reca mediates mgpb and mgpc phase and antigenic variation in mycoplasma genitalium, but plays a...

RecA mediates MgpB and MgpC phase and antigenicvariation in Mycoplasma genitalium, but plays a minor role inDNA repairmmi_8130 1..15

Raul Burgos,1 Gwendolyn E. Wood,1 Lei Young,2

John I. Glass2 and Patricia A. Totten1*1Division of Infectious Diseases, Department ofMedicine, University of Washington, Seattle, WA 98104,USA.2J. Craig Venter Institute, 9704 Medical Center Drive,Rockville, MD 20850, USA.

Summary

Mycoplasma genitalium, a sexually transmitted humanpathogen, encodes MgpB and MgpC adhesins thatundergo phase and antigenic variation through recom-bination with archived ‘MgPar’ donor sequences. Themechanism and molecular factors required for thisgenetic variation are poorly understood. In this study,we estimate that sequence variation at the mgpB/Clocus occurs in vitro at a frequency of > 1.25 ¥ 10-4

events per genome per generation using a quantitativeanchored PCR assay. This rate was dramaticallyreduced in a recA deletion mutant and increased in acomplemented strain overexpressing RecA. Similarly,the frequency of haemadsorption-deficient phase vari-ants was reduced in the recA mutant, but restored bycomplementation. Unlike Escherichia coli, inactiva-tion of recA in M. genitalium had a minimal effect onsurvival after exposure to mitomycin C or UV irradia-tion. In contrast, a deletion mutant for the predictednucleotide excision repair uvrC gene showed growthdefects and was exquisitely sensitive to DNA damage.We conclude that M. genitalium RecA has a primaryrole in mgpB/C–MgPar recombination leading to anti-genic and phase variation, yet plays a minor role inDNA repair. Our results also suggest that M. geni-talium possesses an active nucleotide excision repairsystem, possibly representing the main DNA repairpathway in this minimal bacterium.

Introduction

Mycoplasma genitalium is an emerging sexually transmit-ted pathogen associated with acute and chronic urethritisin men (Jensen, 2004), and cervicitis, endometritis, pelvicinflammatory disease and tubal factor infertility in women(Cohen et al., 2002; Manhart et al., 2003; Haggerty, 2008).When untreated or inappropriately treated, M. genitaliumpersists for months or even years (Iverson-Cabral et al.,2006; Cohen et al., 2007), potentially increasing the risk forsexual transmission and serious upper reproductive tractinfection. Besides its importance as a pathogen, M. geni-talium has the smallest genome (580 kb, encoding 482predicted proteins) described so far for an organismcapable of self-replication (Fraser et al., 1995), and thushas served as a model organism to study the minimalrequirements to sustain life (Carvalho et al., 2005; Glasset al., 2006).

The molecular basis of M. genitalium pathogenesis andthe mechanisms responsible for persistence are poorlyunderstood, yet are thought to be linked in part to itscomplex terminal organelle. This distinctive structure medi-ates adherence, motility and cell division (Burgos et al.,2006; 2007; Lluch-Senar et al., 2010) and is composed ofa complex array of unique proteins (Krause and Balish,2004). Among these proteins are MgpB (also referred to asP140, MgPa or MG_191) and MgpC (also referred to asP110 or MG_192), two surface-exposed proteins essentialfor cell adhesion and terminal organelle development(Burgos et al., 2006). Although MgpB and MgpC areencoded in a single expression site located at the MgPaoperon, certain segments of the mgpB and mgpC genes(referred to herein as mgpB/C) are found in multiple copiesthroughout the genome (Iverson-Cabral et al., 2006; 2007;Ma et al., 2007). These partial copies are known as mgpBrepeat regions B, EF and G, and mgpC repeat region KLM,and are organized in nine distinct chromosomal locationstermed MgPa repeats (MgPars 1 to 9). Overall, the MgParsare 78–90% identical to the corresponding sequenceswithin the expression site and represent 4% of the reducedgenome (Fraser et al., 1995). These observationsprompted the hypothesis that recombination betweensequences in the mgpB/C expression site and the MgPars

Accepted 5 June, 2012. *For correspondence. E-mail [email protected]; Tel. (+1) 206 897 5350; Fax (+1) 206 897 5363.

Molecular Microbiology (2012) � doi:10.1111/j.1365-2958.2012.08130.x

© 2012 Blackwell Publishing Ltd

could mediate MgpB and MgpC antigenic variation (Peter-son et al., 1995). In addition, the distribution and architec-ture of the repetitive sequences within the MgPar sites alsoprovides a mechanism to promote deletions in the mgpB/Cgenes, thus also contributing to MgpB/C phase variation(Burgos et al., 2006). The observation that MgpB andMgpC are among the most immunogenic proteins ofM. genitalium (Svenstrup et al., 2006; Iverson-Cabralet al., 2011) sustains the notion that antigenic variation inthese proteins may enable this microorganism to evade theimmune response and establish persistent infections. Insupport of this hypothesis, we and others have demon-strated that sequence heterogeneity of mgpB/C is exten-sive in vitro and in vivo (Iverson-Cabral et al., 2006; Maet al., 2007) and evolves over time in persistently infectedwomen (Iverson-Cabral et al., 2007).

The isolation and characterization of mgpB/C clonalvariants revealed that mgpB/C gene diversity is achievedthrough segmental and reciprocal recombination with theMgPars (Iverson-Cabral et al., 2007). Of note, this mecha-nism differs from other known antigenic variation systemsbased on DNA recombination, which are generally char-acterized by unidirectional gene conversion events (Vinket al., 2011). Although the specific molecular factors andmechanisms underlying mgpB/C gene variation are notknown, the outcome of this diversity suggests thatthe general recombination apparatus of M. genitaliumcould be involved. However, genome sequence analysisrevealed that many of the genes typically involved inbacterial recombination and DNA repair are apparentlyabsent in this organism (Carvalho et al., 2005). Particu-larly intriguing is the apparent lack of genes involved in theinitiation step of recombination, such as recFOR, recBCDor addAB (Rocha et al., 2005). Thus, how recombinationevents are initiated remains enigmatic and suggests thatadditional and novel factors may be required to elicit andpossibly regulate the mgpB/C recombination. Neverthe-less, the M. genitalium genome contains genes encodinghomologues of the basic recombination enzymes requiredto promote strand exchange, branch migration and reso-lution of the Holliday junction, all of which are essentialsteps in recombination (Kowalczykowski et al., 1994).Among these genes are MG_339 which encodes a RecAhomologue exhibiting DNA strand exchange activity invitro (Sluijter et al., 2009), MG_358 and MG_359 whichencode homologues for the RuvA and RuvB branch migra-tion proteins (Estevao et al., 2011) and MG_352 whichencodes a RecU homologue displaying Holliday junction-resolving activity on synthetic substrates (Sluijter et al.,2010). Although the in vitro activities of these proteinshave been recently analysed (Vink et al., 2011), no geneticstudy elucidating the enzymatic machinery required topromote mgpB/C diversity in M. genitalium cells has beenreported.

