Development and Use of an Efficient System for Random mariner ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2008, p. 3377–3386 0099-2240/08/$08.00⫹0 doi:10.1128/AEM.02665-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 11

Development and Use of an Efficient System for Random mariner Transposon Mutagenesis To Identify Novel Genetic Determinants of Biofilm Formation in the Core Enterococcus faecalis Genome䌤† 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 important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion 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 known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis. Previous studies have identified a small number of genes important for biofilm formation by E. faecalis. Although some are encoded on mobile genetic elements (50), others are chromosomally encoded, suggesting that they belong to the core genome 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 surface proteins to the cell wall (22); and atn, encoding an autolysin (35). Although a role in biofilm formation of E. faecalis has been ascribed to these genes and others, to date no comprehensive genome-wide search has been performed for chromosomally encoded genetic determinants of biofilm formation by E. faecalis. This is due, in part, to the lack of effective genetic tools enabling such a genome-wide search for E. faecalis. In particular, no method for efficient and random transposon mutagenesis has been developed. Biofilm formation is known to be a complex process involving signal transduction systems, transcriptional regulation, and stress responses, among other features (38, 47). We hypothesized that the E. faecalis genome encodes unidentified genetic determinants, in some or all of these functional categories, which promote biofilm formation. We sought to identify these determinants using an unbiased, genome-wide approach, namely, random transposon mutagenesis. At least two transposons have previously been used for mutagenesis of the chromosome in E. faecalis (Tn916 and Tn917). However, these transposons each suffer from significant drawbacks that restrict their utility in the context of a comprehensive genome-scale mutagenesis experiment, including insertion in multiple copies per genome (18) and significantly biased insertion-site prefer-

Enterococcus faecalis is a gram-positive bacterium that primarily associates with humans in a benign manner as a member of the gastrointestinal tract microbial consortium (49). However, enterococci also represent a serious health concern as one of the three most common causes of hospital-acquired infections (39, 42). Enterococci exhibit relatively high-level intrinsic resistance to some antibiotics and share mobile genetic elements carrying additional antibiotic resistance determinants with neighboring bacteria (21), leading to the emergence of multiresistant clones in the hospital setting. Thus, enterococcal infections are an increasingly difficult problem for clinicians given currently available therapeutic agents. In clinical settings, bacteria growing as surface-adherent biofilms have been implicated as etiological agents of chronic infection (10, 12, 34). E. faecalis is known to form robust biofilms in the laboratory as well as in clinically relevant settings (4, 11, 12, 41, 51). E. faecalis biofilms grown in vitro exhibit enhanced tolerance to antibiotics, such as vancomycin and teicoplanin (16). The enhanced antibiotic tolerance resulting from growth in a biofilm state, superimposed on the intrinsic and acquired antibiotic resistances typical of hospital strains of enterococci, suggests that biofilm formation of E. faecalis in a hospital setting may be particularly problematic.

* 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/. 䌤 Published ahead of print on 11 April 2008. 3377

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KRISTICH ET AL. TABLE 1. Strains and plasmids used in this study Strain or plasmid

Relevant characteristic or description

Strains E. faecalis OG1RF CK111 (pCF10-101) E. coli DH5␣ XL1Blue EC1000 Plasmids pMSP3535 pMSP3545 pBursa pWM401 pBADC9 pCJK15 pCJK34 pCJK47 pCJK49 pCJK55 pCJK72 pCJK122 pCJK123 pCJK124 pCJK128

Source or reference

Reference strain Conjugative donor host strain; supplies RepA in trans

14 23

Cloning host Cloning host Cloning host, provides RepA in trans

Lab stock Lab stock 30

Nisin-inducible expression vector; Emr Nisin-inducible expression vector encoding RBS; Emr Source of bursa aurealis transposable element Source of chloramphenicol acetyltransferase gene Source of mariner transposase C9 E. faecalis EF3056 (srtA) cloned into pMSP3535 Mobilizable plasmid; requires RepA for replication; Emr Derivative of pCJK34 carrying pheS* counterselectable marker Intermediate construct carrying modified mini-mariner element mariner transposase C9 cloned into pMSP3545 Mobilizable delivery plasmid carrying EfaMarTn transposable element E. faecalis EF0394 (salB) cloned into pMSP3535 E. faecalis EF0676 cloned into pMSP3535 E. faecalis EF0983 cloned into pMSP3535 E. faecalis EF1090 (ebpR) cloned into pMSP3535

