Nested DNA inversion as a paradigm of programmed gene rearrangement

Abstract

Programmed gene rearrangements are employed by a variety of microorganisms, including viruses, prokaryotes, and simple eukaryotes, to control gene expression. In most instances in which organisms mediate host evasion by large families of homologous gene cassettes, the mechanism of variation is not thought to involve DNA inversion. Here we report that Campylobacter fetus, a pathogenic Gram-negative bacterium, reassorts a single promoter, controlling surface-layer protein expression, and one or more complete ORFs strictly by DNA inversion. Rearrangements were independent of the distance between sites of inversion. These rearrangements permit variation in protein expression from the large surface-layer protein gene family and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.

Figure 1

Schematic representation of serial experiments to use km and cm cassettes to examine SLP gene rearrangement (A). Introduction of cm into strain 23D by marker rescue led to insertion into sapA to create 23D:AC100. This strain was selected on chloramphenicol-containing medium and, as expected, was chloramphenicol (C)-resistant, was S⁻, and not resistant to serum (S) and kanamycin (K), as previously reported (9). Incubation of 23D:AC100 with NHS selected for survivors (at a frequency of 1 × 10⁻⁴; AC200 series) that were S⁺ and serum-resistant but sensitive to kanamycin and chloramphenicol. Strain 23D:AC200, expressing sapA2, was further mutagenized by introduction of km into sapA2 to create the 23D:ACA2K100 series. These strains were S⁻ and serum- and chloramphenicol-sensitive but kanamycin-resistant. Incubation with NHS selected for survivors (at a frequency of 1 × 10⁻⁴) that were S⁺ and serum-resistant but kanamycin- and chloramphenicol-sensitive (ACA2K400 series). The reciprocal relationship between antibiotic- and serum-resistance suggests that only a single promoter for SLP gene cassettes is present. All ACA2K400 series strains must have an SLP gene cassette other than sapA or sapA2 positioned downstream of the single SLP promoter. The predicted genotypes are depicted. P, sapA promoter; bent arrows, location and direction of transcription of SLP gene cassette; ▾, antibiotic resistance gene insertion. (B) Immunoblot of C. fetus strain 23D and selected mutants into which cm or cm and km were inserted into SLP gene cassettes. As expected, all the serum-resistant strains expressed SLP (of 97 or 127 kDa) recognized by antiserum to conserved C. fetus SLP determinants, whereas there was no expression for strains maintained on either antibiotic.

Figure 2

Southern hybridization of HincII (A) or PstI (B) digestions of chromosomal DNA from C. fetus 23D and ACA2K series mutants using probes to km, cm, the promoter region, the sapA-specific 3′ region, the sapA1-middle region, or the sapA1-specific 3′ region. Each probe hybridized to a single fragment regardless of the phenotype of the C. fetus strain. (C) Mapping of SLP gene cassette arrangement by PCR. PCRs were performed with template chromosomal DNA from strains 23D and ACA2K mutants using sapA-specific 3′ region forward (sapA) and km reverse (km) primers (left four lanes), sapA1-specific 3′ region forward (sapA1) and km primers (center four lanes), or sapA and sapA1-specific 3′ region reverse (sapA1) primers (right four lanes). (D) Cumulative restriction maps of the four strains presented in A–C. The location of the probes as indicated from the hybridizations is shown under the map for each strain. sapAx represents an uncharacterized SLP gene cassette; arrows represent the direction of transcription; solid lines represent expressed genes, dashed lines represent silent genes; P over bent arrows represents the sapA promoter; and the heavy line represents the 6.2-kb invertible promoter-containing element, flanked by opposing SLP gene cassettes. The asterisks represent the palindromic putative recombinase recognition sites (TTAAGGAaTCCTTAA) present in the 5′ conserved region of each SLP gene cassette (7), and restriction sites are indicated: H, HincII; N, NdeI; P, PstI.

Figure 3

Proposed model of molecular events involved in SLP gene cassette rearrangement by DNA inversion. DNA inversion between two oppositely oriented cassettes follows DNA strand exchange at the putative recombinase target site (∗) found upstream of each SLP gene cassette within the 5′ conserved region (small shaded box; ref. 8). Patterned boxes represent variable regions of SLP gene cassettes. A 6.2-kb intervening segment is topologically reversed, leading to ordered rearrangement of the SLP gene cassettes. Inversion of DNA segments containing the promoter (P over bent arrow) permits expression of alternate SLP gene cassettes (mRNA, arrow). Illustrated are inversion of the 6.2-kb promoter-containing element alone (Left), and the 6.2-kb element and one (Center) or two (Right) SLP gene cassette ORFs and the resultant genotypes. Each of these genotypes has been observed (Fig. 2D).