In the present study, we examined the role of theMG_339 (recA) gene of M. genitalium on mgpB/C–MgParrecombination and DNA repair. We demonstrate thatRecA is required to promote sequence diversity at themgpB/C locus. Similarly, we show that haemadsorption-deficient (HA-) phase variants arise predominantly by aRecA-dependent mechanism, consistent with MgpB/Cphase variation mediated by mgpB/C–MgPar recombina-tion (Burgos et al., 2006). In contrast to other bacterialspecies, we also show that RecA has a minor role in DNArepair, whereas nucleotide excision repair (NER) could bethe main DNA repair pathway in M. genitalium.

Results

Construction of a M. genitalium recA null mutant

The M. genitalium RecA protein is encoded at the MG_339locus, which is flanked by the MG_338 gene (putativelyencoding a lipoprotein) and the MgPa repeat region 9(Fig. 1A). To investigate the function of RecA in M. geni-talium, we constructed a recA null mutant by gene replace-ment. For this purpose, we produced the suicide plasmidpDMG_339, which contains the chloramphenicol resis-tance gene inserted into regions flanking the MG_339locus. A double crossover event between plasmidpDMG_339 and the Mycoplasma genome replaces bases427113 to 430580 by the antibiotic marker (base co-ordinates refer to GenBank Accession Number NC_000908). The resulting 3469 bp deletion includes the full-length recA open reading frame except for the 3′-terminal23 bp (deletion of the 97.7% coding sequence), and theentire MgPar 9 (Fig. 1A). The intended deletion was con-firmed by PCR amplification with selected primers (Fig. 1B)and Southern blot analyses (Fig. S1). To our knowledge,this is the first report of the successful use of the chloram-phenicol resistance gene to genetically modify M. geni-talium, thus expanding the selective markers available forthis microorganism (Pich et al., 2006a; Algire et al., 2009).

The absence of M. genitalium RecAprotein in the mutantwas further demonstrated by immunoblot analyses usingpolyclonal antibodies raised against M. genitalium RecA(Fig. 1C). Interestingly, we found that these antibodiesrecognized two main bands on immunoblots of wild-typecell lysates (Fig. 1C), even though the protein is encodedby a single gene. One of these bands migrated slightlybelow the predicted molecular size of 37.4 kDa, whereasthe second band was approximately 34 kDa. A third faintband that migrated slightly higher than the 34 kDa bandwas also observed. Remarkably, these bands were absentin the DrecA mutant and were restored upon complemen-tation, thus confirming their RecA origin (Fig. 1C). Tofurther demonstrate the specificity of our antibodies recog-nizing M. genitalium RecA species, we analysed M. geni-

2 R. Burgos et al. �

© 2012 Blackwell Publishing Ltd, Molecular Microbiology

talium and Escherichia coli cell lysates expressing His-tagged and non-tagged RecA variants (Fig. S2). Interest-ingly, only the slow migrating band of ~ 37 kDa wasdetected when M. genitalium RecA protein was expressedin E. coli (Fig. S2D). In contrast, when we analysed celllysates of a M. genitalium DrecA mutant expressing aHis-tagged RecA derivative, we detected the three RecAprotein species with the expected shift in their electro-phoretic mobility (Fig. S2C). Taken together, these resultssuggest that M. genitalium expresses several isoforms ofRecA, although further studies are needed to confirm thishypothesis.

Frequency and rate of mgpB/C gene variation

Several studies have demonstrated that variation withinthe mgpB/C expression site is a result of recombination

with the MgPars (Iverson-Cabral et al., 2006; 2007; Maet al., 2007), yet the frequency of these events is notknown. To address this question, we developed severalqPCR-based assays to estimate the frequency of selectedmgpB/C variants in a given population. As depicted inFig. 2A, this assay is based on the use of a pair of primers,one targeting a conserved region in the mgpB/C expres-sion site, the other targeting a unique sequence in one ormultiple MgPars, resulting in a PCR product only when thetargeted sequence from a MgPar is translocated into theexpression site (generating a mgpB/C variant). Thus, whenthe resulting products are quantified by real-time PCR andnormalized to the number of genomes used in the assay,we can estimate the frequency of variants containing thetargeted MgPar sequences within the population. Basedon this strategy, we designed four different anchoredqPCRs, designated B-24, EF-2478, G-135 and KLM-7

Fig. 1. Gene replacement at the MG_339 locus.A. Genome organization at the MG_339 (recA) locus and schematic representation of the construction of a recA deletion mutant byhomologous recombination. Small arrows represent the primers used for the mutant screening and dotted lines show the predicted PCRproducts for the wild-type (WT) and DrecA mutant.B. PCR confirmation of the intended deletion at the recA locus. Genomic DNA from wild-type, DrecA and DrecA+recA strains were used as atemplate for PCR reactions using primers 5′mg339 and 3′mg340int (PCR 1) and 5′RTMG_339 and 3′mg339 (PCR 2). Primer sequences areshown in Table S1. PCR 1 detects replacement of the recA and MgPar 9 sequences by the chloramphenicol resistance gene (cat). PCR 2demonstrates the absence of recA gene sequences. PCR products were electrophoresed on a 1% agarose gel. Lane M: kb DNA ladder(Stratagene).C. Immunoblot analysis of wild-type, DrecA and DrecA+recA strains with polyclonal antibodies raised against M. genitalium RecA recombinantprotein. Ten micrograms of whole-cell extract was applied to each lane. Apparent molecular weights in kDa are indicated on the left side. Thefull-length blot is presented in Fig. S2B.

M. genitalium RecA in gene variation and DNA repair 3


(see Experimental procedures). In combination, theseqPCR assays detect variants spanning all four of thevariable regions (B, EF, G and KLM) and almost all MgParsites (except for MgPar 9 and the small MgPar 6 site)(Iverson-Cabral et al., 2007).