8 8 2 53 26 25 23 23 This This This This This This This

ences (17, 43, 44) that prevent random coverage of the whole genome. Transposon mutagenesis systems based on the mariner transposable element have been widely used for prokaryotes and are known to yield a random distribution of insertions (1, 20, 29, 32, 33, 40, 46, 48, 54, 55). mariner elements insert at TA dinucleotide pairs in the target sequence and transpose independently of any host factors (27, 28), making them ideal tools for random mutagenesis. To circumvent the problems associated with use of Tn916 and Tn917 in E. faecalis, we developed a substantially improved system for transposon mutagenesis based on a mini-mariner transposable element (EfaMarTn). After transposition by this element, most E. faecalis insertion mutants carried only one EfaMarTn element, and EfaMarTn insertions were found to be distributed around the entire E. faecalis chromosome. Using this mutagenesis system, we constructed a library of EfaMarTn insertion mutants in E. faecalis OG1RF and screened this library to identify genes involved in early stages of biofilm formation. We isolated biofilm-defective mutants carrying transposon insertions in many of the genes previously implicated in biofilm formation by E. faecalis, as well as insertions in new genes not previously known to play a role in biofilm formation, demonstrating the utility of our transposon system as a tool enabling genome-wide genetic analysis of enterococci. MATERIALS AND METHODS Bacterial strains, growth media, and chemicals. Bacterial strains used in this study are listed in Table 1. Unless otherwise indicated, all culture media were purchased from Difco and all chemicals were purchased from Sigma (St. Louis, MO). Bacto brain heart infusion (BHI) and Bacto tryptic soy broth without glucose (TSB) were prepared as described by the manufacturer (Becton Dickinson). Bacto agar was used as a solidifying agent for all semisolid media. Bacteria were routinely stored at ⫺80°C in BHI supplemented with 30% glycerol. When required for selective growth of E. faecalis, erythromycin (Em),

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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 Escherichia coli, Em was used in BHI at 100 ␮g/ml. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Plasmid construction. Plasmids used in this study are listed in Table 1. All derivatives of plasmid pCJK34 were propagated in E. coli strain EC1000 (30), which supplies RepA in trans to allow replication of these plasmids. All other plasmids were propagated in E. coli XL1Blue or DH5␣. The mobilizable delivery plasmid carrying the EfaMarTn transposable element (pCJK72) (see Fig. 1), which is essentially equivalent to a pCJK47 derivative bearing the transposable element on a BamHI fragment, was constructed according to a multistep scheme as follows. The entire 3.2-kb bursa aurealis element was amplified by PCR from plasmid pBursa and cloned into pCJK34 using primer-encoded BamHI sites. Quikchange mutagenesis (Stratagene) was applied to replace the ermB and oriR6K regions from the bursa aurealis element with a short sequence encoding unique SpeI and XhoI restriction sites, creating pCJK49. The pheS* counterselectable marker (23) from pCJK47 was inserted into the plasmid backbone (outside of the transposable element) as a SphI/BglII fragment (note that pheS*-dependent counterselection was not used in this study). The chloramphenicol acetyltransferase gene (cat) from plasmid pWM401 was amplified by PCR and cloned into the SpeI/XhoI sites of the mini-mariner transposable element in the resulting plasmid using primer-encoded restriction sites, thereby creating pCJK72. An expression plasmid for inducible production of mariner transposase was constructed by cloning the mariner transposase C9 from plasmid pBADC9 into the nisin-inducible expression plasmid pMSP3545 as a NcoI/SphI fragment, creating pCJK55. The ribosome binding site (RBS) for the transposase in pCJK55 is provided by pMSP3545. Expression plasmids for complementation analysis of selected EfaMarTn insertion mutants were constructed by first using PCR to amplify the corresponding transposon-disrupted genes from wild-type E. faecalis OG1RF chromosomal DNA. Primers were designed according to the E. faecalis V583 genome sequence. 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 was introduced into the recipient strain by conjugation according to a previously described procedure (23). Briefly, pCJK72 was first introduced by electropora-

