Supplemental Materials for:

Zhang, Z. and Saier, M. H.

Mechanism of Transposon-mediated Directed Mutation
in Escherichia coli

SUPPLEMENTARY TABLES & FIGURES
Figure S1:	Growth of E. coli wild type (), crp (♦),crp Glp+ (•), ihfA () and crp Glp+ ihfA (Δ) cells in liquid glycerol M9 minimal medium.
Figure S2:	Relative mutation rates under different conditions and with different genetic backgrounds. a, The rates of appearance of Glp+ mutations in a crp genetic background when grown on M9 minimal agar plates with 1% glycerol (♦) or 1% sorbitol (). These data were derived from the experiment described in Figure 1b. On glycerol minimal plates, newly formed colonies were counted daily, and the total populations were determined by the method of Cairns and Foster (1991)2. By contrast, for the experiment conducted on minimal sorbitol plates, Glp+ cells and total cells were determined by plating on minimal glycerol medium and on LB agar medium, respectively. The rates of Glp+ mutations in cells grown on M9+glucose agar plates could not be determined due to extremely low mutation rates. b, Effects of losses of GlpR and its operators O1 and O4 on mutation rates when grown in liquid LB medium at 30°C. To determine Glp+ mutation rates, fresh overnight cultures were inoculated into 20 ml of fresh LB in 125 ml flasks (initial OD600 = 0.1). The flasks were shaken at 30°C. Samples were taken at one hour intervals for five hours for determination of total populations (measured on LB plates) and Glp+ cell populations (measured on M9+Glycerol plates). The frequencies of Glp+ mutations relative to the total cell populations were plotted versus time, and the mutation rates were determined from the slopes of the curves.
Figure S3:	Effect of a recA mutation on the Glp+ mutation rate in crp cells without (a) or with (b) glycerol. In a, cells were incubated on LB agar plates, and the numbers of mutants were recorded after 36 hours. In b, cells were incubated on M9 + glycerol plates, and colonies were counted after the time intervals indicated. Error bars represent standard deviations for experiments conducted in triplicate.
Figure S4:	Fluctuation tests of Glp+ mutations in a crp genetic background when plated on M9+1% glycerol agar plates. The data are derived from Figure 1b. a, the distributions of cumulative Glp+ mutations per 103 cells on any given day. b, the distributions of new mutations arising per 103 cells for each day. 130 plates were used for the analysis.
Figure S5:	Analysis of the 5' end of the glpFK message using RNA ligase-mediated RT-PCR, showing cDNA amplification products. The arrow points to the product resulting from a newly initiated message.
Figure S6:	Effect of the loss of GlpR on Glp+ mutation rate in crp cells growing on M9 + glycerol agar plates. Glp+ mutations were assayed as described under "Methods" and the legend of Figure 1. The effect of the loss of GlpR on Glp+ mutation rate in the absence of glycerol is shown in Figure 2.
Figure S7:	Growth of crp, crp glpR, crp O1, crp O4, and crp Glp+ cells and effect of glpR overexpression on growth. a, Growth in LB liquid medium. b, growth in liquid M9 + glycerol medium. c, Effects of glpR overexpression on growth of crp glpR cells in LB with or without inducers (1 mM arabinose + 0.5 mM IAA). All growth experiments were conducted at 30deg;C. In a and b, ♦ = crp, () = crp glpR, () = crp O1, Δ = crp O4, and • = crp Glp+ (only in a). In c, ♦ = pBAD, () = pBAD-glpR, () = pBAD-glpR + arabinose + IAA.
Figure S8:	Fluctuation tests of Glp+ mutations in a crp genetic background when plated on M9+1% glycerol agar plates. The data are derived from Figure 1b. a, the distributions of cumulative Glp+ mutations per 103 cells on any given day. b, the distributions of new mutations arising per 103 cells for each day. 130 plates were used for the analysis.
Figure S9:	Effects of IB and its location on the activity of the lacZYA promoter in crp and crp lacI cells grown in minimal M9 medium + 0.66% CAA + 1% glucose without (left side) or with (right side) IPTG (200 mM). In IB:Plac and IB:Plac', IB is located at -126.5 (the same relative position as in IB:PglpFK) and -178.5 upstream of the lacZ transcriptional start site, respectively.