Using these qPCR assays, we found that the frequencyof variants detected per genome in our M. genitalium clone

G37C (see Experimental procedures) after two passages(seven generations each) was 2.1 ¥ 10-4, 3.4 ¥ 10-4,9.1 ¥ 10-4 and 1.6 ¥ 10-3, for KLM-7, B-24, G-135 andEF-2478 respectively (Figs 2C–F and S3). In these experi-ments, the number of variants detected is well correlatedwith the number of donor sites targeted by each anchoredPCR. Specifically, the frequency of variants for B-24,



G-135 and EF-2478 which target, respectively, 2, 3 and 4MgPars was 1.6, 4.3 and 7.6 times greater than that ofKLM-7 which targets a single MgPar. Furthermore, theanchored PCR products showed a variety of sequencevariants consistent with the occurrence of different recom-bination events between mgpB/C and the targeted MgParsites (data not shown). Importantly, the frequency of vari-ants detected increased over time, demonstrating that newvariants arise during in vitro propagation (Figs 2C–F andS3). Consequently, we also estimated the spontaneousrecombination rate in vitro, by measuring the change infrequency every two passages for a propagation period of10 passages. The calculated rates of gene variation foreach qPCR assay, after normalizing by the estimatednumber of generations, are shown in Table 1. Theseresults indicate that all the variable regions constantlyundergo sequence diversity.After normalizing all the qPCRassays to one donor site, we estimated that the averagerecombination rate in a single variable region was 4.48(� 2.1) ¥ 10-6 events per genome per generation. Sincethere are four variable regions (B, EF, G and KLM) inmgpB/C, each represented in five to eight copies in theMgPars (Iverson-Cabral et al., 2007), we estimate thatthere are a minimum of 28 possible recombination events,assuming a single recombination event per variable regionand possible donor site. Therefore, if all variable regions

recombine at a similar rate and taking this minimumnumber of possible exchange combinations, we hypoth-esize that mgpB/C gene variation occurs at rates higherthan 1.25 ¥ 10-4 events per genome per generation. Theseresults suggest that the potential to generate gene diversityin M. genitalium is high and could be similar to otherwell-characterized systems of bacterial antigenic variation(Turner and Barry, 1989; Criss et al., 2005; Helm andSeifert, 2010).

Mycoplasma genitalium RecA is required for mgpB/Cgene variation

To investigate the role of RecA in mgpB/C gene variation,we determined the occurrence of mgpB/C variants in theDrecA mutant. By applying the qPCR-based assay wefound that loss of RecA drastically reduced the frequencyof mgpB/C variants, although a few recombinants were stilldetected (ranging from 2.6 ¥ 10-5 KLM-7 variants to1.1 ¥ 10-4 EF-2478 variants; Fig. 2C–F). Moreover, in theabsence of RecA we were unable to see accumulation ofvariants during the 10 passages of propagation (Table 1).To examine in more detail whether these variants detectedin the DrecA mutant were genuine or PCR artefacts gen-erated in the absence of recombinant template (Paaboet al., 1989), we adapted our anchored PCR assay to a

Fig. 2. Gene variation at the mgpB/C locus.A. Schematic representation of the qPCR-based assay to monitor mgpB/C gene variation. As an example, the KLM-7-anchored PCR isillustrated. This PCR detects new KLM-7 variants using a primer (P1) targeting a conserved region in mgpC gene and a second primer (P2)matching unique sequences (represented by vertical lines) in MgPar 7. P1 and P2 correspond to 5′MgPar7(KLM) and 3′MgPar7(KLM) primers,listed in Table S1. Thus, the KLM-7-anchored PCR detects specific variants generated by homologous recombination between MgPar 7 andthe KLM variable region of the MgpC expression site. The different variable regions (B, EF, G and KLM) found in mgpB, mgpC and MgPar 7are also indicated.B. Comparison of MG_339 gene expression profiles between G37C and DrecA+recA strains. Relative MG_339 transcript levels weremeasured by quantitative RT-PCR normalized to 16S ribosomal RNA. The mean value � standard deviation is shown from three independentexperiments assayed in duplicate. Differences in MG_339 expression were found to be statistically significant (P = 0.0032, two-tailed Student’st-test).C–F. Frequency and accumulation of KLM-7 (C), B-24 (D), G-135 (E) and EF-2478 (F) variants after propagation of G37C, DrecA andDrecA+recA strains for 2, 4, 6, 8 and 10 passages. Note the different scale for each qPCR assay. The data are presented as themean � standard deviation from two independent experiments, with each sample assayed in duplicate.

Table 1. Recombination rates at the mgpB/C locus in G37C, DrecA and DrecA+recA strains.

Strain

Recombination ratea Fold differenceb

KLM-7 B-24 G-135 EF-2478 KLM-7 B-24 G-135 EF-2478

G37C 5.79 (� 3.48) ¥ 10-6 3.26 (� 4.29) ¥ 10-6 1.22 (� 0.88) ¥ 10-5 2.58 (� 0.41) ¥ 10-5 1.00 1.00 1.00 1.00DrecA ND ND ND ND 0.00 0.00 0.00 0.00DrecA+recA 2.09 (� 1.81) ¥ 10-5 6.80 (� 5.63) ¥ 10-5 2.34 (� 1.93) ¥ 10-4 4.88 (� 3.65) ¥ 10-4 3.62 20.87 19.21 18.90

a. Number of new KLM-7, B-24, G-135 or EF-2478 variants arising per genome per generation � standard deviation. Data were collected fromtwo independent experiments for each strain and represent the mean of the change in frequency every two passages for a total period of cultureof 10 passages and divided by the estimated number of generations.b. For each set of data, the recombination rate of the G37C strain has been set at 1.00, and the recombination rate for each of the other strainshas been normalized to this value.ND, not detected.



nested PCR format as follows. We amplified the KLMvariable region using primers targeting flanking conservedsequences in the mgpC gene, and used this PCR productas a template for the KLM-7-anchored PCR assay. Usingthis strategy, we still detected variants in the DrecA mutant(data not shown), reinforcing the idea that these recombi-nants were genuine.

Growth defects upon recA inactivation have beendescribed for several bacteria (Sciochetti et al., 2001). Toensure that this potential phenotype did not impact therecombination rates, we also compared the growth curvesof the wild-type and DrecA mutant. As shown in Fig. 3, thegrowth characteristics of the DrecA mutant were compa-rable to that of the parental strain, with doubling times ofapproximately 17 h.

Reintroduction of a wild-type copy of the recA gene intothe DrecA mutant restored the frequency of mgpB/C vari-ants, and even increased the recombination rates for all thevariable regions analysed (Fig. 2C–F and Table 1). Toexamine if variations in expression of RecA could bethe cause of this recombination enhancement, we com-pared the quantity of recA transcripts in the G37C andDrecA+recA strains by qRT-PCR. As shown in Fig. 2B, thecomplemented mutant exhibited a ~ 2.5-fold increase inthe expression levels of recA compared to the G37C strain.Southern blot analyses demonstrated that a single trans-poson was inserted in the genome of the DrecA+recAstrain, ruling out the possibility that the increase in expres-sion could be due to the presence of multiple copies of recA(Fig. S4). Alternatively, the genomic environment of theinsertion site may explain the increase in the expression ofthe delivered recA gene. The transposon insertion was

located at repeat region G of the MgPar 4. Importantly, thisinsertion does not disrupt coding regions, and does notaffect any of the variable regions in MgPar 4 targeted by theanchored PCR assays (Fig. S4). Similar results were alsoobtained when we analysed different DrecA+recA transfor-mants also expressing increased levels of recA transcripts(data not shown). Taken together, these results demon-strate that RecA mediates mgpB/C gene diversity andsuggest that variations in the RecA levels influence therates of mgpB/C gene variation.