VOL. 74, 2008 tion into the conjugative donor strain, E. faecalis CK111 (pCF10-101), where it can replicate. The resulting donor strain was used in a conjugation experiment with E. faecalis OG1RF or FA2-2 carrying pCJK55 (or the empty vector control, pMSP3545) as recipients. Stationary-phase overnight cultures of donors and recipients in BHI supplemented with Em (10 ␮g/ml) were independently diluted 20-fold into BHI supplemented with Em (1 ␮g/ml) and incubated at 30°C for 1 h 45 min. The inducer for transpose expression (nisin) was included in the recipient cultures at various concentrations ranging from 0 to 25 ng/ml. Donors and recipients were mixed (1:9), aliquots were plated on BHI agar supplemented with corresponding concentrations of nisin, and the plates were incubated at 30°C for ⬃21 h. Bacteria were recovered by scraping into BHI supplemented with 2 mM EDTA (to prevent aggregation), and aliquots were spread on BHI agar supplemented with Cm, X-Gal, and Fa (to select for transconjugants). Plates were incubated at 37°C. White colonies arose only in the presence of pCJK55, and they arose in greatest numbers when nisin was present at 25 ng/ml. Twenty-seven white colonies were tested for conjugative cotransfer of donor markers as described previously (23), and none was detected. For construction of the EfaMarTn insertion library, the conjugation experiment was repeated as above on a larger scale, using nisin at 25 ng/ml for transposase induction. Transposon mutants were recovered in 22- by 22-cm bioassay trays. Approximately 15,000 white colonies were selected using a Qbot colony picking robot (Genetix) and arrayed into 384-well microtiter dishes (Genetix) preloaded with BHI medium supplemented with Cm, Fa, and 10% glycerol. The 384-well plates were incubated at 37°C for ⬃18 h and subsequently stored at ⫺80°C. Automated colony picking was performed at the High Throughput Biological Analysis Facility at the University of Minnesota. Microtiter-plate biofilm formation assay. The biofilm assay was performed essentially as previously described (24). Briefly, overnight cultures were diluted 100-fold in TSB and incubated for 24 h at 37°C in the wells of 96-well flat-bottom polystyrene microtiter plates (Corning 3595). Planktonic cells were removed, and the plates were washed to remove any nonadherent cells. Adherent biofilms were stained for 20 min at room temperature with 0.1% safranine before the plates were washed and then dried at room temperature. The absorbance of the biofilm on the bottom surface of each well of the dried plates was determined at 490 nm using an enzyme-linked immunosorbent assay microplate reader. All experiments included a blank well (culture medium without any bacteria) and a minimum of seven replicate experimental wells (except for the initial library screening). All experiments were repeated independently on different days a minimum of three times each with similar results. Isolation of biofilm-defective EfaMarTn insertion mutants. For primary screening of the transposon library, overnight precultures were initially prepared in 96-well microtiter plates. This was achieved using a 96-prong replicating device to inoculate fresh BHI medium, supplemented with Cm, from the frozen EfaMarTn transposon library master plates. Biofilm cultures were prepared in Corning 3595 microtiter plates by inoculating 100 ␮l fresh TSB medium with approximately 2 to 3 ␮l of the precultures (using the 96-prong replicating device). Although the precise volume of preculture transferred could not be carefully controlled by this method of inoculation, preliminary control experiments established that the assay produced similar results under the conditions used here with a range of initial dilutions (not shown). Microtiter plates were incubated for 24 h at 37°C and then washed and stained as described above. Isolates exhibiting reduced biofilm accumulation (⬍75% of the wild-type level) in the primary screen were individually retested as described above. For all mutants reproducibly exhibiting a defect in biofilm accumulation, growth kinetics were evaluated by monitoring the increase in culture density at 600 nm as a function of time during growth in TSB after an initial dilution of stationary-phase overnight precultures to an optical density at 600 nm of 0.01. Analysis of E. faecalis biofilms by scanning electron microscopy. Stock solutions of electron microscopy-grade aldehyde fixatives and osmium tetroxide were obtained from Electron Microscopy Sciences (Hatfield, PA). Alcian blue 8GX (CI no. 74240) was obtained from Fluka/Sigma-Aldrich (St. Louis, MO). All reagent solutions were filtered to 0.2 ␮m prior to use. E. faecalis strains OG1RF and 25G5 were cultured in ⬃2.5 ml of TSB in 24-well microtiter plates. Cultures were incubated at 37°C for 8 h with ethylene oxide-sterilized cellulose membrane (Spectrum Laboratories, Rancho Dominguez, CA) coupons of ⬃8 to 10 mm2 in size. Membranes were washed (3⫻) in 140 mM sodium cacodylate (pH 7.4; EM buffer) prior to fixation. Cellulose membranes were fixed for 22 h in a mixture of 2% glutaraldehyde and 2% formaldehyde with 4% sucrose in EM buffer. The polycationic dye alcian blue 8GX was added to the fixation mixture at a concentration of 0.15% to stabilize the biofilm matrix (15). Following primary aldehyde fixation, samples were washed (3⫻) in EM buffer and postfixed in a reduced 1% OsO4 solution containing 1.5% potassium ferricyanide [K3Fe(CN)6] in EM buffer for 90 min. Following postfixation and cacodylate buffer-based rinses (3⫻),

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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 of platinum with an argon ion beam coater (Denton DV-502). Secondary electron imaging was done with a Hitachi S-4700 field emission scanning electron microscope at 2.5 kV; images were collected using Quartz PCI software and stored in uncompressed TIFF format. Complementation analysis of biofilm formation by biofilm-defective EfaMarTn mutants of E. faecalis. Because the insertion mutants might still contain the transposase-encoding plasmid (pCJK55), mutants lacking pCJK55 were obtained by culturing in the absence of Em, plating cell suspensions on BHI agar supplemented with Cm for single colonies, and replica plating to identify Em-sensitive isolates. Expression plasmids (Table 1) or the empty vector control plasmid (pMSP3535) was introduced into the corresponding EfaMarTn insertion mutant by electroporation as previously described (3) with selection on BHI supplemented with Em. For biofilm tests, strains carrying pMSP3535 or expression plasmids were precultured in BHI supplemented with Em and nisin at 25 ng/ml to induce expression of the cloned inserts. The microtiter plate biofilm assay was performed as described above except that the growth medium was TSB supplemented with Em at 10 ␮g/ml and nisin at 25 ng/ml. Preliminary control experiments 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 Biomedical Genomics Center, University of Minnesota. For direct sequencing of transposon insertion sites from genomic DNA, chromosomal DNA from EfaMarTn mutants (2 ml of stationary-phase overnight culture in BHI supplemented with Cm) was purified using the DNEasy blood and tissue kit (Qiagen). The purified DNA was precipitated with ethanol and resuspended in 10 mM Tris, pH 8.0. Approximately 5 to 7 ␮g of DNA was submitted as a template for sequencing with 10 pmol 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°C for 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 insertion mutants 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 sequence with close to 100% identity could be identified. The remaining seven sequences were individually compared to the incomplete genome sequence of E. faecalis OG1RF (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 the genome of strain OG1RF, the partially assembled OG1RF contigs were obtained from the Baylor College of Medicine website. The nucleotide sequences of all annotated V583 open reading frames (ORFs) (obtained from TIGR) were compared with the OG1RF contigs using the Smith-Waterman algorithm computed with the TimeLogic DeCypher software program (Active Motif, Inc.). The topranked hit for each V583 ORF was examined for percent alignment with the OG1RF contig. Hits with greater than 90% alignment were considered matches and scored as present in the OG1RF genome. Tabulation of the results on an ORF-by-ORF basis revealed the presence of multigene gaps missing from the OG1RF genome, some of which were previously known. Gaps larger than 35 kb are depicted in Fig. 3.

