Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

Abstract

Mycoplasma genitalium is an emerging sexually transmitted pathogen associated with reproductive tract disease in men and women, and it can persist for months to years despite the development of a robust antibody response. Mechanisms that may contribute to persistence in vivo include phase and antigenic variation of the MgpB and MgpC adhesins. These processes occur by segmental recombination between discrete variable regions within mgpB and mgpC and multiple archived donor sequences termed MgPa repeats (MgPars). The molecular factors governing mgpB and mgpC variation are poorly understood and obscured by the paucity of recombination genes conserved in the M. genitalium genome. Recently, we demonstrated the requirement for RecA using a quantitative PCR (qPCR) assay developed to measure recombination between the mgpB and mgpC genes and MgPars. Here, we expand these studies by examining the roles of M. genitalium ruvA and ruvB homologs. Deletion of ruvA and ruvB impaired the ability to generate mgpB and mgpC phase and sequence variants, and these deficiencies could be complemented with wild-type copies, including the ruvA gene from Mycoplasma pneumoniae. In contrast, ruvA and ruvB deletions did not affect the sensitivity to UV irradiation, reinforcing our previous findings that the recombinational repair pathway plays a minor role in M. genitalium. Reverse transcription-PCR (RT-PCR) and primer extension analyses also revealed a complex transcriptional organization of the RuvAB system of M. genitalium, which is cotranscribed with two novel open reading frames (ORFs) (termed ORF1 and ORF2 herein) conserved only in M. pneumoniae. These findings suggest that these novel ORFs may play a role in recombination in these two closely related bacteria.W ith a genome of only 580 kb encoding 482 predicted proteins, Mycoplasma genitalium is considered the organism with the smallest known genome capable of self-replication (1). Consequently, M. genitalium has become a model organism for understanding basic biological processes (2-4) and has been used as a platform to identify a minimal gene set required for autonomous cellular life (5). M. genitalium is also a sexually transmitted human pathogen with an implicated reproductive tract disease spectrum very similar to that of Chlamydia trachomatis, including urethritis in men (6) and cervicitis, endometritis, pelvic inflammatory disease (PID), and tubal factor infertility in women (7). M. genitalium infections are often asymptomatic and persistent (8-10), possibly increasing the risk for sexual transmission and sequelae such as PID and infertility. Despite the significance of primary disease and its sequelae, the molecular pathogenesis of M. genitalium has been understudied. Barriers to such investigations include the fastidious nature and slow growth of this bacterium, difficulty of isolating contemporary strains from human specimens, and the limited genetic tools available for molecular investigations (11).In previous studies, we and others have shown that M. genitalium strains gen...

MG428 is a novel positive regulator of recombination that triggers mgpB and mgpC gene variation in Mycoplasma genitalium

“…Blue peaks represent primer extension products and red peaks size standards (GeneScan 400HD ROX; Applied Biosystems). Below the electropherograms are schematics showing the genetic organization of recA and ruvAB locus with mRNA species (dashed arrows) predicted from this work and from a previous study (Burgos and Totten, ). Black bent arrows indicate the positions of TSSs previously described within the ruvAB operon (Burgos and Totten, ), and red bent arrows denotes the position of transcripts induced by MG428.…”

“…Below the electropherograms are schematics showing the genetic organization of recA and ruvAB locus with mRNA species (dashed arrows) predicted from this work and from a previous study (Burgos and Totten, ). Black bent arrows indicate the positions of TSSs previously described within the ruvAB operon (Burgos and Totten, ), and red bent arrows denotes the position of transcripts induced by MG428. The small arrows labelled with PE indicate location of the primers used for primer extension analysis (http://onlinelibrary.wiley.com/doi/10.1111/mmi.12760/suppinfo).…”

“…To identify promoter elements associated with MG428 activation, we mapped the transcriptional start sites (TSSs) of the induced transcripts using a fluorescence‐based primer extension protocol (Lloyd et al ., ; Burgos and Totten, ). Consistent with the observation that deletion of the recA URS does not affect RecA expression, we were unable to detect initiation of transcription upstream of the recA coding region in wild‐type cells (Fig.…”

“…The common DNA motif located between − 10 and − 35 promoter elements is in blue. For reference, both the position of IR‐3 within the recA URS, and an imperfect inverted repeat previously noted within the ORF2 URS (Burgos and Totten, ) are shown by converging arrows.…”

“…In this study, we have identified a novel factor, MG428, which positively regulates recombination and mgpBC gene variation in the human pathogen M. genitalium . Previously, we demonstrated that mgpBC gene diversity results from reciprocal‐recombination events (Iverson‐Cabral et al ., ) involving key recombination factors such as RecA, RuvA and RuvB (Burgos et al ., ; Burgos and Totten, ). Now, we show that overexpression of MG428 enhances the frequency of genetic variation and this increase correlates with the transcriptional activation of recA , ruvA and ruvB .…”