Generation of spontaneous non-adherent phasevariants is dependent on RecA expression

It has been previously shown that M. genitalium HA- vari-ants arise spontaneously at high rates (Mernaugh et al.,1993). Further characterization of these variants revealedthat they originated by deletions in mgpB and mgpC genesas a consequence of recombination events with the MgParsites, suggesting a possible mechanism for MgpB/Cphase variation (Burgos et al., 2006). These observationssuggest that, in addition to promoting gene variation, RecAmight also be required for the generation of non-adherentphase variants. To test this hypothesis, we examined thepresence of HA- variants in the G37C and DrecA strains.We found that the frequency of phase variants in G37C was0.6% at passage 1 and increased to 5% after 10 passages(Fig. 4). In contrast, the DrecA mutant exhibited a fre-quency of 0.04% throughout the experiment. Complemen-tation of the DrecA mutant restored its capacity to generateHA- variants at a frequency that was threefold higher thanG37C at passage 1, consistent with the previous findingof enhancement of mgpB/C gene variation (Fig. 2 andTable 1).After 10 passages, the DrecA+recA strain showeda similar frequency of HA- variants as the G37C strain,likely indicating the achievement of equilibrium. Theseresults demonstrate that HA- phase variants are predomi-nantly derived by a RecA-dependent mechanism.

Mycoplasma genitalium RecA protein plays a minor rolein DNA repair

Given the central role that RecA plays in DNA repair inother organisms, we also examined the DNA repair capa-bilities of the M. genitalium DrecA mutant. First, we com-pared the sensitivity of wild-type and DrecA strains to DNAdamage induced by UV light. Surprisingly, no differenceswere observed even at the highest dose (Fig. 5A). Sub-sequently, we tested the sensitivity to increasing levels ofmitomycin C (MMC). This DNA-damaging agent causesDNA strand cross-links, resulting in single- and double-strand breaks, the latter requiring homologous recombi-nation for repair. After 24 h of treatment with increasingconcentrations of MMC, we observed a progressive

Fig. 3. Growth curves of wild-type (WT) G37 and its derivativesstrains, DrecA, DrecA+recA, DuvrC and DuvrC+uvrC. Growth wasmonitored over time by measuring the number of genomes byqPCR. Data are shown in a semi-logarithmic plot and represent themean � standard deviation of three separate experiments.



decrease in cell viability for both strains (Fig. 5B).Although the sensitivity exhibited by the DrecA mutantwas greater than the parental strain at higher dosages,these differences were not statistically significant. Takentogether, these results suggest that RecA plays a minorrole in DNA repair in M. genitalium.

Nucleotide excision repair may be the major DNA repairpathway in M. genitalium

Nucleotide excision repair is an important mechanism thatcan repair UV-induced DNA damage and other bulkylesions. In E. coli NER is executed by the cooperative

Fig. 4. Role of RecA in generating haemadsorption-deficient (HA-) phase variants.A. Frequency and accumulation of HA- phase variants after propagation of G37C strain, DrecA and DrecA complemented mutant for 10passages. The frequency of phase variants was calculated by dividing the number of colonies that failed to adsorb erythrocytes by the totalnumber of colonies analysed. The data are presented as the mean � standard deviation from two independent experiments assayed induplicate. The asterisk indicates a statistically significant difference when compared to G37C strain (P < 0.001, two-tailed Student’s t-test).B. Representative image showing M. genitalium colonies after the HA assay. Arrows indicate phase variant colonies, which fail to adsorberythrocytes.

Fig. 5. Survival of wild-type (WT) G37, DrecA, DuvrC and DuvrC complemented strains after (A) UV irradiation and (B) mitomycin C (MMC)treatment. Survivals are shown in semi-logarithmic plots and represent the mean � standard deviation of three independent experiments.Survival of wild-type and DuvrC mutant strains differed significantly after UV and MMC treatment with P-values of P < 0.05 and P < 0.1respectively (two-tailed Student’s t-test). Differences between wild-type and DrecA mutant did not achieve statistical significance for any of thematched UV or MMC treatments.



action of the encoded products of the uvrA, uvrB, uvrC anduvrD genes (Van Houten et al., 2005). Homologues ofthese genes have also been identified in the M. genitaliumgenome, representing the only complete DNA repairpathway identified in this genomically limited pathogen(Carvalho et al., 2005). The minor role of RecA in DNArepair in M. genitalium led us to further characterize theDNA repair capabilities of this microorganism. To accom-plish this, we constructed a null mutant of the MG_206gene, which is predicted to encode a protein with 27%amino acid identity to E. coli UvrC. To do so, we con-structed the suicide plasmid pDMG_206, engineered topromote the replacement of 88.1% of the coding sequenceof the MG_206 gene with the tetM438 antibiotic marker byhomologous recombination (Fig. 6A). The intended allelicexchange was confirmed by PCR amplification withselected primers (Fig. 6B) and Southern blot analyses(Fig. S1).

When propagating the DuvrC mutant we noticed thatcolonies were smaller compared to the wild-type strain(data not shown) and that the mutant also grew slower inliquid media. Comparison of the growth curves of theDuvrC mutant with the wild-type strain revealed a similargrowth rate but a delay in the onset of the logarithmic phase(Fig. 3). This growth defect was partially complemented bythe reintroduction of a wild-type uvrC copy (Fig. 3).

To test a possible role of UvrC in gene variation, weanalysed the frequency of mgpB/C variants in the DuvrCmutant, but no significant differences were observedwhen compared to G37C (data not shown). The DuvrCmutant was then tested for survival following DNAdamage induced by UV irradiation and MMC. Asexpected, this mutant was extremely sensitive to UVlight, exhibiting a ~ 104-fold decrease in survival relativeto the wild-type strain at a UV dose of 10 J m-2 (Fig. 5A).Similarly, the DuvrC mutant also exhibited a greater sen-sitivity to MMC (Fig. 5B). For example, after treatmentwith 80 ng ml-1 MMC, the viability of the wild-type strainand the DrecA mutant was, respectively, 150- and 46-foldgreater than that of the DuvrC mutant. Complementationof the DuvrC mutant with a wild-type copy of uvrCrestored the UV and MMC survival to wild-type levels(Fig. 5). Indeed, an enhanced UV survival was observedin the complemented mutant strain (Fig. 5A), reinforcingthe important role of NER pathway in repairingUV-induced damage. We conclude that M. genitaliumhas the capacity to repair its DNA and that the uvrC geneis critical to M. genitalium survival after DNA damage.These observations support the hypothesis that M. geni-talium possess an active NER system, which could rep-resent the main DNA repair pathway in this minimalbacterium.

