Development and Use of an Efficient System for Random mariner Transposon Mutagenesis To Identify...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2008, p. 3377–3386 Vol. 74, No. 110099-2240/08/$08.00�0 doi:10.1128/AEM.02665-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development and Use of an Efficient System for Random marinerTransposon Mutagenesis To Identify Novel Genetic Determinants

of Biofilm Formation in the Core Enterococcus faecalis Genome�†Christopher J. Kristich,1,2 Vy T. Nguyen,1 Thinh Le,1 Aaron M. T. Barnes,1

Suzanne Grindle,1 and Gary M. Dunny1*Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455,1 and Department of

Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532262

Received 14 November 2007/Accepted 28 March 2008

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an importantopportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the geneticanalysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation andanalysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of thegenetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify suchdeterminants, we developed a substantially improved system for transposon mutagenesis in E. faecalis basedon a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded pre-dominantly mutants carrying a single copy of the transposable element, and insertions were distributed aroundthe entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposoninsertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation.Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously knownto play a role in biofilm formation and in new genes lacking any known function; for several genes identifiedin the screen, complementation analysis confirmed a direct role in biofilm formation. These results providesignificant new information about the genetics of enterococcal biofilm formation and demonstrate the generalutility of our transposon system for functional genomic analysis of E. faecalis.

Enterococcus faecalis is a gram-positive bacterium that pri-marily associates with humans in a benign manner as a memberof the gastrointestinal tract microbial consortium (49). How-ever, enterococci also represent a serious health concern asone of the three most common causes of hospital-acquiredinfections (39, 42). Enterococci exhibit relatively high-levelintrinsic resistance to some antibiotics and share mobile ge-netic elements carrying additional antibiotic resistance deter-minants with neighboring bacteria (21), leading to the emer-gence of multiresistant clones in the hospital setting. Thus,enterococcal infections are an increasingly difficult problem forclinicians given currently available therapeutic agents.

In clinical settings, bacteria growing as surface-adherent bio-films have been implicated as etiological agents of chronicinfection (10, 12, 34). E. faecalis is known to form robustbiofilms in the laboratory as well as in clinically relevant set-tings (4, 11, 12, 41, 51). E. faecalis biofilms grown in vitroexhibit enhanced tolerance to antibiotics, such as vancomycinand teicoplanin (16). The enhanced antibiotic tolerance result-ing from growth in a biofilm state, superimposed on the intrin-sic and acquired antibiotic resistances typical of hospital strainsof enterococci, suggests that biofilm formation of E. faecalis ina hospital setting may be particularly problematic.

Previous studies have identified a small number of genesimportant for biofilm formation by E. faecalis. Although someare encoded on mobile genetic elements (50), others are chro-mosomally encoded, suggesting that they belong to the coregenome of the species. Some examples include the ebp locus,encoding endocarditis- and biofilm-associated pili (37); srtA,encoding the major sortase responsible for anchoring cell sur-face proteins to the cell wall (22); and atn, encoding an auto-lysin (35). Although a role in biofilm formation of E. faecalishas been ascribed to these genes and others, to date no com-prehensive genome-wide search has been performed for chro-mosomally encoded genetic determinants of biofilm formationby E. faecalis. This is due, in part, to the lack of effectivegenetic tools enabling such a genome-wide search for E. fae-calis. In particular, no method for efficient and random trans-poson mutagenesis has been developed.

Biofilm formation is known to be a complex process involv-ing signal transduction systems, transcriptional regulation, andstress responses, among other features (38, 47). We hypothe-sized that the E. faecalis genome encodes unidentified geneticdeterminants, in some or all of these functional categories,which promote biofilm formation. We sought to identify thesedeterminants using an unbiased, genome-wide approach,namely, random transposon mutagenesis. At least two trans-posons have previously been used for mutagenesis of the chro-mosome in E. faecalis (Tn916 and Tn917). However, thesetransposons each suffer from significant drawbacks that restricttheir utility in the context of a comprehensive genome-scalemutagenesis experiment, including insertion in multiple copiesper genome (18) and significantly biased insertion-site prefer-

* Corresponding author. Mailing address: 420 Delaware St. SE,MMC196, Minneapolis, MN 55455. Phone: (612) 625-9930. Fax: (612)626-0623. E-mail: [email protected].

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 11 April 2008.

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ences (17, 43, 44) that prevent random coverage of the wholegenome.

Transposon mutagenesis systems based on the mariner trans-posable element have been widely used for prokaryotes andare known to yield a random distribution of insertions (1, 20,29, 32, 33, 40, 46, 48, 54, 55). mariner elements insert at TAdinucleotide pairs in the target sequence and transpose inde-pendently of any host factors (27, 28), making them ideal toolsfor random mutagenesis. To circumvent the problems associ-ated with use of Tn916 and Tn917 in E. faecalis, we developeda substantially improved system for transposon mutagenesisbased on a mini-mariner transposable element (EfaMarTn).After transposition by this element, most E. faecalis insertionmutants carried only one EfaMarTn element, and EfaMarTninsertions were found to be distributed around the entire E.faecalis chromosome. Using this mutagenesis system, we con-structed a library of EfaMarTn insertion mutants in E. faecalisOG1RF and screened this library to identify genes involved inearly stages of biofilm formation. We isolated biofilm-defectivemutants carrying transposon insertions in many of the genespreviously implicated in biofilm formation by E. faecalis, aswell as insertions in new genes not previously known to play arole in biofilm formation, demonstrating the utility of ourtransposon system as a tool enabling genome-wide geneticanalysis of enterococci.

