JOURNAL OF BACTERIOLOGY, Aug. 2008, p. 5690–5698
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 16
Regulation of Autolysis-Dependent Extracellular DNA Release by
Enterococcus faecalis Extracellular Proteases Influences
Vinai Chittezham Thomas, Lance R. Thurlow, Dan Boyle, and Lynn E. Hancock*
Division of Biology, Kansas State University, Manhattan, Kansas 66502
Received 2 February 2008/Accepted 5 June 2008
Enterococci are major contributors of hospital-acquired infections and have emerged as important reser-
voirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an
important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has
been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been
hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm
formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental
process. Confocal imaging of biofilms suggested that GelE?mutants were significantly reduced in biofilm
biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression
of biofilm development. The phenotype observed in a SprE?mutant was linked to an observed increase in
autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy con-
firmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm
cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm
matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role
for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and
coregulated proteases—GelE and SprE—is responsible for regulating autolysis and the release of high-
molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.
Bacteria are often found in nature as communities of sessile
surface-adherent populations covered in a slimy matrix com-
posed of exopolysaccharides, protein, and DNA (9, 19, 23).
Bacteria present within these communities (also referred to as
biofilms) exhibit social behavior analogous to that found in
higher organisms in that they can communicate and rapidly
adapt to changing growth environments (5, 23, 55).
The gram-positive opportunistic pathogen, Enterococcus fae-
calis develops persistent biofilm-like vegetations on implant
devices, including orthopedic implants, urethral stents, cathe-
ters, and heart valves, making it a leading cause of nosocomial
infection (29). Enterococci are becoming increasingly resistant
to many conventional antibiotics (22). Compounding the drug
resistance phenotypes displayed by clinical isolates is the ob-
servation that enterococci growing as biofilms are more resis-
tant to vancomycin, ampicillin, and linezolid than their plank-
tonic counterparts (44). Epidemiological data also suggest
enterococci to be important reservoirs for the transmission of
antibiotic resistance genes among different species of bacteria
Of the factors reported to be important for E. faecalis bio-
film formation (29), the enterococcal surface protein (Esp) and
the secreted metalloprotease, gelatinase (GelE), are known to
be expressed as variable traits (33, 47). More recently, Ten-
dolkar et al. (51a) identified a locus from a clinical E. faecalis
urinary tract isolate that they termed biofilm enhancer in En-
terococcus (bee locus). The genes from this locus resemble the
pilin biosynthetic genes identified by Nallapareddy et al. (33a)
and have been shown to contribute to biofilm formation, but
were found to be present in less than 5% of clinical isolates. It
is noteworthy that Arciola et al. (3) recently correlated the
presence of the esp gene and high phenotypic expression of
gelatinase with the ability of E. faecalis epidemic clones from
orthopedic implant infections to form biofilms. The esp gene
that encodes the surface-associated Esp is located on a 153-kb
pathogenicity island, and its expression significantly increases
the bacterial cell surface hydrophobicity and attachment on a
substratum (51, 52). The expression of GelE is dependent on
the fsr regulatory system (38, 39) and is known to vary among
strains of E. faecalis due to a defined 23.9-kb deletion in the
genome that encompasses the fsr genes (33). The fsr locus
consists of four genes, designated fsrA, fsrB, fsrC, and fsrD (32).
The fsrC and fsrA genes encode a two-component sensor ki-
nase-response regulator pair (39). The fsrD codes for a peptide
lactone that functions in a cell-density-dependent manner (31).