Fig. 6. Gene replacement at the MG_206 locus.A. Genome organization at the MG_206 (uvrC) locus and schematic representation of the construction of a uvrC deletion mutant byhomologous recombination. Small arrows represent the primers used for the mutant screening and dotted lines show the predicted PCRproducts for the wild-type (WT) and DuvrC mutant.B. PCR confirmation of the intended deletion at the uvrC locus. Genomic DNA from wild-type, DuvrC and DuvrC+uvrC strains were used as atemplate for PCR reactions using primers 5′mg206 and 3′mg206 (PCR 1) and 5′mg206int and 3′mg206int (PCR 2). Primer sequences areshown in Table S1. PCR 1 detects replacement of the uvrC gene by the tetM438 resistance gene. Note that the primers used in PCR 1 arethe same as those employed for the complementation test, and thus two bands are detected in the DuvrC+uvrC strain. PCR 2 demonstratesthe absence of uvrC gene sequences. PCR products were electrophoresed on a 1% agarose gel. Lane M: kb DNA ladder (Stratagene).



Discussion

Phase and antigenic variation are common mechanismsemployed by pathogenic bacteria to overcome immunepressure or to respond to different niches and environ-ments between or within hosts (Henderson et al., 1999;van der Woude and Baumler, 2004). Mycoplasmaspossess a number of sophisticated systems to diversifytheir surfaces, including switching of gene expression byDNA slippage or site-specific recombination events, orpromoting antigenic variation by unidirectional gene con-version events as occurs with the VlhA protein of Myco-plasma synoviae (Citti et al., 2010). Despite its reducedgenome, M. genitalium also undergoes phase and anti-genic variation in two of its surface proteins (MgpB andMgpC), but accomplishes this by a unique mechanismbased on reciprocal recombination events (Iverson-Cabralet al., 2007). To understand the mechanisms governingMgpB/C phase and antigenic variation, we defined the roleof the M. genitalium RecA protein, a pivotal enzymerequired for recombination and DNA repair in other organ-isms. We found that RecA is required for mgpB/Csequence diversity as well as MgpB/C phase variation, yetplays a minor role in DNA repair in M. genitalium.

To undertake these studies, we developed an anchoredqPCR-based assay that allows the straightforward deter-mination of mgpB/C diversity within the population. Usingthis assay, we estimated that variation within mgpB/Cgenes occurs at rates of at least 1.25 ¥ 10-4 events pergenome per generation. This value is expected to be muchhigher, since our assay and rate estimates only consider asubset of the possible recombination events between theexpression locus and donor sites. Therefore, to measurethe full potential of mgpB/C gene variation, other unbiasedapproaches should be considered, such as those previ-ously described based on large-scale DNA sequencing(Criss et al., 2005; Helm and Seifert, 2010). Regardless,our results suggest that M. genitalium may undergo genevariation at rates similar to that exhibited by other specieshaving recombination-based antigenic variation systems.For example, Neisseria meningitidis and Neisseria gonor-rhoeae exhibit rates of 1.6 ¥ 10-3 and 4 ¥ 10-3 events percell per generation respectively (Criss et al., 2005; Helmand Seifert, 2010). Trypanosoma brucei, a eukaryoticparasite transmitted through insect vectors, possessesa similar degree of sequence diversity (Turner and Barry,1989), although switching rates are drastically reduced inlaboratory-adapted strains (Turner, 1997).

Our anchored qPCR-based assay has proven to beparticularly useful to compare mgpB/C gene variationbetween wild-type and mutant strains. Using this assay wehave found that mgpB/C gene variation is drasticallydecreased in a DrecA mutant, demonstrating the involve-ment of RecA in mgpB/C diversity. Given the recombina-

torial nature of the MgPar system, this result is notsurprising, since RecA is the central player in homologousrecombination, promoting genetic exchange by pairinghomologous DNA substrates in an ATP-dependent fashion(Kuzminov, 1999; Sluijter et al., 2009). Similarly, antigenicvariation systems from N. gonorrhoeae and T. brucei arealso dependent, respectively, on the action of RecA andRad 51, the eukaryotic homologue of RecA(Koomey et al.,1987; McCulloch and Barry, 1999). However, a differentscenario is found in Borrelia burgdorferi, in which theRuvAB helicase, but not RecA, is required for vlsE anti-genic variation (Liveris et al., 2008; Dresser et al., 2009;Lin et al., 2009). An intriguing observation of this study wasthat, despite the significant reduction in recombinationrate, a small but detectable number of mgpB/C variantswere still detected in the DrecA mutant. Therefore,although a RecA-dependent mechanism appears to be themain pathway, we cannot exclude the existence of analternative mechanism to generate mgpB/C gene variationin M. genitalium. Of note, evidence for RecA-independentrecombination events has been previously reported inE. coli (Dutra et al., 2007). Similarly, we also determinedthat spontaneous HA- phase variants arise principally by aRecA-dependent mechanism, yet a limited number of HA-

variants were detected in the absence of RecA. Theseresults are consistent with our previous work showing thatHA- variants (designated class I and class II mutants) werethe result of large deletions affecting the MgpB/C locus,driven by recombination between the expression site andthe MgPar sequences (Burgos et al., 2006). The origin ofthe few HA- variants found in the recA background could bedue to spontaneous point mutations in the mgpB/C locusor other cytadherence-related genes. However, we alsocannot rule out the possibility that these variants wereoriginated by a RecA-independent recombination mecha-nism involving the MgPar sequences. Further analysis ofthese HA- variants is warranted to more clearly elucidatethe mechanisms involved.

Our observation that phase and antigenic variants accu-mulate in vitro suggests that the mechanism triggeringmgpB/C gene variation is stochastic and independent ofselective pressure. However, we cannot rule out the pos-sibility that gene variation could be enhanced by specificconditions. In this regard, we found that differences in theexpression levels of recA have a direct impact on genevariation rates, indicating that gene diversity can poten-tially be enhanced. It is tempting to speculate that M. geni-talium may be able to increase mgpB/C recombination invivo by adjusting the RecA levels in response to the hostenvironment. For instance, B. burgdorferi gene variation atthe vlsE locus occurs exclusively in the mammalian host,suggesting the involvement of host factors in inducing orselecting variants in this microorganism (Zhang and Norris,1998).



Another interesting observation from our study wasthe detection of three different RecA protein species inM. genitalium cell lysates. The possibility that these addi-tional bands are the result of artefacts during proteinelectrophoresis or cell lysate preparation seems unlikely,because only one M. genitalium RecA species is detectedwhen expressed in E. coli. Thus, the generation of theseRecA species may be dependent on the M. genitalium cellmetabolism. Although further work is needed to confirmthis view, the expression of these potential RecA isoformscould be explained by specific posttranslational cleavageevents on the full-length RecA protein. Alternatively, theymay originate from additional translational start codonspresent in the recA coding sequence, which are recog-nized by the M. genitalium translational machinery. Sup-porting this hypothesis is the presence of two in-frametriplets downstream from the proposed ATG start codon.These triplets are ATT and TTG, encoding residues I19and L24, and may serve as alternative initiation codons(Golderer et al., 1995), resulting in predicted productswith the observed molecular sizes. These hypotheses arecurrently being tested and work is in progress to deter-mine the functional relevance of the expression of theseputative isoforms.