RESULTS Development of a mariner-based system for transposon mutagenesis in E. faecalis. mariner-based transposons have been constructed for use in diverse bacterial species. In Staphylococcus aureus, random mutagenesis on a genome-wide scale was achieved using the mariner-based bursa aurealis transposon system, a two-plasmid system in which the transposable element is delivered separately from mariner transposase (2). Initial attempts to isolate insertions in the genome of E. faecalis OG1RF using the bursa aurealis system were unsuccessful. Since the cause of these failures was unknown and could be due to many factors, we completely reengineered the system for use in E. faecalis. The result is a two-plasmid system featuring a 2.1-kb transposable element (EfaMarTn) encoding a

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

FIG. 1. Relevant features of the EfaMarTn transposable element and delivery plasmid for transposon mutagenesis in E. faecalis. (A) Schematic of the EfaMarTn element. The element is defined by terminal inverted repeats (IR), encoding recognition sites for the mariner transposase, and carries two ORFs (a cat gene encoding chloramphenicol resistance in gram-positive bacteria and a promoterless gfp gene). The horizontal black rectangle represents the site of hybridization for the Southern blot probe used in this study. A unique SpeI site used for Southern analysis is indicated. Primer GFPR2, used to sequence sites of EfaMarTn insertion in genomic DNA, is depicted below. (B) Schematic of the EfaMarTn conjugative delivery plasmid, pCJK72. This plasmid is essentially a derivative of pCJK47 carrying the EfaMarTn element on a BamHI fragment. A cloned origin of transfer (oriT) enables conjugative delivery of pCJK72 to recipient E. faecalis cells targeted for mutagenesis, while a constitutively expressed lacZ gene renders pCJK72-containing cells blue on medium supplemented with X-Gal. Note that pCJK72 lacks a copy of the essential replication protein RepA and therefore requires that RepA be provided in trans for plasmid replication.

promoterless gfp gene and a Cm resistance determinant flanked by inverted repeat sequences recognized by the mariner transposase (Fig. 1). The EfaMarTn element is carried on a delivery plasmid (pCJK72) (Fig. 1) that is essentially a derivative of pCJK47 (23), enabling the previously described high-frequency conjugative delivery system for E. faecalis to be used as an efficient means of introducing EfaMarTn into cells targeted for mutagenesis. For transposition to occur, the target cells must carry a separate expression plasmid encoding a nisin-inducible mariner transposase (pCJK55), which acts in trans on the EfaMarTn element. Because the EfaMarTn delivery plasmid cannot replicate in target cells, Cm-resistant transconjugants arising after delivery of pCJK72 are expected to carry EfaMarTn insertions. Subsequent growth of target cells occurs in the absence of nisin and of selection for pCJK55, enabling plasmid segregation. We note that although the EfaMarTn element carries a promoterless gfp gene whose expression could be activated by transcriptional readthrough from adjacent chromosomal DNA, preliminary analysis by flow cytom-

etry of several EfaMarTn-containing insertion mutants revealed little fluorescence (not shown), suggesting that the suboptimal RBS upstream of gfp is not efficiently recognized in E. faecalis. Isolation of E. faecalis OG1RF mutants carrying EfaMarTn. To test our transposon mutagenesis system, the EfaMarTn element was introduced by conjugation of pCJK72 into E. faecalis OG1RF carrying either the expression plasmid for the mariner transposase, pCJK55, or an empty control plasmid. A range of nisin concentrations was tested during conjugation to modulate the abundance of transposase in recipient cells. Conjugation was followed by selection for Cm-resistant transconjugants on medium supplemented with X-Gal. The constitutively expressed lacZ gene found on the plasmid backbone of pCJK72 should be lost after a successful transposition event, thereby yielding white, Cm-resistant colonies on medium containing X-Gal. We found that appearance of such colonies was dependent on both the presence of the transposase in recipient cells and induction of transposase expression with nisin (not shown). However, blue colonies were recovered from all conjugation experiments, regardless of the addition of the inducer or the presence of the transposase, suggesting that the entire pCJK72 plasmid was retained in some recipient cells by an unknown mechanism. These blue colonies were not investigated further. Genomic DNA was prepared from 28 EfaMarTn insertion mutants (Cm-resistant, white isolates), digested with SpeI, and subjected to Southern blot analysis using a probe (Fig. 1) specific for gfp in the EfaMarTn element. For most insertion mutants (27/28), a single hybridization signal was detected (Fig. 2), indicating that most EfaMarTn mutants carried only one transposon insertion. Furthermore, the variability in size of the hybridizing genomic fragment indicated that EfaMarTn insertions 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 transposon insertions in a strain of E. faecalis other than OG1RF, the completely independent strain FA2-2 (9) was used as a recipient in a conjugation experiment as described above. We found that FA2-2 transposon mutants appeared in a transposon-dependent manner and at a frequency similar to that seen with OG1RF (not shown).

