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.
Subject(s)
DNA Restriction-Modification Enzymes/genetics , Gene Rearrangement , Genes, Bacterial , Mycoplasma/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Mycoplasma/enzymologyABSTRACT
Two paralogous, site-specific invertible loci, designated hsd1 and hsd2 (host specificity determinant), have been identified in the Mycoplasma pulmonis genome. They encode putative type I restriction and modification (R-M) systems with maximum sequence homology to the type IC family, which includes EcoR124II and EcoDXXI. Each locus encodes an endonuclease subunit (HsdR), a methylase subunit (HsdM) and two DNA specificity subunits (HsdS). The gene organization at each locus is such that hsdR and hsdM are flanked by two hsdS genes. Within each locus, one of the hsdS genes, hsdR and hsdM, is encoded in tandem by the same DNA strand, while the second hsdS gene is encoded by the complementary strand but without overlap with the other three hsd genes. The hsdR and hsdM sequences of one locus are almost identical to their counterparts in the other. The four hsdS genes (two per locus) are highly homologous at their 5' ends and also share sequence similarities in the 3' ends of their corresponding coding regions. Owing to the disposition of and sequence similarities among the hsdS genes, they form inverted repeats at each locus. Analysis by polymerase chain reaction (PCR) has shown that both loci behave as site-specific DNA invertible elements with multiple inversion sites, termed 'vipareetus', occurring within the hsdS genes. The inversions lead to a reassortment of hsdS sequences, generating an array of recombinant genes that probably encode S subunits possessing alternative DNA-binding specificities. Sequence information obtained from the analysis of hsd2 transcripts by 5' RACE (rapid amplification of cDNA ends) indicates that inversion induces the transcription of alternative hsdS genes by the relocation of coding sequences downstream of a promoter and ribosome-binding site (RBS) situated at one end of each locus.
Subject(s)
Bacterial Proteins/genetics , DNA Restriction-Modification Enzymes , Deoxyribonucleases, Type I Site-Specific , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Mycoplasma/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , Cloning, Molecular , DNA Primers , Electrophoresis, Agar Gel , Gene Rearrangement , Genes, Bacterial/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
The effect of an Ayurvedic preparation consisting of Swasakutara, Curcuma longa (Haridra) and Withania somnifera (Asvagandha) is accessed in 12 patients of tropical eosinphilia. The modern control drug Hetrazan is used in another batch of 11 patients for comparison. The Ayurvedic compound causes complete relief of most of the clinical signs and symptoms associated with the disease and reduces E.S.R. significantly. However the drug has no effect on the level of circulating eosinophils.