MATERIALS AND METHODS

Bacterial strains, growth media, and chemicals. Bacterial strains used in thisstudy are listed in Table 1. Unless otherwise indicated, all culture media werepurchased from Difco and all chemicals were purchased from Sigma (St. Louis,MO). Bacto brain heart infusion (BHI) and Bacto tryptic soy broth withoutglucose (TSB) were prepared as described by the manufacturer (Becton Dick-inson). Bacto agar was used as a solidifying agent for all semisolid media.Bacteria were routinely stored at �80°C in BHI supplemented with 30% glyc-erol. When required for selective growth of E. faecalis, erythromycin (Em),

tetracycline, and chloramphenicol (Cm) were used at 10 �g/ml; fusidic acid (Fa)at 25 �g/ml; and spectinomycin at 1,000 �g/ml. When required for E. faecalis,5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-Gal) was added at 250�g/ml and 5-fluorouracil at 1 mM. When required for selective growth of Esch-erichia coli, Em was used in BHI at 100 �g/ml. Oligonucleotides were purchasedfrom Integrated DNA Technologies (Coralville, IA).

Plasmid construction. Plasmids used in this study are listed in Table 1. Allderivatives of plasmid pCJK34 were propagated in E. coli strain EC1000 (30),which supplies RepA in trans to allow replication of these plasmids. All otherplasmids were propagated in E. coli XL1Blue or DH5�.

The mobilizable delivery plasmid carrying the EfaMarTn transposable ele-ment (pCJK72) (see Fig. 1), which is essentially equivalent to a pCJK47 deriv-ative bearing the transposable element on a BamHI fragment, was constructedaccording to a multistep scheme as follows. The entire 3.2-kb bursa aurealiselement was amplified by PCR from plasmid pBursa and cloned into pCJK34using primer-encoded BamHI sites. Quikchange mutagenesis (Stratagene) wasapplied to replace the ermB and oriR6K regions from the bursa aurealis elementwith a short sequence encoding unique SpeI and XhoI restriction sites, creatingpCJK49. The pheS* counterselectable marker (23) from pCJK47 was insertedinto the plasmid backbone (outside of the transposable element) as a SphI/BglIIfragment (note that pheS*-dependent counterselection was not used in thisstudy). The chloramphenicol acetyltransferase gene (cat) from plasmid pWM401was amplified by PCR and cloned into the SpeI/XhoI sites of the mini-marinertransposable element in the resulting plasmid using primer-encoded restrictionsites, thereby creating pCJK72.

An expression plasmid for inducible production of mariner transposase wasconstructed by cloning the mariner transposase C9 from plasmid pBADC9 intothe nisin-inducible expression plasmid pMSP3545 as a NcoI/SphI fragment,creating pCJK55. The ribosome binding site (RBS) for the transposase inpCJK55 is provided by pMSP3545.

Expression plasmids for complementation analysis of selected EfaMarTn in-sertion mutants were constructed by first using PCR to amplify the correspond-ing transposon-disrupted genes from wild-type E. faecalis OG1RF chromosomalDNA. Primers were designed according to the E. faecalis V583 genome se-quence. The amplicons, which included the putative RBS for each of the genes,were cloned into pMSP3535 using primer-encoded SpeI (upstream) and XhoI(downstream) restriction sites, thereby creating pCJK122, pCJK123, pCJK124,and pCJK128.

Transposon mutagenesis of E. faecalis OG1RF. For mutagenesis, pCJK72 wasintroduced into the recipient strain by conjugation according to a previouslydescribed procedure (23). Briefly, pCJK72 was first introduced by electropora-

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant characteristic or description Source or reference

StrainsE. faecalis

OG1RF Reference strain 14CK111 (pCF10-101) Conjugative donor host strain; supplies RepA in trans 23

E. coliDH5� Cloning host Lab stockXL1Blue Cloning host Lab stockEC1000 Cloning host, provides RepA in trans 30

PlasmidspMSP3535 Nisin-inducible expression vector; Emr 8pMSP3545 Nisin-inducible expression vector encoding RBS; Emr 8pBursa Source of bursa aurealis transposable element 2pWM401 Source of chloramphenicol acetyltransferase gene 53pBADC9 Source of mariner transposase C9 26pCJK15 E. faecalis EF3056 (srtA) cloned into pMSP3535 25pCJK34 Mobilizable plasmid; requires RepA for replication; Emr 23pCJK47 Derivative of pCJK34 carrying pheS* counterselectable marker 23pCJK49 Intermediate construct carrying modified mini-mariner element This workpCJK55 mariner transposase C9 cloned into pMSP3545 This workpCJK72 Mobilizable delivery plasmid carrying EfaMarTn transposable element This workpCJK122 E. faecalis EF0394 (salB) cloned into pMSP3535 This workpCJK123 E. faecalis EF0676 cloned into pMSP3535 This workpCJK124 E. faecalis EF0983 cloned into pMSP3535 This workpCJK128 E. faecalis EF1090 (ebpR) cloned into pMSP3535 This work

3378 KRISTICH ET AL. APPL. ENVIRON. MICROBIOL.

tion into the conjugative donor strain, E. faecalis CK111 (pCF10-101), where itcan replicate. The resulting donor strain was used in a conjugation experimentwith E. faecalis OG1RF or FA2-2 carrying pCJK55 (or the empty vector control,pMSP3545) as recipients. Stationary-phase overnight cultures of donors andrecipients in BHI supplemented with Em (10 �g/ml) were independently diluted20-fold into BHI supplemented with Em (1 �g/ml) and incubated at 30°C for 1 h45 min. The inducer for transpose expression (nisin) was included in the recipientcultures at various concentrations ranging from 0 to 25 ng/ml. Donors andrecipients were mixed (1:9), aliquots were plated on BHI agar supplemented withcorresponding concentrations of nisin, and the plates were incubated at 30°C for�21 h. Bacteria were recovered by scraping into BHI supplemented with 2 mMEDTA (to prevent aggregation), and aliquots were spread on BHI agar supple-mented with Cm, X-Gal, and Fa (to select for transconjugants). Plates wereincubated at 37°C. White colonies arose only in the presence of pCJK55, andthey arose in greatest numbers when nisin was present at 25 ng/ml. Twenty-sevenwhite colonies were tested for conjugative cotransfer of donor markers as de-scribed previously (23), and none was detected.

