Diversity and strength of internal outward-oriented promoters in group IIC-attC introns

Abstract

Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (P(out)). Bioinformatic analyses indicate that one or two putative P(out), which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that P(out) with different versions of the -35 and -10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. P(out) in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron.

Figure 1.

Class 1 integron and cassette arrays. (A) Schematic diagrams of the general structure of a class 1 integron. P, promoters; intI1, integrase gene; qacEΔ1, antiseptic resistance gene; sul1, sulfonamide resistance gene; orf5, gene of unknown function. (B) Schematic diagrams of the variable region (gene cassettes) of class 1 integrons found in S. marcescens SCH909, E. coli 702, K. pneumoniae and Acinetobacter genomospecies genomes. The gray arrows indicate cassette ORFs; the gray boxes indicate cassette attC sites; the white rectangles and arrows indicate group IIC-attC introns with their intron encoded proteins (IEP); and P_out indicates a putative outward-oriented promoter within the intron.

Figure 2.

Thin-layer chromatography of the [¹⁴C]chloramphenicol (Cm) CAT assay products in order to determine the effect of S.ma.I2 insertion in the integron #2 from S. marcescens. The pKK232 clones used for this assay are described in Table 1. The TLC plate was exposed to a Kodak BioMax MR film to obtain this image. CAT activity was assayed as described in ‘Materials and Methods’ section. (a) indicates the origin; (b) indicates non-acetylated Cm; (c) indicates 1-acetoxy-Cm; (d) indicates 3-acetoxy-Cm.

Figure 3.

Phylogenetic tree for group IIC-attC intron IEP amino acid sequences from various organisms. Evolutionary distances were computed using the neighbor-joining algorithm of the MEGA4 software (‘Materials and Methods’ section).

Figure 4.

Agarose gel (2%) of the 5′-RACE-PCR products. 5′-RACE assays were performed as described in ‘Materials and Methods’ section in order to find the transcription initiation site of cat in the indicated pKK232 clones (see Table 1 for description).

Figure 5.

Alignments of the 5′-RACE product sequences with their corresponding intron DNA sequences. ts and bs indicate the top strand and bottom strand sequences; −35 and −10, components of the promoter. +1, transcription initiation site. Reversed and complemented 5′-RACE sequences are indicated (asterisks).

Figure 6.

Thin-layer chromatography of the [¹⁴C]chloramphenicol (Cm) CAT assay products for determination of the relative strengths of group IIC-attC intron promoters (P_out). The TLC plate was exposed to a Kodak BioMax MR film to obtain this image. CAT activity was assayed as described in ‘Materials and Methods’ section. The pKK232 clones used for this assay are described in Table 1. pLQ872 and pLQ876 contain the weak and strong versions of integron Pc promoters (respectively) cloned into pKK232-8 (1). (a) indicates the origin; (b) indicates non-acetylated Cm; (c) indicates 1-acetoxy-Cm; (d) indicates 3-acetoxy-Cm.