mutation four GlpR binding sites and two CRP α deoxyglucose or 0.5% -methyl glucoside. relieves, but until recently, in no case had binding sites in the control region of the Not surprisingly, IS5 insertional directed such a mechanism been established. We glpFK operon. Our studies also provide the mutants could be isolated under these have demonstrated that mutations in the rationale for the evolution of this elaborate conditions (Zhang and Saier, unpublished glpFK control region, allowing growth of mechanism of gene activation. results). Abolition of the CRP binding E. coli crp or cya mutants on glycerol plates, sites in the glpFK upstream regulatory or wt cells on glycerol plus 2-deoxyglucose ACKNOWLEDGMENT region greatly enhanced the frequency of or α-methyl glucoside plates, are specif- We thank Fengyi (Andy) Tang for these mutants in an otherwise wt genetic ically induced by the presence of these assistance with the preparation of this background, even in the absence of a non- compounds. The glycerol regulon repres- manuscript, and the NIH for financial metabolizable glucose analog, showing sor, GlpR, which binds to its four operators support (GM077402). that binding of the cAMP-CRP complex (O1–O4)infrontoftheglpFK operon to the glpFK control region negatively reg- (Figure 1A) and is displaced from these REFERENCES ulates IS5 insertion. These experiments sites when α-glycerol phosphate binds Cairns, J., Overbaugh, J., and Miller, S. (1988). 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