Our studies also revealed notable differences betweenM. genitalium and the E. coli paradigm in DNA repair. Wefound that inactivation of recA in M. genitalium has noeffect on cell survival after UV irradiation. Similarly, only amoderate but not significant reduction in cell viability wasobserved after DNAdamage induced by MMC to the DrecAmutant. Such findings are in contrast to results obtainedwith E. coli as well as other species, in which recA mutantsexhibit an exquisite sensitivity to these DNA-damagingtreatments (Kuzminov, 1999). In E. coli, RecA performstwo major functions in DNA repair: (i) it mediates homolo-gous recombination, a process particularly important forrestoring stalled replication forks and for repairing double-strand breaks (Lusetti and Cox, 2002) and (ii) it plays a keyregulatory role inducing the SOS response (Kuzminov,1999). In the presence of DNA damage, the co-proteaseactivity of RecA becomes activated and triggers the auto-catalytic cleavage of the LexA repressor, leading to theco-ordinated induction of different SOS genes, includingrecA and genes from the NER pathway. It is unknownwhether M. genitalium RecA protein conserves thisco-protease activity, but genome analysis does not revealorthologues of the lexA gene. Despite the apparent lack ofa SOS system, we found that M. genitalium is proficient inrepairing photoproducts induced by UV irradiation, prob-ably through the action of the NER pathway, as suggestedby the high UV sensitivity exhibited by the DuvrC mutant.Although we cannot exclude the possibility that NER genesare induced through a RecA-independent mechanism,our results suggest that the basal expression level of the

uvr genes is sufficient to guarantee the genome stabilityin M. genitalium. This hypothesis is favoured by theextremely slow growth rate of this microorganism (~ 17 h).As previously suggested (Hanawalt, 1966), a reducedgrowth rate may allow the NER pathway sufficient time torepair DNA damage and prevent the formation of stalledreplication forks without the need to induce a rapid SOSresponse. This scenario would minimize the requirement ofthe recombinational DNA repair pathway mediated byRecA. Consistent with this idea, it has been shown that inMycoplasma pulmonis, which has a faster doubling time(~ 2 h) (Dybvig et al., 1989), a recA mutant is sensitive toUV irradiation (French et al., 2008).

Recombinational DNA repair is a complex pathway thatrequires, in addition to RecA, the action of multiple proteins(Kuzminov, 1999). Since M. genitalium apparently lacksmany of these genes (Carvalho et al., 2005), it is alsoconceivable that recombinational DNA repair is not effi-cient in this organism. How M. genitalium deals with inter-strand cross-links (ICLs) induced by MMC exposure is alsounclear. The commonly accepted pathway to repair thissevere damage is the combined action of NER and recom-binational DNA repair, although an alternative mechanisminvolving NER and translation synthesis polymerases hasalso been suggested (Dronkert and Kanaar, 2001). Theobservation that the DuvrC mutant is sensitive to MMCreinforces the important role of NER in repairing MMC-induced DNA damage. However, the finding is differentthan that observed in E. coli, where a recombination-deficient, SOS-proficient recA142 mutant shows a similarsensitivity to a uvrB mutant and greater than a uvrC mutant(Vidal et al., 2006). One explanation for these differencescould be that ICLs’ repair in M. genitalium is biased tothe polymerase translation repair pathway. Alternatively,M. genitalium may be unable to efficiently repair ICLs, andthe higher sensitivity observed in the DuvrC mutant is dueto the action of NER repairing other MMC-induced DNAlesions different than ICLs (Dronkert and Kanaar, 2001).

Remarkably, in a study of saturating whole-genometransposon mutagenesis, no transposon insertions wereidentified in uvrA, uvrB and uvrC, suggesting that the corecomponents of the NER pathway are essential for growthin M. genitalium (Glass et al., 2006). Our successfuldeletion of the uvrC gene suggests that is not the case,at least for the uvrC endonuclease. Nevertheless, thegrowth defects observed in the DuvrC mutant and itssevere DNA repair-deficient phenotype support thehypothesis that NER is the main DNA repair pathway toguarantee the genomic stability in M. genitalium.

In conclusion, to our knowledge, this study is the firstreport of the molecular requirements for mgpB/C phaseand antigenic variation and provides fundamental insightson DNA repair in the context of a minimal genome.Studies are underway to investigate the role of other



putative recombination enzymes in these processes, pos-sible regulatory mechanisms for M. genitalium RecAexpression, and the derivation and function of putativeRecA isoforms.

Experimental procedures

Bacterial strains and growth conditions

Wild-type M. genitalium strain G37 (ATCC 33530) and itsderivatives were grown in SP-4 broth (Tully et al., 1979) at37°C under 5% CO2 in tissue culture flasks (Corning), unlessotherwise indicated. SP-4 medium was supplemented with0.8% agar (Difco) for colony development, and with chloram-phenicol (15 mg ml-1), tetracycline (2 mg ml-1) or gentamicin(100 mg ml-1) during the selection of transformants. For theanalysis of mgpB/C recombination we used a G37 singlecolony-filtered clone designated G37C, previously describedelsewhere (Iverson-Cabral et al., 2007). The G37C strain ishomogenous and contains mgpB/C sequences identical tothose published for the G37 strain. E. coli strains TOP10 andBL21(DE3) (Invitrogen), used for cloning and protein overex-pression, respectively, were grown at 37°C in LB broth or LBagar plates containing ampicillin (100 mg ml-1) and X-Gal(40 mg ml-1) as needed.

Construction of M. genitalium mutants andcomplementation experiments

General DNA manipulations were performed following stan-dard procedures (Sambrook and Russell, 2001). All PCRproducts were obtained from genomic DNA of strain G37using the primers summarized in Table S1.

To generate a recA null mutant, the suicide plasmidpDMG_339 was engineered as follows. A 1100 bp fragmentspanning the 5′ flanking sequence of MG_339 gene wasamplified with primers 5′RAKOmg339 and 3′RAKOmg339.Similarly, a 1035 bp fragment from the 3′ flanking region wasamplified with primers 5′LAKOmg339 and 3′LAKOmg339.We then amplified the chloramphenicol resistance gene (cat)under control of the Spiroplasma citri spiralin promoter (SpPr)(Lartigue et al., 2002) from a plasmid containing that antibi-otic resistance gene expression cassette (sequence avail-able from GenBank, Accession Number JX02666) usingprimers 5′cat-(SpPr) and 3′cat-(SpPr). The three PCR frag-ments generated overlapped each other by 50 bp and werejoined by polymerase chain assembly (PCA) (Stemmer et al.,1995). The PCA product was amplified again using the ter-minal primers 5′LAKOmg339 and 3′RAKOmg339, andcloned into the pCR-Blunt II-Topo vector (Invitrogen).