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FIG. 3. Distribution of randomly chosen EfaMarTn insertions in the E. faecalis chromosome. Because the genome assembly for E. faecalis OG1RF is not yet complete, insertion sites of the EfaMarTn transposable element in 53 transposon mutants of E. faecalis OG1RF were mapped onto a circular representation of the E. faecalis V583 chromosome. Positions of the EfaMarTn insertions are indicated by tick marks. Black boxes represent segments of the V583 genome that are absent from the genome of strain OG1RF, determined as described in Materials and Methods.

Construction of an EfaMarTn insertion library for E. faecalis OG1RF. To attempt comprehensive genome-wide transposon mutagenesis of the E. faecalis OG1RF genome, the transposon mutagenesis experiment was repeated on a larger scale. Approximately 15,000 white, Cm-resistant colonies were selected, cultured in 384-well microtiter-plate arrays, and stored at ⫺80°C to create a library of EfaMarTn mutants. Genomic DNA was isolated from ⬃80 randomly chosen isolates and subjected to DNA sequencing using primer GFPR2 (Fig. 1), successfully generating sequence data that extend off the end of the EfaMarTn element into adjacent chromosomal DNA for 60 of the isolates tested. BLAST analysis of the sequence data against the complete E. faecalis V583 genome sequence (available at The Institute for Genomic Research; see URL above) was used to determine the site of insertion of the EfaMarTn element. Consistent with mariner-based mutagenesis systems used for other bacterial species, all EfaMarTn insertions in E. faecalis were found to have occurred at a TA dinucleotide. Corresponding sequences could not be identified in the V583 genome for seven of the EfaMarTn mutants. However, BLAST analysis of those seven

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sequences against the incomplete E. faecalis OG1RF genome sequence (available from Baylor College of Medicine; see URL above) revealed the presence of corresponding sequences in that strain of E. faecalis, indicating that the EfaMarTn element inserted into OG1RF-specific DNA in these seven isolates. Insertion sites for the remaining 53 isolates could be identified in the V583 genome, indicating that EfaMarTn insertion occurred in a segment of the OG1RF genome that is common to both strains. These insertions are mapped onto a circular representation of the V583 genome in Fig. 3, revealing a genome-wide distribution of insertion sites that are found both in ORFs (47 insertions) and in intergenic regions (5 insertions). Large gaps that are apparent in the insertion site map of Fig. 3 largely correspond to genomic regions that are V583 specific, such as the 150-kb E. faecalis pathogenicity island (45) and the locus encoding high-level vancomycin resistance, explaining the absence of insertions in those regions (V583-specific segments are depicted in Fig. 3). Collectively, the data shown in Fig. 2 and 3 indicate that transposition of the EfaMarTn element in E. faecalis produces predominantly mutants with single insertions that are distributed around the chromosome in an apparently random fashion. Isolation of biofilm-defective EfaMarTn insertion mutants. To identify genetic determinants in the E. faecalis chromosome that contribute to its ability to form biofilms, we screened the EfaMarTn insertion library using an established microtiter plate procedure that is suited to a relatively high-throughput format (see Materials and Methods). Biofilm formation by E. faecalis has previously been evaluated with this approach (or slight 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 exhibiting a reduced ability to form biofilms. Twenty-five unique mutants that 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 subjected to DNA sequencing using primer GFPR2. BLAST analysis of the DNA sequences flanking the EfaMarTn element revealed that these mutants carried EfaMarTn insertions at 10 distinct loci (Table 2). Many of these mutations occurred in loci previously reported to be involved in E. faecalis biofilm formation, thereby validating the experimental approach. For example, the ebp locus, encoding endocarditis- and biofilm-

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

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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 17M8 38L2 28G1 30M10 25G5 32M20 20K19 11I11 30B8 1D21 16P14 12F13 1M21 21O19 12F3 21H10 34N3 16P3 34H22 1O23 20M2 23D4 39F15 39C17

EF0394 EF0676 EF0721 EF0798/EF0799 EF0799 EF0799 EF0799 EF0983 EF0983 EF0999 EF1090 EF1090 EF1090 EF1090/EF1091 EF1091 EF1092 EF1093 EF1094 EF1305/EF1306 EF1305/EF1306 EF1307 EF1308 EF1310 EF1718 EF3056

Secreted antigen (salB) Transcriptional regulator, ArgR family ATP-dependent DNA helicase (pcrA) Intergenic region Autolysin (atn) Autolysin (atn) Autolysin (atn) Transcriptional regulator, ArgR family Transcriptional regulator, ArgR family Conserved hypothetical protein Endocarditis and biofilm pilus regulator (ebpR) Endocarditis and biofilm pilus regulator (ebpR) Endocarditis and biofilm pilus regulator (ebpR) ebpRA intergenic region Endocarditis and biofilm pilus subunit (ebpA) Endocarditis and biofilm pilus subunit (ebpB) Endocarditis and biofilm pilus subunit (ebpC) srtC sortase (bps) Intergenic region Intergenic region Heat shock protein (grpE) Heat shock protein (dnaK) Heat shock protein (dnaJ) Dihydroorotase (pyrC) Major sortase (srtA)