For construction of the EfaMarTn insertion library, the conjugation experi-ment was repeated as above on a larger scale, using nisin at 25 ng/ml fortransposase induction. Transposon mutants were recovered in 22- by 22-cmbioassay trays. Approximately 15,000 white colonies were selected using a Qbotcolony picking robot (Genetix) and arrayed into 384-well microtiter dishes (Ge-netix) preloaded with BHI medium supplemented with Cm, Fa, and 10% glyc-erol. The 384-well plates were incubated at 37°C for �18 h and subsequentlystored at �80°C. Automated colony picking was performed at the High Through-put Biological Analysis Facility at the University of Minnesota.

Microtiter-plate biofilm formation assay. The biofilm assay was performedessentially as previously described (24). Briefly, overnight cultures were diluted100-fold in TSB and incubated for 24 h at 37°C in the wells of 96-well flat-bottompolystyrene microtiter plates (Corning 3595). Planktonic cells were removed, andthe plates were washed to remove any nonadherent cells. Adherent biofilms werestained for 20 min at room temperature with 0.1% safranine before the plateswere washed and then dried at room temperature. The absorbance of the biofilmon the bottom surface of each well of the dried plates was determined at 490 nmusing an enzyme-linked immunosorbent assay microplate reader. All experi-ments included a blank well (culture medium without any bacteria) and a min-imum of seven replicate experimental wells (except for the initial library screen-ing). All experiments were repeated independently on different days a minimumof three times each with similar results.

Isolation of biofilm-defective EfaMarTn insertion mutants. For primaryscreening of the transposon library, overnight precultures were initially preparedin 96-well microtiter plates. This was achieved using a 96-prong replicating deviceto inoculate fresh BHI medium, supplemented with Cm, from the frozenEfaMarTn transposon library master plates. Biofilm cultures were prepared inCorning 3595 microtiter plates by inoculating 100 �l fresh TSB medium withapproximately 2 to 3 �l of the precultures (using the 96-prong replicating device).Although the precise volume of preculture transferred could not be carefullycontrolled by this method of inoculation, preliminary control experiments estab-lished that the assay produced similar results under the conditions used here witha range of initial dilutions (not shown). Microtiter plates were incubated for 24 hat 37°C and then washed and stained as described above. Isolates exhibitingreduced biofilm accumulation (�75% of the wild-type level) in the primaryscreen were individually retested as described above. For all mutants reproduc-ibly exhibiting a defect in biofilm accumulation, growth kinetics were evaluatedby monitoring the increase in culture density at 600 nm as a function of timeduring growth in TSB after an initial dilution of stationary-phase overnightprecultures to an optical density at 600 nm of 0.01.

Analysis of E. faecalis biofilms by scanning electron microscopy. Stock solu-tions of electron microscopy-grade aldehyde fixatives and osmium tetroxide wereobtained from Electron Microscopy Sciences (Hatfield, PA). Alcian blue 8GX(CI no. 74240) was obtained from Fluka/Sigma-Aldrich (St. Louis, MO). Allreagent solutions were filtered to 0.2 �m prior to use. E. faecalis strains OG1RFand 25G5 were cultured in �2.5 ml of TSB in 24-well microtiter plates. Cultureswere incubated at 37°C for 8 h with ethylene oxide-sterilized cellulose membrane(Spectrum Laboratories, Rancho Dominguez, CA) coupons of �8 to 10 mm2 insize. Membranes were washed (3�) in 140 mM sodium cacodylate (pH 7.4; EMbuffer) prior to fixation. Cellulose membranes were fixed for 22 h in a mixture of2% glutaraldehyde and 2% formaldehyde with 4% sucrose in EM buffer. Thepolycationic dye alcian blue 8GX was added to the fixation mixture at a concen-tration of 0.15% to stabilize the biofilm matrix (15). Following primary aldehydefixation, samples were washed (3�) in EM buffer and postfixed in a reduced 1%OsO4 solution containing 1.5% potassium ferricyanide [K3Fe(CN)6] in EMbuffer for 90 min. Following postfixation and cacodylate buffer-based rinses (3�),

the membranes were chemically dehydrated in an ethanol series [50%, 70, 85, 95(2�), 100% (2�)]; ethanol was removed via critical point drying with CO2.Samples were mounted on conductive carbon tabs and coated with 1 to 2 nm ofplatinum with an argon ion beam coater (Denton DV-502). Secondary electronimaging was done with a Hitachi S-4700 field emission scanning electron micro-scope at 2.5 kV; images were collected using Quartz PCI software and stored inuncompressed TIFF format.

Complementation analysis of biofilm formation by biofilm-defective EfaMarTnmutants of E. faecalis. Because the insertion mutants might still contain thetransposase-encoding plasmid (pCJK55), mutants lacking pCJK55 were obtainedby culturing in the absence of Em, plating cell suspensions on BHI agar supple-mented with Cm for single colonies, and replica plating to identify Em-sensitiveisolates. Expression plasmids (Table 1) or the empty vector control plasmid(pMSP3535) was introduced into the corresponding EfaMarTn insertion mutantby electroporation as previously described (3) with selection on BHI supple-mented with Em. For biofilm tests, strains carrying pMSP3535 or expressionplasmids were precultured in BHI supplemented with Em and nisin at 25 ng/mlto induce expression of the cloned inserts. The microtiter plate biofilm assay wasperformed as described above except that the growth medium was TSB supple-mented with Em at 10 �g/ml and nisin at 25 ng/ml. Preliminary control experi-ments established that inclusion of Em and nisin, under the conditions used here,did not affect biofilm formation of wild-type E. faecalis.

