A novel mechanism of transposon-mediated gene activation

Abstract

Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3' end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms.

Figure 1. Schematic diagram of IS5 and its various regions upstream of the glpFK promoter (PglpFK) region used in this study (A), the 177-bp region (IB) at the 3′ end of IS5 and the glpFK promoter region (B).

In (A), the numbers are relative to the 5′ end of IS5. IS5, P-less IS5, IB, and 178 bp refer to the entire IS5, IS5 deleted for the 98 bps from the 5′ end, the 177 bp 3′ end region of IS5 (IB), and the 178 bp region between the 226th and the 403rd nucleotides of IS5, respectively. In (B), the junction between IB and PglpFK is marked by an *. In IB, A-tracts are capitalized and numbered, and the IHF binding site is underlined. Mutations introduced into the IHF binding site or the A-tracts are labeled with letters above the sequence. In PglpFK, the +1, the promoter region (−10 and −35), the ribosome binding site (RBS) and the start codon for glpF are shaded. The GlpR binding sites are overlined and the Crp binding sites are underlined. The IS5 insertion site (CTAA) is shaded.

Figure 2. Real time PCR analysis of glpFK expression (A,B) and determination of glycerol kinase (GlpK) activity (C).

(A) glpFK mRNA levels in wt, crp and crp Glp⁺ cells. (B) glpFK mRNA levels in glpR, crp glpR and crp glpR Glp⁺ cells. (C) Glycerol kinase (GlpK) activities in wt, crp, and crp Glp⁺ cells. Cells were grown in LB with or without 1% glycerol (Gly). Radioactivity was measured by scintillation counting with 10 ml of Bio-safe II fluid.

Figure 3. Analysis of the 5′ end of the glpFK message.

(A) Gel electrophoresis of PCR products of the 5′ end of the glpFK cDNA. RNA ligase-mediated RT–PCR was employed to amplify the 5′ end of the glpFK mRNA. cDNA was synthesized using an Invitrogen superscript first-strand synthesis kit. The arrow points to the product resulting from a newly initiated message. The other band is a nonspecific PCR product. (B) Chromatogram of a part of the DNA sequence showing the junction between the 5′ end of the glpFK cDNA and the reverse transcribed adaptor. The amplified 5′ end of the glpFK cDNA was sequenced using the oligo PglpFK-extn-R (see Table S2) that binds to the ∼210 bp region downstream of +1. The arrow points to the transcriptional start site (+1) on the complementary strand.

Figure 4. Control of glpFK operon promoter activity.

(A) Effects of IS5, promoter-less IS5 and IB on expression of the downstream glpFK promoter in crp cells. ‘IS’, ‘P-less’, ‘IB’, ‘178’, ‘none’, and ‘IBZ’ refer to transcriptional lacZ fusions for IS5:PglpFK, promoter-less IS5:PglpFK, IB:PglpFK, 178 bp:PglpFK, native PglpFK, and IB alone, respectively (see Figure 1A). (B) IB:PglpFK activity in wt, crp, glpR and crp glpR cells. In both A and B, wild-type and mutant cells were grown with shaking in LB with or without 1% glycerol (Gly) or 1% glucose (Glu). (C) Effect of a 5- or 10-oligonucleotide insertion between IB and PglpFK on promoter activity. A 5 bp (TACCT) or a 10 bp (TACCTTACCT) fragment was inserted between −117 and −118 relative to +1 of PglpFK (see Figure 1B). β-Galactosidase activities of these promoters were measured in crp and crp glpR cells grown in minimal M9 medium + 0.66% casamino acids (CAA) + 1% glucose. ‘IB’, ‘IB:5’, ‘IB:10’, and ‘none’ refer to IB:PglpFK, IB:5 bp:PglpFK, IB:10 bp:PglpFK and PglpFK alone, respectively.

Figure 5. Dependency of glpFK promoter activation on IHF binding to IB (A–C) as well as the A-tract-promoting permanent bend in IB (D).

(A) Growth of crp Glp⁺ (•), ihfA (σ) and crp Glp⁺ ihfA (▒) cells in liquid glycerol (1%) M9 minimal medium. (B) Effect of a host ihfA null mutation on IB:PglpFK activity. (C) Effect of IHF binding site mutations in IB on IB:PglpFK activity. ‘None’, ‘single’, and ‘double’ refer to no mutation, mutation of TCAA (−221 to −218 relative to +1 of PglpFK) to GTCT, and mutation of TCAA to GTCT as well as TT (−213 to −212) to GC in the IHF binding site located in IB, respectively (see Figure 1B). (D) lacZ expression measured by β-galactosidase assay for crp cells carrying an IB:PglpFK-lacZ fusion with various A-tract mutations in IB. The strain bearing altered A-tracts 4–8 includes the mutations shown in A-tracts 4–8. ‘none’, ‘A-tracts 4–8’, ‘A-tract 3’ and ‘A-tracts 1–3’ refer to no mutation, mutations in A-tracts 4 to 8, mutation in A-tract 3 and mutations in A-tracts 1 to 3, respectively (see Figure 1B).

Figure 6. Effects of IB and its location on the activity of the lacZYA promoter (Plac).

(A) Effect on Plac in the absence of Crp. (B) Effect on Plac in the presence of Crp. crp, crp lacI, and wt cells were grown in M9 minimal medium + 0.66% CAA + 1% glucose without or with IPTG (200 µM). 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.