FsrB is thought to be responsible for the proteolytic cleavage
and cyclization of FsrD (32). It is likely that FsrC sensor his-
tidine kinase senses the accumulation of the FsrD peptide in
the extracellular space, leading to activation of the response
regulator FsrA. The gene encoding GelE is located immedi-
ately adjacent to the 3? end of fsrC and is cotranscribed with
sprE, which encodes a secreted serine protease (38, 39). Mu-
tations in the fsr locus and its downstream target gelE resulted
in poor biofilm-forming capabilities, indicating that biofilm
formation in Enterococcus is dependent on quorum sensing
(20, 30, 36). Mutants defective in fsr quorum signaling were
restored to wild-type biofilm levels by the addition of purified
* Corresponding author. Mailing address: Division of Biology, Kan-
sas State University, 116 Ackert Hall, Manhattan, KS 66506. Phone:
(785) 532-6122. Fax: (785) 532-6653. E-mail: firstname.lastname@example.org.
?Published ahead of print on 13 June 2008.
GelE, indicating that GelE alone is a major contributor to
biofilm development (20).
The mechanism by which GelE positively regulates biofilm
formation has hitherto been unknown. It was hypothesized that
GelE, like Esp, may be able to modify the bacterial cell surface
hydrophobicity by virtue of its ability to cleave substrates at
hydrophobic residues (6, 27, 28). An alternate hypothesis in-
volves the ability of GelE to activate cell wall autolysins (48,
54). SprE has also been shown to be an important virulence
factor since an sprE gene disruption resulted in decreased
virulence in a mouse peritonitis model (39, 50), a Caenorhab-
ditis elegans model (15, 49), and a rabbit endophthalmitis
In the present study, we investigated the role of both extra-
cellular secreted proteases in biofilm formation by comparing
isogenic single ?gelE and ?sprE and double protease ?gelE-
sprE deletion mutants of E. faecalis V583. Further, the ability
to regulate autolysis with the concomitant release of extracel-
lular DNA (eDNA) was shown to be a key contributor to the
overall development of E. faecalis biofilms.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The different strains and
plasmids used in the present study are listed in Tables 1 and 2, respectively.
Strains were cultured in Todd-Hewitt broth (THB) or M17 medium (Difco
Laboratories) and grown at 37°C unless otherwise indicated. Escherichia coli
EC1000 was used for plasmid constructions. The antibiotics used for selection in
E. coli were chloramphenicol, kanamycin, and spectinomycin at concentrations
of 10, 50, and 150 ?g/ml, respectively, and those used for E. faecalis included
chloramphenicol, tetracycline, and spectinomycin at concentrations of 15, 15,
and 750 ?g/ml, respectively.
Construction of E. faecalis V583 in-frame protease deletion mutants. In-frame
deletions of gelE, sprE, and gelE-sprE were constructed by using pLT06, an E. coli
enterococcal temperature-sensitive cloning vector that had selectable and coun-
terselectable markers that aided in the selection of mutants containing the
targeted deletions (L. Thurlow and L. E. Hancock, unpublished results). The
vector pLT06 is a derivative of pCJK47 (25), retaining the counterselection
properties on DL-p-chlorophenylalanine containing agar due to the presence of
the pheS dominant-negative allele (25). In addition, pLT06 contains a chloram-
phenicol resistance marker and origin of replication from pWV01 (26).
Flanking regions (?1 kb) from both the 5? and the 3? ends of the targeted
proteases were PCR amplified with the primers listed in Table 3. For the con-
struction of pVT01 (gelE deletion), the primers GelEP1 and GelEP2 were used
to amplify the region 5? to gelE on the V583 genome. The primers GelEP3 and
GelEP4 were used to amplify the region 3? to gelE. GelEP1 and GelEP2 con-
tained EcoRI and XhoI sites, respectively, and GelEP3 and GelEP4 contained
SalI and BamHI restriction sites to facilitate cloning. Each PCR product was
digested with the corresponding restriction enzymes, and both products were
ligated into pLT06 cut with EcoRI and BamHI, prior to electroporation into E.
coli EC1000. The correct constructs were identified by selection on LB agar
plates containing chloramphenicol at 10 ?g/ml, screened by restriction digest
analysis, and further sequenced for verification. A similar approach was used in
the construction of pVT02 (sprE deletion) using the primer pairs SprEP1 and
SprEP2, as well as SprEP3 and SprEP4; and for pVT03 (gelE-sprE deletion) the
primer pairs GelEP1 and GelEP2 were used, along with SprEP3 and SprEP4.