DNA sequencing. All DNA sequencing was performed at the BiomedicalGenomics Center, University of Minnesota. For direct sequencing of transposoninsertion sites from genomic DNA, chromosomal DNA from EfaMarTn mutants(2 ml of stationary-phase overnight culture in BHI supplemented with Cm) waspurified using the DNEasy blood and tissue kit (Qiagen). The purified DNA wasprecipitated with ethanol and resuspended in 10 mM Tris, pH 8.0. Approxi-mately 5 to 7 �g of DNA was submitted as a template for sequencing with 10pmol of primer GFPR2 using Big Dye terminator cycle sequencing (ABI).Cycling parameters were as follows: 95°C for 5 min, followed by 60 cycles of 95°Cfor 30 s, 55°C for 20 s, and 60°C for 4 min.

Bioinformatic analyses. To identify the site of EfaMarTn insertion in the E.faecalis genome, DNA sequences obtained from genomic DNA of insertionmutants were individually compared to the complete genome sequence of E.faecalis V583 (available at The Institute for Genomic Research [http://www.tigr.org]) using the BLASTN algorithm. In 53 out of 60 cases, a matching sequencewith close to 100% identity could be identified. The remaining seven sequenceswere individually compared to the incomplete genome sequence of E. faecalisOG1RF (available from Baylor College of Medicine [http://www.hgsc.bcm.tmc.edu]), using the BLASTN algorithm to identify matching sequences.

To identify segments of the E. faecalis V583 genome that are absent from thegenome of strain OG1RF, the partially assembled OG1RF contigs were obtainedfrom the Baylor College of Medicine website. The nucleotide sequences of allannotated V583 open reading frames (ORFs) (obtained from TIGR) were com-pared with the OG1RF contigs using the Smith-Waterman algorithm computedwith the TimeLogic DeCypher software program (Active Motif, Inc.). The top-ranked hit for each V583 ORF was examined for percent alignment with theOG1RF contig. Hits with greater than 90% alignment were considered matchesand scored as present in the OG1RF genome. Tabulation of the results on anORF-by-ORF basis revealed the presence of multigene gaps missing from theOG1RF genome, some of which were previously known. Gaps larger than 35 kbare depicted in Fig. 3.

RESULTS

Development of a mariner-based system for transposon mu-tagenesis in E. faecalis. mariner-based transposons have beenconstructed for use in diverse bacterial species. In Staphylo-coccus aureus, random mutagenesis on a genome-wide scalewas achieved using the mariner-based bursa aurealis transposonsystem, a two-plasmid system in which the transposable ele-ment is delivered separately from mariner transposase (2). Ini-tial attempts to isolate insertions in the genome of E. faecalisOG1RF using the bursa aurealis system were unsuccessful.Since the cause of these failures was unknown and could bedue to many factors, we completely reengineered the systemfor use in E. faecalis. The result is a two-plasmid system fea-turing a 2.1-kb transposable element (EfaMarTn) encoding a

VOL. 74, 2008 mariner MUTAGENESIS IN ENTEROCOCCUS FAECALIS 3379

promoterless gfp gene and a Cm resistance determinant flankedby inverted repeat sequences recognized by the mariner trans-posase (Fig. 1). The EfaMarTn element is carried on a deliveryplasmid (pCJK72) (Fig. 1) that is essentially a derivative ofpCJK47 (23), enabling the previously described high-frequencyconjugative delivery system for E. faecalis to be used as anefficient means of introducing EfaMarTn into cells targeted formutagenesis. For transposition to occur, the target cells mustcarry a separate expression plasmid encoding a nisin-induciblemariner transposase (pCJK55), which acts in trans on theEfaMarTn element. Because the EfaMarTn delivery plasmidcannot replicate in target cells, Cm-resistant transconjugantsarising after delivery of pCJK72 are expected to carryEfaMarTn insertions. Subsequent growth of target cells occursin the absence of nisin and of selection for pCJK55, enablingplasmid segregation. We note that although the EfaMarTnelement carries a promoterless gfp gene whose expressioncould be activated by transcriptional readthrough from adja-cent chromosomal DNA, preliminary analysis by flow cytom-

etry of several EfaMarTn-containing insertion mutants re-vealed little fluorescence (not shown), suggesting that thesuboptimal RBS upstream of gfp is not efficiently recognized inE. faecalis.

Isolation of E. faecalis OG1RF mutants carrying EfaMarTn.To test our transposon mutagenesis system, the EfaMarTnelement was introduced by conjugation of pCJK72 into E.faecalis OG1RF carrying either the expression plasmid for themariner transposase, pCJK55, or an empty control plasmid. Arange of nisin concentrations was tested during conjugation tomodulate the abundance of transposase in recipient cells. Con-jugation was followed by selection for Cm-resistant transcon-jugants on medium supplemented with X-Gal. The constitu-tively expressed lacZ gene found on the plasmid backbone ofpCJK72 should be lost after a successful transposition event,thereby yielding white, Cm-resistant colonies on medium con-taining X-Gal. We found that appearance of such colonies wasdependent on both the presence of the transposase in recipientcells and induction of transposase expression with nisin (notshown). However, blue colonies were recovered from all con-jugation experiments, regardless of the addition of the induceror the presence of the transposase, suggesting that the entirepCJK72 plasmid was retained in some recipient cells by anunknown mechanism. These blue colonies were not investi-gated further.

Genomic DNA was prepared from 28 EfaMarTn insertionmutants (Cm-resistant, white isolates), digested with SpeI, andsubjected to Southern blot analysis using a probe (Fig. 1)specific for gfp in the EfaMarTn element. For most insertionmutants (27/28), a single hybridization signal was detected(Fig. 2), indicating that most EfaMarTn mutants carried onlyone transposon insertion. Furthermore, the variability in sizeof the hybridizing genomic fragment indicated that EfaMarTninsertions occurred at a variety of distinct locations in the E.faecalis genome (see Fig. 2 for representative data).

