A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements

Abstract

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

Figure 1

Organization of the M. pulmonis hsd1 (Upper) and hsd2 (Lower) loci. To better illustrate features within the hsdS genes, the hsdM and hsdR genes were shortened and were not drawn to scale. Homologous regions within hsdS are shown by like shading. The sites labeled α, β, γ, and δ refer to vip DNA inversion sites. The sites labeled hrs1, hrs2, and hrs3 are additional sites for DNA inversion. The single promoter driving hsd transcription is labeled by P. The orientations of primer binding sites for PCR analysis are indicated by arrows.

Figure 2

Southern blot analysis of HindIII-digested M. pulmonis chromosomal DNA hybridized with the hsdS-specific probe described in Materials and Methods. Strain abbreviations: 15, KD735–15; 16, KD735–16H; 17, KD117; 129, KD129; etc.

Figure 3

Schematic diagrams of the hsdS genes associated with each restriction group. Shaded segments refer to regions of homology as shown in Fig. 1. Recombination sites (vip and hrs) and promoter (P) sites are as in Fig. 1.

Figure 4

Alignment of the vip and hrs DNA inversion sites of the hsd2 locus. Sequences are from strains KD735–15 or KD129 with the abbreviated strain designation in parenthesis. Boxes are drawn around the 12-nt vip sequences and the 20-nt hrs sequences.