366051 628777 677469 758940 759651 759961 760262 941186 941379 957433 1056825 1056565 1055842 1057183 1057891 1060920 1063381 1064624 1272779 1272774 1274261 1274777 1277419 1668694 2930221

36 None None 35 35 35 None None None 7 7 7 37 37 37 22, 37 None None None None None None 22

a

EF locus designation assigned by TIGR (from the completely annotated genome, EF0001 to EF3333) for the ORF disrupted by the EfaMarTn transposon. Annotation assigned by TIGR or, where available, in the literature. 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. b c

associated pili, has been implicated in biofilm formation (7, 37). We isolated mutants carrying insertions in all three genes encoding structural components of the pili (ebpABC), in the gene encoding the adjacent sortase presumably responsible for biogenesis of the pili (srtC/bps), and in the gene (ebpR) encoding the divergently transcribed regulatory protein responsible for activating ebp gene expression. We also isolated a mutant carrying an EfaMarTn insertion in the ebpR-ebpA intergenic region that presumably affects expression of one or both of the adjacent genes, although this hypothesis has not been tested experimentally. Several other loci that have been previously implicated in E. faecalis biofilm formation were also identified in our screen and are indicated in Table 2. Importantly, our screen yielded 11 mutants with insertions in 6 genetic loci that were not previously known to have a role in biofilm formation (Table 2), suggesting a role for the corresponding gene products in early stages of biofilm formation and validating the EfaMarTn transposon as a discovery tool enabling genomewide analysis of the E. faecalis chromosome. To test the possibility that general defects in growth of the EfaMarTn mutants contribute to the observed reduction in biofilm formation, we performed kinetic analyses of growth. Most of the insertion mutants grew with kinetics similar to that of wild-type E. faecalis OG1RF (not shown), indicating that the transposon insertion confers a biofilm-specific defect for these mutants. A few mutants did exhibit a growth defect compared to the wild type: the insertion in EF0394 (salB), mutation of which was previously reported to cause a growth defect (36); the insertion in EF0721, which, to the best of our knowledge, has not been studied for E. faecalis; and insertions in the locus encoding heat shock proteins (EF1306 to EF1310).

The possibility that the general growth defects of these insertion mutants contribute to their biofilm-defective phenotype cannot be excluded. However, a salB mutant of E. faecalis has previously been reported to exhibit a defect in biofilm formation (36), and a connection between heat shock genes and biofilm formation has been reported for other species of bacteria (6, 13, 31), suggesting that these gene products may indeed have a biofilm-specific role that extends beyond their requirement for wild-type growth. Additional work is required to elucidate the role of these gene products in biofilm formation by E. faecalis. Comparative analysis of biofilm formation by wild-type E. faecalis and an EF0799 (autolysin) mutant by scanning electron microscopy. While a comprehensive analysis of biofilm formation by strains containing mutations in all of the putative biofilm loci described above is beyond the scope of this study, we wanted to examine the potential utility of high-resolution field emission scanning electron microscopy (FESEM) for this purpose; 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 conducive for FESEM work, we cultivated biofilms for various time periods on submerged cellulose membrane coupons (Materials and Methods) and compared appearances of the resulting adherent populations using FESEM. Growth of the wild-type strain for 8 h resulted in the appearance of numerous attached 2- to 6-cell chains, with frequent “microcolonies” ranging in number from 10 to 20 cells to multilayered masses of thousands of adherent cells (Fig. 5). In addition, a substantial portion of the membrane surface, including regions that did not contain attached bacteria, was covered with filamentous mate-

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FIG. 5. Scanning electron microscopic analysis of biofilm formation by E. faecalis OG1RF. Biofilm cultures were generated by growth for 8 h on cellulose membrane coupons, fixed, stained with alcian blue, and examined by field emission scanning electron microscopy as described 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).

rial of various lengths and thicknesses; this material seemed to result from growth of the bacteria, since it was not found in identically prepared membranes incubated in sterile medium (not shown). In the case of the EF0799 mutant (Fig. 6), attached bacterial cells were also observed on the surface (although examination of numerous fields suggested that the frequency of attached cells per unit of surface area was reduced relative to that of the wild type), as was some adherent extracellular filamentous material. The apparent chain length of the cells was longer than that of the wild type, as has been previously observed with autolysis-defective E. faecalis (52). The most striking difference between the two strains was that development of multilayered microcolonies such as those shown in Fig. 5 was clearly impaired in the mutant strain and essentially no multilayered masses were observed in examining dozens of fields from multiple membrane biofilm cultures (see the additional micrographs of 8-h biofilm cells presented in the supplemental material). A detailed, testable hypothesis for the role of the E. faecalis autolysin in biofilm development is presented below (see Discussion). We anticipate that FESEM will be a useful experimental tool in further analysis of the mutants identified in this study. Complementation analysis of biofilm-defective EfaMarTn mutants. To test whether EfaMarTn insertion or some other unknown mutation was responsible for the biofilm-defective phenotype of the EfaMarTn mutants, we performed complementation analysis on selected mutants. Expression of EF0676,