To explore the ability of EfaMarTn to generate transposoninsertions in a strain of E. faecalis other than OG1RF, thecompletely independent strain FA2-2 (9) was used as a recip-ient in a conjugation experiment as described above. We foundthat FA2-2 transposon mutants appeared in a transposon-de-pendent manner and at a frequency similar to that seen withOG1RF (not shown).

FIG. 1. Relevant features of the EfaMarTn transposable elementand delivery plasmid for transposon mutagenesis in E. faecalis.(A) Schematic of the EfaMarTn element. The element is defined byterminal inverted repeats (IR), encoding recognition sites for the mar-iner transposase, and carries two ORFs (a cat gene encoding chloram-phenicol resistance in gram-positive bacteria and a promoterless gfpgene). The horizontal black rectangle represents the site of hybridiza-tion for the Southern blot probe used in this study. A unique SpeI siteused for Southern analysis is indicated. Primer GFPR2, used to se-quence sites of EfaMarTn insertion in genomic DNA, is depictedbelow. (B) Schematic of the EfaMarTn conjugative delivery plasmid,pCJK72. This plasmid is essentially a derivative of pCJK47 carrying theEfaMarTn element on a BamHI fragment. A cloned origin of transfer(oriT) enables conjugative delivery of pCJK72 to recipient E. faecaliscells targeted for mutagenesis, while a constitutively expressed lacZgene renders pCJK72-containing cells blue on medium supplementedwith X-Gal. Note that pCJK72 lacks a copy of the essential replicationprotein RepA and therefore requires that RepA be provided in transfor plasmid replication.

FIG. 2. Southern blot analysis of randomly chosen E. faecalisEfaMarTn insertion mutants. Genomic DNA from Cm-resistant mu-tants forming white colonies on agar supplemented with X-Gal wasdigested with SpeI and hybridized to a digoxigenin-labeled probe spe-cific for gfp (lanes 1 to 14). Results are representative of 28 isolatestested. As a positive control, pCJK72 plasmid DNA was included (laneP). Digoxigenin-labeled molecular size markers were included as sizestandards (lane L). The four clearly visible size standards are (top tobottom) 5.1 kb, 4.9 kb, 2.0 kb, and 1.9 kb.


Construction of an EfaMarTn insertion library for E. fae-calis OG1RF. To attempt comprehensive genome-wide trans-poson mutagenesis of the E. faecalis OG1RF genome, thetransposon mutagenesis experiment was repeated on a largerscale. Approximately 15,000 white, Cm-resistant colonies wereselected, cultured in 384-well microtiter-plate arrays, andstored at �80°C to create a library of EfaMarTn mutants.Genomic DNA was isolated from �80 randomly chosen iso-lates and subjected to DNA sequencing using primer GFPR2(Fig. 1), successfully generating sequence data that extend offthe end of the EfaMarTn element into adjacent chromosomalDNA for 60 of the isolates tested. BLAST analysis of thesequence data against the complete E. faecalis V583 genomesequence (available at The Institute for Genomic Research;see URL above) was used to determine the site of insertion ofthe EfaMarTn element. Consistent with mariner-based mu-tagenesis systems used for other bacterial species, allEfaMarTn insertions in E. faecalis were found to have oc-curred at a TA dinucleotide. Corresponding sequences couldnot be identified in the V583 genome for seven of theEfaMarTn mutants. However, BLAST analysis of those seven

sequences against the incomplete E. faecalis OG1RF genomesequence (available from Baylor College of Medicine; seeURL above) revealed the presence of corresponding se-quences in that strain of E. faecalis, indicating that theEfaMarTn element inserted into OG1RF-specific DNA inthese seven isolates. Insertion sites for the remaining 53 iso-lates could be identified in the V583 genome, indicating thatEfaMarTn insertion occurred in a segment of the OG1RFgenome that is common to both strains. These insertions aremapped onto a circular representation of the V583 genome inFig. 3, revealing a genome-wide distribution of insertion sitesthat are found both in ORFs (47 insertions) and in intergenicregions (5 insertions). Large gaps that are apparent in theinsertion site map of Fig. 3 largely correspond to genomicregions that are V583 specific, such as the 150-kb E. faecalispathogenicity island (45) and the locus encoding high-levelvancomycin resistance, explaining the absence of insertions inthose regions (V583-specific segments are depicted in Fig. 3).Collectively, the data shown in Fig. 2 and 3 indicate that trans-position of the EfaMarTn element in E. faecalis produces pre-dominantly mutants with single insertions that are distributedaround the chromosome in an apparently random fashion.

Isolation of biofilm-defective EfaMarTn insertion mutants.To identify genetic determinants in the E. faecalis chromosomethat contribute to its ability to form biofilms, we screened theEfaMarTn insertion library using an established microtiterplate procedure that is suited to a relatively high-throughputformat (see Materials and Methods). Biofilm formation by E.faecalis has previously been evaluated with this approach (orslight variations thereof) by several groups (19, 24, 37, 51).Using this assay, the entire EfaMarTn insertion library(�15,000 isolates) was screened to identify mutants exhibitinga reduced ability to form biofilms. Twenty-five unique mutantsthat formed biofilms more poorly than the parental strain(OG1RF) under our assay conditions were isolated (Fig. 4).Genomic DNA was prepared from these 25 isolates and sub-jected to DNA sequencing using primer GFPR2. BLAST anal-ysis of the DNA sequences flanking the EfaMarTn elementrevealed that these mutants carried EfaMarTn insertions at 10distinct loci (Table 2). Many of these mutations occurred inloci previously reported to be involved in E. faecalis biofilmformation, thereby validating the experimental approach. Forexample, the ebp locus, encoding endocarditis- and biofilm-

FIG. 3. Distribution of randomly chosen EfaMarTn insertions inthe E. faecalis chromosome. Because the genome assembly for E.faecalis OG1RF is not yet complete, insertion sites of the EfaMarTntransposable element in 53 transposon mutants of E. faecalis OG1RFwere mapped onto a circular representation of the E. faecalis V583chromosome. Positions of the EfaMarTn insertions are indicated bytick marks. Black boxes represent segments of the V583 genome thatare absent from the genome of strain OG1RF, determined as de-scribed in Materials and Methods.