To generate a null uvrC mutant the suicide plasmidpDMG_206 was constructed as follows. A 981 bp PCR frag-ment spanning the 5′ flanking sequence of the MG_206gene was amplified using primers 5′LAKOmg206 and3′LAKOmg206, which incorporate Acc65I and EcoRI restric-tion sites at their 5′ ends respectively. A second 993 bp frag-ment containing the 3′ flanking sequence of the MG_206gene was amplified using primers 5′RAKOmg206 and3′RAKOmg206. These primers contained BamHI and XbaIrestriction sites at their 5′ ends respectively. Both PCR frag-ments were cloned into an EcoRV-digested pBE (Pich et al.,

2006a), excised with the corresponding restriction enzymes,and ligated together with a 2 kb fragment containing thetetM438 selectable marker and an Acc65I/XbaI-digestedpBSKII+ (Invitrogen). The tetM438 selectable marker wasreleased from the pMTnTetM438 plasmid (Pich et al., 2006a)by digestion with EcoRI and BamHI.

For the complementation of the DrecA mutant, the mini-transposon plasmid pMTnTetM438–MG_339 containing awild-type recA gene was constructed as follows. A fragmentencompassing the first 219 bp upstream of the MG_339 geneand its coding sequence was amplified with primers 3′mg339and 5′mg339, which contain XhoI and EcoRI restriction sites attheir 5′ ends respectively. The PCR product was cloned into anEcoRV-digested pBE (Pich et al., 2006a), excised with thecorresponding restriction enzymes, and cloned into a XhoI/EcoRI-digested pMTnTetM438 vector (Pich et al., 2006a).

For the complementation of the DuvrC mutant, the mini-transposon plasmid pMTnGm–MG_206 containing a wild-type uvrC gene was constructed as follows. A fragmentencompassing the first 503 bp upstream of the MG_206 gene,and its coding sequence was amplified with primers 5′mg206and 3′mg206, which contain XhoI and EcoRI restriction sites attheir 5′ ends respectively. The PCR product was cloned into anEcoRV-digested pBE (Pich et al., 2006a), excised with thecorresponding restriction enzymes, and cloned into a XhoI/EcoRI-digested pMTnGm vector (Pich et al., 2006a).

Transformation of strain G37 was accomplished by elec-troporation as previously described (Pich et al., 2006a) withfew modifications. Briefly, 30 mg of knockout construct DNA(pDMG_339 or pDMG_206) dissolved in 30 ml of electropora-tion buffer (8 mM HEPES pH 7.2, 272 mM sucrose) wasmixed with 90 ml of cells (approximately 109 cells per millilitre)also resuspended in electroporation buffer. The mixture wastransferred to a 2 mm gapped electroporation cuvette (Bio-Rad), kept on ice for 15 min and electroporated using theGene Pulser® II electroporation system (Bio-Rad) with thePulse Controller set at 2.5 kV and 100 W. The cuvette wasplaced on ice for 15 min and then 900 ml of SP-4 medium wasadded. After 3 h of incubation at 37°C, transformants wereselected onto SP-4 plates containing the appropriate antibi-otic. After 2 weeks of incubation at 37°C and 5% CO2, singlecolonies were picked, propagated in 5 ml of SP-4 containingthe appropriate antibiotic and stored at -80°C. The intendedgenetic modifications were then confirmed by PCR usingselected primers (Figs 1 and 6) and Southern blot analyses(Fig. S1). Complementation of the resulting null mutants wasachieved via transposition, by electroporating 5 mg ofpMTnTetM438–MG_339 or pMTnGm–MG_206 plasmids intothe DrecA or DuvrC mutants, respectively, using similar tech-niques as described above. The insertion of a single transpo-son in the genome of the DrecA and DuvrC complementedstrains was confirmed by Southern blot analysis (Fig. S4). Thespecific insertion sites were further determined by sequencingthe flanking genomic DNA with 5′ Tc_seq and 3′ Tn_seqprimers respectively (Table S1, Fig. S4).

Expression and purification of M. genitalium RecA andantibody production

The MG_339 coding sequence was amplified from genomicDNA of strain G37 using primers 5′ExpRecA and 3′ExpRecA,



which contain XhoI and NcoI restriction sites at their 5′ endsrespectively. The resulting PCR product was cloned into anEcoRV-digested pBE (Pich et al., 2006a), excised with thecorresponding restriction enzymes, cloned into a XhoI/NcoI-digested pET21-d expression vector (Novagen) and trans-formed into E. coli BL21(DE3). Transformant cells weregrown in 400 ml of LB broth at 37°C to an optical density at600 nm of 0.6. After 3 h of induction with 1 mM IPTG, cellswere harvested, resuspended in 20 ml of binding buffer(5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), lysedby sonication in the presence of 1 mM PMSF and clarifiedby centrifugation. The supernatant was filtered through a0.45 mm filter and applied to a column containing Ni-NTA-Agarose (Qiagen) pre-equilibrated with binding buffer. Thecolumn was washed with 60 mM imidazole, 0.5 M NaCl,20 mM Tris-HCl (pH 7.9) and proteins were eluted in 1 Mimidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9). The elutedfraction was dialysed against 2 l of PBS and purity was esti-mated by SDS-PAGE and Coomassie blue staining to be> 95% (Fig. S2A).

Polyclonal antisera against M. genitalium RecA was com-mercially produced by immunization of rabbits with the puri-fied recombinant RecA protein (Pacific Immunology, CA,USA). Antibody specificity was further assessed by immuno-blot using E. coli and M. genitalium cell extracts expressingHis-tagged and non-tagged M. genitalium RecA variants(Fig. S2C and D).

Immunoblot analyses

Cell extracts were subjected to electrophoresis through a12% SDS-polyacrylamide gel, transferred electrophoreticallyto a nitrocellulose membrane (Protran BA 85) and probed for2 h at room temperature with a 1:1000 dilution of rabbit anti-M. genitalium RecA antibodies, following standard proce-dures (Sambrook and Russell, 2001). Goat anti-rabbit IgGconjugated to alkaline phosphatase (Sigma) was used assecondary antibody. Antigen–antibody complexes were visu-alized using nitroblue tetrazolium (Bio-Rad) and 5-bromo-4-chloro-3-indolyl-phosphate (Roche) reagents according tothe supplier’s instructions.

Assessment of growth rates

Growth curves were obtained by monitoring the number ofgenomes over time by real-time quantitative PCR (qPCR). Todo so, the M. genitalium G37 and mutant strains were grown in20 ml of SP-4 broth in 50 ml conical tubes (Corning) for 10days at 37°C under 5% CO2. The screw caps of the tubes wereloosened to permit gas exchange. Cultures grown to a similardensity of approximately 1 ¥ 108 cfu ml-1 were used as inocu-lum for each strain. The qPCR confirmed that a similar numberof bacterial cell equivalents were inoculated. Aliquots of 0.5 mlwere taken every 48 h from each culture, centrifuged at20 000 g for 15 min and the resulting pellets were maintainedat -20°C until needed. DNA extraction from each sample wasperformed using the MasterPureTM DNA purification kit (Epi-center) according to the manufacture’s instructions. Thenumber of genomes in each sample was quantified by real-time PCR as described below for the mgpB/C gene variationassay.

mgpB/C gene variation assay

To monitor gene variation over time, G37C, DrecA andDrecA+recA strains were propagated as follows. Five millili-tres of SP-4 were inoculated and cultivated for 5 days(seven generations). Cells were scraped off from the flasksin the same culture media, thereby harvesting both adher-ent and non-adherent cells. One millilitre from this 5 ml cellsuspension was taken and centrifuged, and the pellet wasstored at -20°C for further DNA extraction. Another 1 mlwas kept as a frozen stock at -80°C for the haemadsorptionassay (see below). Twenty-five microlitres of culture wasthen used to inoculate the next culture and the process wasrepeated for 10 passages. Each passage was performed induplicate.