EF0983, EF1090 (ebpR), and EF3056 (srtA) rescued the biofilm formation defect of the corresponding EfaMarTn mutants (Fig. 7), demonstrating that disruption of these genes with EfaMarTn was indeed responsible for the biofilm defect of the mutants. We note that complementation with the regulatory factor EF1090 (ebpR), known to control expression of the genes encoding endocarditis- and biofilm-associated pili (7), reproducibly resulted in enhanced production of biofilm relative to that of the wild type under our conditions, suggesting that pili production may be a limiting factor during biofilm formation by wild-type E. faecalis OG1RF, at least under the conditions used here. We also attempted a similar complementation experiment for EF0394 (salB), but for unknown reasons we did not observe rescue of the biofilm defect upon expression of the salB ORF (not shown). DISCUSSION Genetic analysis of enterococci has so far been limited by inefficient or nonexistent tools for genetic manipulation, including effective approaches for transposon mutagenesis. The problems inherent in previous approaches for transposon mutagenesis of the E. faecalis chromosome, such as the biased insertion site preference exhibited by Tn917 (17), severely limit research probing the genetic basis of E. faecalis physiology. We now report the development of an efficient system for genomewide, random transposon mutagenesis in E. faecalis that over-

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

comes limitations plaguing previous approaches to transposon mutagenesis in this organism. Our approach uses a two-plasmid scheme based on the mariner transposon, in which the EfaMarTn transposable element is delivered at high frequency by conjugation into recipient cells that harbor an expression plasmid encoding an inducible copy of the mariner transposase.

FIG. 7. Complementation analysis of biofilm formation by biofilmdefective EfaMarTn mutants of E. faecalis. The experiment was performed as described in Materials and Methods. The mean absorbance of a minimum of seven microtiter wells each for a given transposon mutant carrying either empty expression vector (⫺) or expression vector plus the indicated E. faecalis ORF (⫹) is expressed relative to that of parental wild-type E. faecalis OG1RF carrying the empty expression vector (which typically gave a mean absorbances of ⬃0.18). Results are representative of a minimum of three trials conducted on different days. Error bars represent standard errors of the means. Transposon mutants tested for complementation were 17M8 (EF0676), 20K19 (EF0983), 1D21 (EF1090), and 39C17 (EF3056).

mariner-based transposable elements have been adapted for mutagenesis in a wide variety of prokaryotes (1, 2, 20, 29, 32, 33, 40, 46, 48, 54, 55), largely because they offer the advantage of host-factor-independent, random insertion distributions that enable a relatively comprehensive discovery of genetic determinants underlying a phenotype of interest. Our results indicate that the EfaMarTn transposable element described here inserts in an apparently random fashion at sites distributed throughout the entire genome of E. faecalis (Fig. 2 and 3), suggesting this transposon represents the first such tool enabling effective genome-wide genetic analysis in this organism. As proof of principle, we used the EfaMarTn element to isolate E. faecalis mutants exhibiting a reduced ability to form biofilms relative to that of wild-type E. faecalis OG1RF. Several genes involved in the process of biofilm formation by E. faecalis OG1RF had previously been identified using targeted mutagenesis approaches, but given the complexity and multifactorial nature of the biofilm formation phenotype of other bacteria, we hypothesized that additional, unknown genetic determinants contribute to biofilm formation by E. faecalis. Using a microtiter-based biofilm assay, we screened ⬃15,000 EfaMarTn insertion mutants for a defect in biofilm formation and identified insertions in many of the genes known to contribute to growth of E. faecalis in a biofilm state, as expected (Table 2). However, we also identified 11 insertions in 6 previously unknown genetic loci, suggesting a role for the corresponding gene products in E. faecalis biofilm formation.

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Homologs of some of the previously unknown genes we identified have been linked to biofilm formation by other organisms. For example, we identified a cluster of EfaMarTn insertions in heat shock genes (EF1306 to EF1310), homologs of which are overexpressed in mature biofilms of E. coli (6). Heat shock genes have also been linked to biofilm formation by Pseudomonas putida (13) and Streptococcus mutans (31). Other previously unknown loci we identified encode gene products that have not been implicated in biofilm formation elsewhere and might lead to new insights into the regulation of biofilm formation. For example, to the best of our knowledge no homologs of pcrA or pyrC are known to play a role in biofilm formation in other bacteria. While the role of these gene products in biofilm formation is not yet clear, one possibility is that the extent of DNA supercoiling (in the case of the pcrA DNA helicase) or changes in the levels of pyrimidine nucleotide pools (in the case of pyrC dihydroorotase) could alter the expression of genes controlling biofilm formation. More directly, two of the previously unknown loci encode putative transcriptional regulatory factors of unknown function (EF0676 and EF0983; Table 2) that may modulate expression of genes that play a role in initial adaptation of E. faecalis to surface-associated growth. One noteworthy feature of our results is the fact that we obtained multiple—but nonsibling—EfaMarTn insertions in several of the loci identified as important for biofilm formation (Table 2). For example, we obtained three distinct insertions in both EF0799 (atn) and EF1090 (ebpR). Additionally, we obtained insertions in each of the other four genes known to comprise the ebp gene cluster (EF1091 to 1094) required for biogenesis of pili, as well as insertions at multiple sites within the heat shock locus (EF1306 to EF1310). These observations suggest that our transposon mutagenesis screen for biofilmdefective insertion mutants has essentially reached saturation, at least in the context of the specific set of experimental conditions we used here. A well-known limitation of the microtiter-plate biofilm assay is that it reflects primarily contributions from initial adherence and early stages of biofilm formation. Thus, although our screen may have identified all of the genetic determinants that are important for biofilm formation of E. faecalis OG1RF under these specific conditions, it likely has not revealed many other determinants that play a role in later stages of biofilm formation. For example, exopolysaccharide biosynthesis is required for formation of mature biofilms by many bacteria, because the exopolysaccharides form critical components of the extracellular matrix defining biofilm architecture. E. faecalis likely also relies on exopolysaccharides for mature biofilm formation, yet we did not identify any genes for exopolysaccharide biosynthesis in our screen, suggesting that such genes are required much later in the pathway leading to mature biofilms. Nonetheless, the genes identified here may play critical roles in regulating the initial transition of E. faecalis from a planktonic to a sessile lifestyle. Initial scanning electron microscopic analysis of the effects of inactivation of the EF0799 locus on biofilm formation was carried out because of previous evidence for a role of the E. faecalis autolysin (33) and because autolysis and release of DNA into the biofilm matrix have been suggested to be important in biofilm development for many organisms (5). Scanning electron microscopy analysis (Fig. 5 and 6) suggested that