FIG. 4. Biofilm formation by biofilm-defective EfaMarTn mutants of E. faecalis in the wells of microtiter plates. The experiment was performedas described in Materials and Methods. The mean absorbance for seven microtiter wells each for a given transposon mutant is expressed relativeto that for the parental wild-type E. faecalis OG1RF (which typically gave mean absorbances of �0.18). Results are representative of a minimumof three trials conducted on different days. Error bars represent the standard errors of the means.


associated pili, has been implicated in biofilm formation (7,37). We isolated mutants carrying insertions in all three genesencoding structural components of the pili (ebpABC), in thegene encoding the adjacent sortase presumably responsible forbiogenesis of the pili (srtC/bps), and in the gene (ebpR) encod-ing the divergently transcribed regulatory protein responsiblefor activating ebp gene expression. We also isolated a mutantcarrying an EfaMarTn insertion in the ebpR-ebpA intergenicregion that presumably affects expression of one or both of theadjacent genes, although this hypothesis has not been testedexperimentally. Several other loci that have been previouslyimplicated in E. faecalis biofilm formation were also identifiedin our screen and are indicated in Table 2. Importantly, ourscreen yielded 11 mutants with insertions in 6 genetic loci thatwere not previously known to have a role in biofilm formation(Table 2), suggesting a role for the corresponding gene prod-ucts in early stages of biofilm formation and validating theEfaMarTn transposon as a discovery tool enabling genome-wide analysis of the E. faecalis chromosome.

To test the possibility that general defects in growth of theEfaMarTn mutants contribute to the observed reduction inbiofilm formation, we performed kinetic analyses of growth.Most of the insertion mutants grew with kinetics similar to thatof wild-type E. faecalis OG1RF (not shown), indicating thatthe transposon insertion confers a biofilm-specific defect forthese mutants. A few mutants did exhibit a growth defectcompared to the wild type: the insertion in EF0394 (salB),mutation of which was previously reported to cause a growthdefect (36); the insertion in EF0721, which, to the best of ourknowledge, has not been studied for E. faecalis; and insertionsin the locus encoding heat shock proteins (EF1306 to EF1310).

The possibility that the general growth defects of these inser-tion mutants contribute to their biofilm-defective phenotypecannot be excluded. However, a salB mutant of E. faecalis haspreviously been reported to exhibit a defect in biofilm forma-tion (36), and a connection between heat shock genes andbiofilm formation has been reported for other species of bac-teria (6, 13, 31), suggesting that these gene products may in-deed have a biofilm-specific role that extends beyond theirrequirement for wild-type growth. Additional work is requiredto elucidate the role of these gene products in biofilm forma-tion by E. faecalis.

Comparative analysis of biofilm formation by wild-type E.faecalis and an EF0799 (autolysin) mutant by scanning elec-tron microscopy. While a comprehensive analysis of biofilmformation by strains containing mutations in all of the putativebiofilm loci described above is beyond the scope of this study,we wanted to examine the potential utility of high-resolutionfield emission scanning electron microscopy (FESEM) for thispurpose; for our initial study, we chose to examine strain 25G5,containing an insertion in the E. faecalis autolysin gene(EF0799). Since the microtiter plate system is not conducivefor FESEM work, we cultivated biofilms for various time pe-riods on submerged cellulose membrane coupons (Materialsand Methods) and compared appearances of the resulting ad-herent populations using FESEM. Growth of the wild-typestrain for 8 h resulted in the appearance of numerous attached2- to 6-cell chains, with frequent “microcolonies” ranging innumber from 10 to 20 cells to multilayered masses of thou-sands of adherent cells (Fig. 5). In addition, a substantial por-tion of the membrane surface, including regions that did notcontain attached bacteria, was covered with filamentous mate-

TABLE 2. Complete list of independent biofilm-defective EfaMarTn transposon mutants isolated in this study

Mutant EF locusa Annotation (gene name)b Coordinatec Referenced

24C19 EF0394 Secreted antigen (salB) 366051 3617M8 EF0676 Transcriptional regulator, ArgR family 628777 None38L2 EF0721 ATP-dependent DNA helicase (pcrA) 677469 None28G1 EF0798/EF0799 Intergenic region 75894030M10 EF0799 Autolysin (atn) 759651 3525G5 EF0799 Autolysin (atn) 759961 3532M20 EF0799 Autolysin (atn) 760262 3520K19 EF0983 Transcriptional regulator, ArgR family 941186 None11I11 EF0983 Transcriptional regulator, ArgR family 941379 None30B8 EF0999 Conserved hypothetical protein 957433 None1D21 EF1090 Endocarditis and biofilm pilus regulator (ebpR) 1056825 716P14 EF1090 Endocarditis and biofilm pilus regulator (ebpR) 1056565 712F13 EF1090 Endocarditis and biofilm pilus regulator (ebpR) 1055842 71M21 EF1090/EF1091 ebpRA intergenic region 105718321O19 EF1091 Endocarditis and biofilm pilus subunit (ebpA) 1057891 3712F3 EF1092 Endocarditis and biofilm pilus subunit (ebpB) 1060920 3721H10 EF1093 Endocarditis and biofilm pilus subunit (ebpC) 1063381 3734N3 EF1094 srtC sortase (bps) 1064624 22, 3716P3 EF1305/EF1306 Intergenic region 1272779 None34H22 EF1305/EF1306 Intergenic region 1272774 None1O23 EF1307 Heat shock protein (grpE) 1274261 None20M2 EF1308 Heat shock protein (dnaK) 1274777 None23D4 EF1310 Heat shock protein (dnaJ) 1277419 None39F15 EF1718 Dihydroorotase (pyrC) 1668694 None39C17 EF3056 Major sortase (srtA) 2930221 22

a EF locus designation assigned by TIGR (from the completely annotated genome, EF0001 to EF3333) for the ORF disrupted by the EfaMarTn transposon.b Annotation assigned by TIGR or, where available, in the literature.c Coordinate of the EfaMarTn insertion, mapped onto the E. faecalis V583 genome.d In the indicated reference, a biofilm defect was reported for an E. faecalis strain carrying a mutation in the corresponding gene.


