Precise excision of IS5 from the intergenic region between the fucPIK and the fucAO operons and mutational control of fucPIK operon expression in Escherichia coli

Abstract

Excision of transposable genetic elements from host DNA occurs at low frequencies and is usually imprecise. A common insertion sequence element in Escherichia coli, IS5, has been shown to provide various benefits to its host by inserting into specific sites. Precise excision of this element had not previously been demonstrated. Using a unique system, the fucose (fuc) regulon, in which IS5 insertion and excision result in two distinct selectable phenotypes, we have demonstrated that IS5 can precisely excise from its insertion site, restoring the wild-type phenotype. In addition to precise excision, several "suppressor" insertion, deletion, and point mutations restore the wild-type Fuc(+) phenotype to various degrees without IS5 excision. The possible bases for these observations are discussed.

FIG. 1.

The intergenic region between the fucPIK operon and the fucAO operon. In the fucPIK promoter region, the transcriptional initiation site (+1) is capitalized. The CTAG IS5 target site, the −10 and −35 hexamers, the ribosome binding site (RBS) and the ATG start codon for fucP are shaded. The two Crp binding sites (CrpI and CrpII) are underlined. These annotations correspond to those of Podolny et al. (20) and Regulon DB (http://regulondb/ccg.unam.mx). The black backward arrow above the sequence indicates the location of IS5 upstream of the fucPIK operon in PPD⁺ Fuc⁻ cells. The arrow shows the orientation of IS5 with the defined left end linked to the downstream fucAO promoter. In the fucAO promoter region, the ribosome binding site and the start codon for fucA are shaded. The promoter region for the fucAO operon has not been characterized, and the Crp binding sites have not been confirmed by experimentation.

FIG. 2.

PCR products of the fucPIK promoter regions from wild-type cells, the IS5 excision mutants, and Fuc⁺ pseudorevertants that are derived from the Fuc⁻ PPD⁺ cells. Lane “kb” shows DNA size markers. Lane “wt” shows the PCR products of the promoter region from wild-type cells. Lanes 6 and 12 show the PCR products of the promoter region from IS5 excision revertants, and the others show the PCR products from the pseudorevertants. The primers used for PCR amplification are listed in Table 1.

FIG. 3.

Three predicted promoters created by three types of mutations (A) and a diagram showing the predicted secondary structure of the region between the transcriptional start site (+1) and the start codon of the fucP gene (B). In panel A, the −35 hexamer is the same for all three potential promoters; only the −10 hexamer and the spacings between them differ. In panel B, the structure was predicted using the CONTRAfold program. The mutations present downstream of the +1 position that give rise to a Fuc⁺ phenotype in a PPD⁺ background are labeled.

FIG. 4.

Growth of the E. coli wild type and Fuc⁺ revertants derived from a PPD⁺ strain on l-fucose in minimal M9 medium. Cells from fresh LB cultures were washed once with M9 medium lacking a carbon source and resuspended in the same medium. These cell suspensions were used to inoculate M9 media (6 ml/50-ml glass tube) containing 0.5% l-fucose to an optical density at 600 nm (OD₆₀₀) of 0.1. The tubes were shaken at 37°C. ⧫, wild type; ⋄, Fuc⁺ revertant with IS5 excised; ▴, Fuc⁺ pseudorevertant with the Crp(K53N) mutation; •, Fuc⁺ pseudorevertant with a 13-bp deletion between +1 and +15 in PfucPIK; ○ Fuc⁺ pseudorevertant with a G-to-T substitution at position −12 in PfucPIK; and ▪, the original PPD⁺ mutant (Fuc⁻).

FIG. 5.

Growth of E. coli wild type and Fuc⁺ revertants derived from a PPD⁺ strain at 37°C in 1% l-1,2-propandiol in M9 minimal medium. ⧫, wild type; ⋄, a Fuc⁺ revertant with IS5 precisely excised; ▴, a Fuc⁺ pseudorevertant with the Crp(K53N) mutation; •, a Fuc⁺ pseudorevertant with a 13-bp deletion between +1 and +15 in PfucPIK; ○, a Fuc⁺ pseudorevertant with a G-to-T substitution at position −12 in PfucPIK; ▪, the original PPD⁺ mutant (Fuc⁻).