Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions

Abstract

Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5 upstream region (avg-B 2 ) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B 2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5 upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.Mycoplasma agalactiae belongs to the genus Mycoplasma, which comprises more than 180 wall-less species that represent the smallest self-replicating life forms on earth and phyogenetically are related to gram-positive eubacteria (28, 29). Most mycoplasmas have been identified as infectious agents of human or other animals (29, 36). M.agalactiae, commonly known as the cause of contagious agalactia of sheep and goats, is one of the most devastating mycoplasmal pathogens of small ruminants (3). The disease is expressed clinically as mastitis, arthritis, and keratoconjunctivitis and is of a considerable economic importance worldwide, particularly in the Mediterranean basin (3). The pathogen is present in milk and other body fluids of infected animals and also in animals that do not exhibit clinical signs. These asymptomatic carrier animals play an important role in transmission of the disease (3). The successful persistence of M. agalactiae within hosts indicates that frequently the host defense mechanisms fail to eliminate the bacterial pathogen. Recent advances in the molecular biology of mycoplasmas have indicated that high-frequency variation of surface components, particularly lipoproteins, plays an important role in establishing th...

Avoidance of the Host Immune System through Phase Variation in Mycoplasma pulmonis

“…Subpopulations containing fewer tandem repeats were found equally in isolates from wild-type and immunodeficient animals. on May 10, 2018 by guest http://iai.asm.org/ plasma agalactiae (10,11), the P35 family of proteins from Mycoplasma penetrans (16), and the Vsa proteins of M. pulmonis all use DNA inversion as a mechanism for phase variation. The MB antigen of Ureaplasma urealyticum likely uses a similar mechanism because it has been shown to phase vary (22), and a gene encoding a predicted site-specific DNA recombinase is present near the locus that encodes the MB antigen (http://cbi .labri.fr/outils/molligen/).…”

Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

“…These surface-associated proteins vary in expression at an unusually high frequency, and only one vpma gene at a time is transcribed from a single promoter present in that locus, while all other genes are silent (9,10). An open reading frame (ORF) with homology to the -integrase family of site-specific recombinases was found in the vicinity of the vpma locus and was predicted to mediate DNA inversions responsible for switching the promoter from an active vpma gene to a silent one, resulting in alteration of vpma expression (9,10). This recombinase, designated Xer1, was indeed recently demonstrated to be responsible for phase variation of Vpma proteins (4).…”

“…The so-called vpma locus constitutes a family of six distinct but related genes that encode major immunodominant membrane lipoproteins, the Vpmas (variable proteins of Mycoplasma agalactiae) (10,11). These surface-associated proteins vary in expression at an unusually high frequency, and only one vpma gene at a time is transcribed from a single promoter present in that locus, while all other genes are silent (9,10). An open reading frame (ORF) with homology to the -integrase family of site-specific recombinases was found in the vicinity of the vpma locus and was predicted to mediate DNA inversions responsible for switching the promoter from an active vpma gene to a silent one, resulting in alteration of vpma expression (9,10).…”

“…The important ruminant pathogen Mycoplasma agalactiae causes contagious agalactia in sheep and goats and exhibits antigenic diversity by site-specific DNA rearrangements within a pathogenicity island-like gene locus (9,10,26). The so-called vpma locus constitutes a family of six distinct but related genes that encode major immunodominant membrane lipoproteins, the Vpmas (variable proteins of Mycoplasma agalactiae) (10,11).…”

Occurrence, Plasticity, and Evolution of the vpma Gene Family, a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae

“…Usually, these systems combine a set of contingency genes with a molecular switch for turning expression on or off that is based on either (i) spontaneous mutation (slipped-strand mispairing), (ii) gene conversion, or (iii) specific DNA rearrangements (9). While high-frequency phenotypic variation using the two first mechanisms has been described thoroughly for other bacteria (47), switching of surface components by shuffling of silent genes at a particular single expression locus has been studied mainly in mycoplasmas (3,8,14,16,23,39,43).…”