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the inactivation of EF0799 impaired the ability of the organism to transition from a single chain of adherent bacteria to a multilayered mass of bacteria typical of more mature biofilms. It can be hypothesized that autolysin-enhanced release of DNA by bacteria during the first few hours of surface growth contributes to an extracellular matrix that facilitates development of the mature biofilm. Future research will test this hypothesis and will probe the specific functions of the other ORFs identified here in promoting biofilm formation of E. faecalis. Importantly, these results emphasize the utility of an unbiased genome-wide approach, such as EfaMarTn mutagenesis, to probe the genetic basis of E. faecalis physiology and behavior. ACKNOWLEDGMENTS We thank David Lampe, Olaf Schneewind, Dominique Missiakas, and Taeok Bae for providing plasmids; Laura Case for flow cytometry on selected transposon mutants; and Dawn Manias for technical assistance and support. Preliminary sequence data for E. faecalis OG1RF were obtained from the Baylor College of Medicine Human Genome Sequencing Center website at http://www.hgsc.bcm.tmc.edu. Computational resources and support services were provided by the Center for Biomedical Research Informatics at the University of Minnesota (http://cbri.umn.edu). Sequencing of E. faecalis OG1RF was supported by grant R21 AI64470 from NIAID. Parts of this work were carried out in the University of Minnesota I.T. Characterization Facility, which receives partial support from NSF through the NNIN program. C.J.K. was supported during a portion of this work by NRSA fellowship F32AI56684 from NIAID. A.M.T.B. received support from Medical Scientist Training Grant 5T32-GM008244 from the NIH. This work was supported by grant AI058134 from NIH to G.M.D. REFERENCES 1. Ashour, J., and M. K. Hondalus. 2003. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis. J. Bacteriol. 185:2644–2652. 2. Bae, T., A. K. Banger, A. Wallace, E. M. Glass, F. Aslund, O. Schneewind, and D. M. Missiakas. 2004. Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. USA 101:12312–12317. 3. Bae, T., B. Kozlowicz, and G. M. Dunny. 2002. Two targets in pCF10 DNA for PrgX binding: their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation. J. Mol. Biol. 315:995–1007. 4. Baldassarri, L., R. Cecchini, L. Bertuccini, M. G. Ammendolia, F. Iosi, C. R. Arciola, L. Montanaro, R. Di Rosa, G. Gherardi, G. Dicuonzo, G. Orefici, and R. Creti. 2001. Enterococcus sp. produces slime and survives in rat peritoneal macrophages. Med. Microbiol. Immunol. (Berlin) 190:113–120. 5. Bayles, K. 2007. The biological role of death and lysis in biofilm development. Nat. Rev. Microbiol. 5:721–726. 6. Beloin, C., J. Valle, P. Latour-Lambert, P. Faure, M. Kzreminski, D. Balestrino, J. A. Haagensen, S. Molin, G. Prensier, B. Arbeille, and J. M. Ghigo. 2004. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol. Microbiol. 51:659–674. 7. Bourgogne, A., K. V. Singh, K. A. Fox, K. J. Pflughoeft, B. E. Murray, and D. A. Garsin. 2007. EbpR is important for biofilm formation by activating expression of the endocarditis and biofilm-associated pilus operon (ebpABC) of Enterococcus faecalis OG1RF. J. Bacteriol. 189:6490–6493. 8. Bryan, E. M., T. Bae, M. Kleerebezem, and G. M. Dunny. 2000. Improved vectors for nisin-controlled expression in gram-positive bacteria. Plasmid 44:183–190. 9. Clewell, D. B., P. K. Tomich, M. C. Gawron-Burke, A. E. Franke, Y. Yagi, and F. Y. An. 1982. Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J. Bacteriol. 152:1220– 1230. 10. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322. 11. Donlan, R. M. 2001. Biofilm formation: a clinically relevant microbiological process. Clin. Infect. Dis. 33:1387–1392. 12. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15:167–193. 13. Dubern, J. F., E. L. Lagendijk, B. J. Lugtenberg, and G. V. Bloemberg. 2005. The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of

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