rial of various lengths and thicknesses; this material seemed toresult from growth of the bacteria, since it was not found inidentically prepared membranes incubated in sterile medium(not shown). In the case of the EF0799 mutant (Fig. 6), at-tached bacterial cells were also observed on the surface (al-though examination of numerous fields suggested that the fre-quency of attached cells per unit of surface area was reducedrelative to that of the wild type), as was some adherent extra-cellular filamentous material. The apparent chain length of thecells was longer than that of the wild type, as has been previ-ously observed with autolysis-defective E. faecalis (52). Themost striking difference between the two strains was that de-velopment of multilayered microcolonies such as those shownin Fig. 5 was clearly impaired in the mutant strain and essen-tially no multilayered masses were observed in examining doz-ens of fields from multiple membrane biofilm cultures (see theadditional micrographs of 8-h biofilm cells presented in thesupplemental material). A detailed, testable hypothesis forthe role of the E. faecalis autolysin in biofilm development ispresented below (see Discussion). We anticipate that FESEMwill be a useful experimental tool in further analysis of themutants identified in this study.

Complementation analysis of biofilm-defective EfaMarTnmutants. To test whether EfaMarTn insertion or some otherunknown mutation was responsible for the biofilm-defectivephenotype of the EfaMarTn mutants, we performed comple-mentation analysis on selected mutants. Expression of EF0676,

EF0983, EF1090 (ebpR), and EF3056 (srtA) rescued the bio-film formation defect of the corresponding EfaMarTn mutants(Fig. 7), demonstrating that disruption of these genes withEfaMarTn was indeed responsible for the biofilm defect of themutants. We note that complementation with the regulatoryfactor EF1090 (ebpR), known to control expression of thegenes encoding endocarditis- and biofilm-associated pili (7),reproducibly resulted in enhanced production of biofilm rela-tive to that of the wild type under our conditions, suggestingthat pili production may be a limiting factor during biofilmformation by wild-type E. faecalis OG1RF, at least under theconditions used here. We also attempted a similar complemen-tation experiment for EF0394 (salB), but for unknown reasonswe did not observe rescue of the biofilm defect upon expres-sion of the salB ORF (not shown).

DISCUSSION

Genetic analysis of enterococci has so far been limited byinefficient or nonexistent tools for genetic manipulation, in-cluding effective approaches for transposon mutagenesis. Theproblems inherent in previous approaches for transposon mu-tagenesis of the E. faecalis chromosome, such as the biasedinsertion site preference exhibited by Tn917 (17), severely limitresearch probing the genetic basis of E. faecalis physiology. Wenow report the development of an efficient system for genome-wide, random transposon mutagenesis in E. faecalis that over-

FIG. 5. Scanning electron microscopic analysis of biofilm formation by E. faecalis OG1RF. Biofilm cultures were generated by growth for8 h on cellulose membrane coupons, fixed, stained with alcian blue, and examined by field emission scanning electron microscopy asdescribed in Materials and Methods. The same microcolony is shown in all four images. Magnifications, �880 (A), �2,640 (B), �8,800 (C),and �22,000 (D).


comes limitations plaguing previous approaches to transposonmutagenesis in this organism. Our approach uses a two-plas-mid scheme based on the mariner transposon, in which theEfaMarTn transposable element is delivered at high frequencyby conjugation into recipient cells that harbor an expressionplasmid encoding an inducible copy of the mariner transposase.

mariner-based transposable elements have been adapted formutagenesis in a wide variety of prokaryotes (1, 2, 20, 29, 32,33, 40, 46, 48, 54, 55), largely because they offer the advantageof host-factor-independent, random insertion distributionsthat enable a relatively comprehensive discovery of geneticdeterminants underlying a phenotype of interest. Our resultsindicate that the EfaMarTn transposable element describedhere inserts in an apparently random fashion at sites distrib-uted throughout the entire genome of E. faecalis (Fig. 2 and 3),suggesting this transposon represents the first such tool en-abling effective genome-wide genetic analysis in this organism.

As proof of principle, we used the EfaMarTn element toisolate E. faecalis mutants exhibiting a reduced ability to formbiofilms relative to that of wild-type E. faecalis OG1RF. Sev-eral genes involved in the process of biofilm formation by E.faecalis OG1RF had previously been identified using targetedmutagenesis approaches, but given the complexity and multi-factorial nature of the biofilm formation phenotype of otherbacteria, we hypothesized that additional, unknown geneticdeterminants contribute to biofilm formation by E. faecalis.Using a microtiter-based biofilm assay, we screened �15,000EfaMarTn insertion mutants for a defect in biofilm formationand identified insertions in many of the genes known to con-tribute to growth of E. faecalis in a biofilm state, as expected(Table 2). However, we also identified 11 insertions in 6 pre-viously unknown genetic loci, suggesting a role for the corre-sponding gene products in E. faecalis biofilm formation.