A real-time qPCR-based assay was used to determine thefrequency of mgpB/C variants in each passage. Based onthe genome sequence of G37 strain (GenBank AccessionNumber NC_000908), we designed four different anchoredPCR assays designated B-24, EF-2478, G-135 and KLM-7,with letters and numbers indicating, respectively, the variableregion within mgpB or mgpC genes and the MgPar sitestargeted (e.g. B-24 targets recombinants between region Band MgPar sites 2 or 4). These anchored PCR assays detectspecific variants in which the MgPar sequences targeted bythe primers have translocated into the MgpB/C expressionsite. The set of primers used for each PCR assay are listedin Table S1. To normalize the number of variants to thenumber of genomes in each sample, we used a secondqPCR targeting the 16S ribosomal RNA gene which ispresent in a single copy by using primers 5′RT16SrRNA and3′RT16SrRNA (Table S1). Real-time qPCR was performed ina LightCycler instrument (Roche), using the LightCycler-FastStart DNA Master SYBR Green I Kit (Roche). PCR reac-tions were performed in a final volume of 20 ml containing2 ml of DNA (approximately 1 ¥ 107 genomes), 0.5 mM ofeach primer and 2 ml of 10¥ SYBR Green mix. The MgCl2concentration was optimized for all reactions at 3 mM,except for KLM-7 and G-135 qPCRs, which required 4.5 and4 mM respectively. After an initial 10 min incubation at 95°C,each qPCR assay was performed using the following ampli-fication conditions: (i) KLM-7: 40 cycles of 95°C for 10 s,52°C for 10 s and 72°C for 18 s, (ii) B-24 and G-135: 40cycles of 95°C for 10 s, 56°C for 10 s and 72°C for 16 s and(iii) 16SrRNA and EF-2478: 40 cycles of 95°C for 10 s, 54°Cfor 10 s and 72°C for 10 s. Fluorescence readings wereacquired at 79°C for KLM-7 and 77°C for the other qPCRassays. Each set of primers generated amplification reac-tions with efficiencies ranging from 90% to 100%. Specificityof PCR products was verified by melting curve analysis andagarose gel electrophoresis. PCR products were also clonedinto an EcoRV-digested pBE (Pich et al., 2006a) and theexpected sequence from five clones for each anchored PCRwas confirmed by sequencing. Absolute quantification wasperformed by creating standard curves using ScaI-linearizedpBE (Pich et al., 2006a) plasmids harbouring the PCRproduct. The plasmid copy number was determined spectro-photometrically using an ND-1000 spectrophotometer(NanoDrop Technologies). The qPCR reactions were per-formed in duplicate and output data were analysed by usingthe LightCycler 3.5 software (Roche).



RNA isolation and quantitative reverse transcriptasePCR (qRT-PCR)

Total RNA was extracted from 20 ml of mid-logarithmic phasecultures using the RNAaqueous Kit (Ambion) followingthe manufacturer’s instructions. One microgram of DNAse-treated RNA (TURBO DNAse, Ambion) was reverse-transcribed by using the SuperScript III First-Strand synthesiskit (Invitrogen) with random hexamers according to the manu-facturer’s instructions. Parallel reactions were performed withno reverse transcriptase to control for contaminating DNA.Real-time qPCR analysis of the resulting cDNAs was per-formed in duplicate as described for the mgpB/C gene varia-tion assay using 2 ml of > 1:10 diluted cDNA. Primers used foramplification of MG_339 transcripts were 5′RTMG_339 and3′RTMG_339 (Table S1). PCR conditions were the same asthose used for the reference gene (16S ribosomal RNA),which were previously described in the mgpB/C gene variationassay (see above). Arbitrary quantification of target and refer-ence genes was determined from standard curves generatedby five serial dilutions of the cDNA. The relative abundance ofMG_339 transcripts for each sample was then normalized tothe 16S ribosomal RNA.

Haemadsorption (HA) assay

G37C, DrecA and DrecA+recA strains were serially passagedas described above for the mgpB/C gene variation assay.Serial dilutions of cells from the first and 10th passage weregrown on SP-4 plates and the resulting colonies were testedfor their capacity to adsorb sheep erythrocytes (Remel) aspreviously described (Pich et al., 2006b). Colonies were visu-alized by light microscopy and the frequency of spontaneousHA-deficient variants was calculated by dividing the numberof colonies that failed to adsorb erythrocytes by the totalnumber of colonies analysed. Approximately 3000 coloniesper passage were analysed for G37C and DrecA+recAstrains, whereas ~ 15 000 colonies were examined for theDrecA mutant.

UV and mitomycin C sensitivity assays

Mycoplasma genitalium G37 and mutant strains were grown toexponential phase in 20 ml of SP-4 broth. For the UV sensi-tivity assay, cells were washed three times in phosphate-buffered saline (PBS), scraped off the tissue culture flasksand diluted in 10 ml of PBS to obtain approximately1 ¥ 108 cfu ml-1. One millilitre of this cell suspension was thendispensed into several 35 ¥ 10 mm culture dishes, which wereseparately exposed to increasing doses of UV radiation usinga CL-1000 Ultraviolet Crosslinker (UVP). After UV exposure,cells were serially diluted and 10 ml of each dilution wasspotted in duplicate onto SP-4 plates. Although no photolyaseenzymes have been identified in M. genitalium, light wasminimized during the whole procedure. After incubation for 14days, colonies were counted and survival rates were calcu-lated as compared to untreated samples.

For the MMC sensitivity assay, cells were scraped off thetissue culture flasks, suspended in SP-4 broth and aliquotedinto tubes containing serial twofold dilutions of MMC (Sigma).

After 24 h of MMC exposure at 37°C under 5% CO2, serialdilutions of each treatment were plated in duplicate onto SP-4plates and survival rates were analysed as described abovefor the UV sensitivity assay.

Acknowledgements

This work was supported by NIH Grant R56 AI071175 ARRAto P. A. T. and the US Department of Energy CooperativeAgreement No. DE-FC02-02ER63453 at the J. Craig VenterInstitute. We thank Nacyra Assad-Garcia and Carole Lartiguefor assistance making the DrecA mutant and StefanieIverson-Cabral for helpful discussions and critical reading ofthe manuscript.

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reca mediates mgpb and mgpc phase and antigenic variation in mycoplasma genitalium, but plays a...

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