A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Although mycoplasmas lack cell walls, they are in many respects similar to the gram-positive bacteria with which they share a common ancestor. The molecular biology of mycoplasmas is intriguing because the chromosome is uniquely small (< 600 kb in some species) and extremely A-T rich (as high as 75 mol% in some species). Perhaps to accommodate DNA with a lower G + C content, most mycoplasmas do not have the "universal" genetic code. In these species, TGA is not a stop codon; instead it encodes tryptophan at a frequency 10 times greater than TGG, the usual codon for this amino acid. Because of the presence of TGA codons, the translation of mycoplasmal proteins terminates prematurely when cloned genes are expressed in other eubacteria, such as Escherichia coli. Many mycoplasmas possess strikingly dynamic chromosomes in which high-frequency changes result from errors in DNA repair or replication and from highly active recombination systems. Often, high-frequency changes in the mycoplasmal chromosome are associated with antigenic and phase variation, which regulate the production of factors critical to disease pathogenesis.

Microorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high-frequency DNA rearrangements which are the subject of this review.

The chromosome of the murine pathogen Mycoplasma pulmonis undergoes rearrangements at a high frequency. We show that some of these rearrangements regulate the phase-variable expression of a cluster of genes (the vsa locus) that encode the variable V-1 surface antigens. Only one vsa gene is associated with an expression site; the other vsa genes are transcriptionally silent. The silent genes lack the 5' end region (promoter and ribosome-binding site) that is present in the expressed gene, and DNA rearrangements regulate gene expression by reassorting the 5' end region from an expressed gene with the 3' end region from a previously silent gene. All vsa rearrangements identified so far are site-specific DNA inversions that occur between copies of a specific 34 bp sequence that is conserved in each vsa gene. Interestingly, DNA inversions within the vsa locus apparently occur in concert with inversion of the hsd1 element, which regulates restriction and modification activity in M. pulmonis.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identi®ed and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

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...

SummaryTo obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn4001T was determined for 1700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared with those reported for Mycoplasma genitalium and Bacillus subtilis. Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA, uvrA, uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N-acetylglucosamine was identified.

BackgroundMycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40 % of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England.ResultsIllumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99 % identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes.ConclusionsThese data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1801-0) contains supplementary material, which is available to authorized users.