FIG. 6. Scanning electron microscopic analysis of biofilm formation by E. faecalis mutant 25G5. Strain 25G5, containing an EfaMarTn insertionin EF0799, was cultivated for 8 h on cellulose membrane coupons and prepared for FESEM analysis using the same procedure as that describedabove for OG1RF. The same attached chain is shown. Magnifications, �880 (A), �2,640 (B), �8,800 (C), and �22,000 (D).

FIG. 7. Complementation analysis of biofilm formation by biofilm-defective EfaMarTn mutants of E. faecalis. The experiment was per-formed as described in Materials and Methods. The mean absorbanceof a minimum of seven microtiter wells each for a given transposonmutant carrying either empty expression vector (�) or expressionvector plus the indicated E. faecalis ORF (�) is expressed relative tothat of parental wild-type E. faecalis OG1RF carrying the empty ex-pression vector (which typically gave a mean absorbances of �0.18).Results are representative of a minimum of three trials conducted ondifferent days. Error bars represent standard errors of the means.Transposon mutants tested for complementation were 17M8(EF0676), 20K19 (EF0983), 1D21 (EF1090), and 39C17 (EF3056).


Homologs of some of the previously unknown genes weidentified have been linked to biofilm formation by other or-ganisms. For example, we identified a cluster of EfaMarTninsertions in heat shock genes (EF1306 to EF1310), homologsof which are overexpressed in mature biofilms of E. coli (6).Heat shock genes have also been linked to biofilm formationby Pseudomonas putida (13) and Streptococcus mutans (31).Other previously unknown loci we identified encode geneproducts that have not been implicated in biofilm formationelsewhere and might lead to new insights into the regulation ofbiofilm formation. For example, to the best of our knowledgeno homologs of pcrA or pyrC are known to play a role in biofilmformation in other bacteria. While the role of these gene prod-ucts in biofilm formation is not yet clear, one possibility is thatthe extent of DNA supercoiling (in the case of the pcrA DNAhelicase) or changes in the levels of pyrimidine nucleotidepools (in the case of pyrC dihydroorotase) could alter theexpression of genes controlling biofilm formation. More directly,two of the previously unknown loci encode putative transcrip-tional regulatory factors of unknown function (EF0676 andEF0983; Table 2) that may modulate expression of genes that playa role in initial adaptation of E. faecalis to surface-associatedgrowth.

One noteworthy feature of our results is the fact that weobtained multiple—but nonsibling—EfaMarTn insertions inseveral of the loci identified as important for biofilm formation(Table 2). For example, we obtained three distinct insertions inboth EF0799 (atn) and EF1090 (ebpR). Additionally, we ob-tained insertions in each of the other four genes known tocomprise the ebp gene cluster (EF1091 to 1094) required forbiogenesis of pili, as well as insertions at multiple sites withinthe heat shock locus (EF1306 to EF1310). These observationssuggest that our transposon mutagenesis screen for biofilm-defective insertion mutants has essentially reached saturation,at least in the context of the specific set of experimental con-ditions we used here. A well-known limitation of the micro-titer-plate biofilm assay is that it reflects primarily contributionsfrom initial adherence and early stages of biofilm formation.Thus, although our screen may have identified all of the ge-netic determinants that are important for biofilm formation ofE. faecalis OG1RF under these specific conditions, it likely hasnot revealed many other determinants that play a role in laterstages of biofilm formation. For example, exopolysaccharidebiosynthesis is required for formation of mature biofilms bymany bacteria, because the exopolysaccharides form criticalcomponents of the extracellular matrix defining biofilm archi-tecture. E. faecalis likely also relies on exopolysaccharides formature biofilm formation, yet we did not identify any genes forexopolysaccharide biosynthesis in our screen, suggesting thatsuch genes are required much later in the pathway leading tomature biofilms. Nonetheless, the genes identified here mayplay critical roles in regulating the initial transition of E. fae-calis from a planktonic to a sessile lifestyle.

Initial scanning electron microscopic analysis of the effectsof inactivation of the EF0799 locus on biofilm formation wascarried out because of previous evidence for a role of the E.faecalis autolysin (33) and because autolysis and release ofDNA into the biofilm matrix have been suggested to be im-portant in biofilm development for many organisms (5). Scan-ning electron microscopy analysis (Fig. 5 and 6) suggested that

the inactivation of EF0799 impaired the ability of the organismto transition from a single chain of adherent bacteria to amultilayered mass of bacteria typical of more mature biofilms.It can be hypothesized that autolysin-enhanced release ofDNA by bacteria during the first few hours of surface growthcontributes to an extracellular matrix that facilitates develop-ment of the mature biofilm. Future research will test this hy-pothesis and will probe the specific functions of the otherORFs identified here in promoting biofilm formation of E.faecalis. Importantly, these results emphasize the utility of anunbiased genome-wide approach, such as EfaMarTn mutagen-esis, to probe the genetic basis of E. faecalis physiology andbehavior.

ACKNOWLEDGMENTS

We thank David Lampe, Olaf Schneewind, Dominique Missiakas,and Taeok Bae for providing plasmids; Laura Case for flow cytometryon selected transposon mutants; and Dawn Manias for technical as-sistance and support. Preliminary sequence data for E. faecalis OG1RFwere obtained from the Baylor College of Medicine Human GenomeSequencing Center website at http://www.hgsc.bcm.tmc.edu. Compu-tational resources and support services were provided by the Centerfor Biomedical Research Informatics at the University of Minnesota(http://cbri.umn.edu).

Sequencing of E. faecalis OG1RF was supported by grant R21AI64470 from NIAID. Parts of this work were carried out in theUniversity of Minnesota I.T. Characterization Facility, which receivespartial support from NSF through the NNIN program. C.J.K. wassupported during a portion of this work by NRSA fellowship F32-AI56684 from NIAID. A.M.T.B. received support from Medical Sci-entist Training Grant 5T32-GM008244 from the NIH. This work wassupported by grant AI058134 from NIH to G.M.D.

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