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.Bacterial restriction and modification (R-M) systems function as a defense mechanism conferring protection from phage infection and other types of DNA invasion. Incoming DNA lacking the appropriate base modifications is cleaved by the restriction endonuclease while host DNA is protected by the presence of sequence-specific base modifications introduced by the activity of the DNA methyltransferase (MTase). These systems usually confer incomplete protection from phage infection because some phage DNA molecules are modified by the MTase before the endonucleolytic cleavage can occur, giving rise to progeny phage containing DNA modifications identical to that of the host and, therefore, resistant to restriction activity.The type I restriction enzymes are considerably more complex than the more prevalent type II enzymes. Type II restriction activity and MTase activity are performed by two distinct enzymes encoded by gene pairs. The endonuclease reaction is sequence-specific and occurs at or very near to the site modified by the MTase. In the type I systems, the nuclease and MTase activities are performed by the same holoenzyme, which consists of three types of subunits. The S subunit dictates the sequence specificity of the MTase activity. Both the S and M subunits are necessary and sufficient for MTase activity, which occurs at specific adenine residues. All three types of subunits (S, R, and M) are required for the nuclease reaction, which occurs at essentially random sites up to 7 kilobases from the recognition sequence (1). Translocation of the holoenzyme from the recognition site to the cleavage site occurs through the ATP-dependent helicase activity of the R subunit.It has been proposed that some restriction systems are selfish beca...

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…Pathogenic bacteria use the phase variability of gene expression to evade the host immune system. Based on sequence analysis and biochemical evidence, many type I and III enzymes of Bacteroides fragilis as well as Haemophilus, Helicobacter, Mycoplasma, and Neisseria species were found to be potentially phase variable (176)(177)(178)(179)(180)(181). The biological significance of R-M systems that exhibit phase variability is not completely understood.…”

“…On the other hand, in a restriction-on phase (r ϩ ), REase would limit invasive xenogeneic DNA. It was proposed that the phase-variable expression of R-M systems might fine-tune these two phases in the population (179,185). The finetuning of foreign DNA uptake might be an important process in naturally competent bacteria, viz., B. subtilis, H. influenzae, H. pylori, N. gonorrhoeae, and S. pneumoniae.…”

Reannotation of the Corynebacterium diphtheriae NCTC13129 genome as a new approach to studying gene targets connected to virulence and pathogenicity in diphtheria

“…This mechanism is seen as a preventive action taken by the cell against invasion by foreign DNA, principally by bacteriophages. 37,38 In C. diphtheriae, there is a complete operon with the three genes present: hsdR-S-M (DIP2312, DIP2313, and DIP2314, respectively), and the interrupted gene hsdM (DIP2081) in another region of the genome. In many populations of Mycoplasma pulmonis, the presence of these enzymes was not detected, and even with intact genes, the bacterium is susceptible to infection by bacteriophages.…”

Restriction-modification systems in Mycoplasma spp

“…Since mycoplasmas possess only one sigma factor and a low number of transcription factors, other mechanisms are proposed for regulation of gene expression. Phase variation is an important mechanism for generating protein variability in several microorganisms, including mycoplasmas (Dybvig et al, 1998;GumulakSmith et al, 2001). Interestingly, short tandem repeated sequences have been observed in genes encoding proteins of the R-M system in bacteria (Ryan and Lo, 1999;Saunders et al, 1998;De Bolle et al, 2000).…”

“…Interestingly, phase variation was observed in some genes encoding Type I and Type III enzymes (Dybvig et al, 1998;Rocha and Blanchard, 2002). Although the importance of this mechanism for the biology of mycoplasmas is not clear, a possible relationship to virulence has been proposed for Mycoplasma pulmonis (Gumulak-Smith et al, 2001).…”