The isolated plasmids were electroporated into electrocompetent E. faecalis
V583 (10). E. faecalis V583 transformants were selected by growth at 28°C on
THB agar containing chloramphenicol at 15 ?g/ml and X-Gal (5-bromo-4-
chloro-3-indolyl-?-D-galactopyranoside) at 120 ?g/ml. Blue colonies were inoc-
TABLE 1. E. faecalis strains used in this study
Strain Relevant genotype Complementation in pAT28Relevant phenotypea
gelE promoter gelE
gelE promoter gelEsprE
gelE promoter sprE
aSpecr, spectinomycin resistance; Tetr, tetracycline resistance.
TABLE 2. Plasmids used in this study
Plasmid Description Source or reference
pLT06Integration vector, Cmrderivative of pCJK47
L. Thurlow, unpublished
This studypVT01 pLT06 containing a 2.0-kb EcoRI/BamHI fragment containing engineered
pLT06 containing a 2.0-kb EcoRI/BamHI fragment containing engineered
pLT06 containing a 2.0-kb EcoRI/BamHI fragment containing engineered
gelE sprE deletion
Broad-host-range shuttle vector, spectinomycin resistance
pAT28 containing 1748-bp EcoRI/XhoI fragment containing the native
gelE promoter along with full-length gelE
pAT28 containing 2687-bp EcoRI/BamHI fragment containing the native
gelE promoter and full-length gelE sprE
pAT28 containing 2123-bp EcoRI/BamHI fragment containing the native
gelE promoter, a truncated gelE, and full-length sprE
Gram-positive replicative vector expressing a Gfp reporter
pVT02 This study
VOL. 190, 2008EXTRACELLULAR PROTEASES MODULATE BIOFILM DEVELOPMENT 5691
ulated into fresh THB containing chloramphenicol at 15 ?g/ml. Cultures were
grown overnight at 28°C, diluted 1:100 into fresh medium, and grown for an
additional 2.5 h at 28°C and then shifted to 42°C for an additional 2.5 h to favor
single-site integration of plasmids into the E. faecalis V583 genome. Serial
dilutions of the integrants were plated onto THB agar plates supplemented with
15 ?g of chloramphenicol/ml. Colony PCR was used to confirm single-site inte-
gration for each construct using vector-specific primers OriF or SeqR, along with
primers targeted to regions 5? or 3? to the site of insertion (GelEUp, GelEDown,
SprEUp, or SprEDown). A positive colony was then cultured in the absence of
selection until the culture reached stationary phase (?2 ? 109CFU/ml). Serial
dilutions were prepared, and fresh medium (THB) was inoculated such that it
contained 100 CFU/ml. Serial dilutions (1:500 and 1:1,000) were plated on
MM9YEG agar supplemented with 10 mM DL-p-Cl-Phe and X-Gal at 120 ?g/ml.
Counterselection using DL-p-Cl-Phe has been shown to favor the selection of
colonies that have lost the plasmid (25). Colony PCR using the primers GelEUp
and GelEDown for VT01, SprEUp and SprEDown for VT02, and GelEUp and
SprEDown for VT03 were used to confirm the gene deletion in the genome.
Phenotypic confirmation of the protease deletions were also visualized on THB
agar containing 1.5% skim milk.
Complementation of E. faecalis V583 in-frame protease deletion mutants.
In-frame protease deletions of E. faecalis V583 were complemented with full-
length gelE, sprE, and gelE-sprE, each with the native gelE promoter region in a
pAT28 vector (53) and were denoted pVT05, pVT08, and pVT07, respectively.
The gelE complement insert was cloned by PCR amplification of V583 genome
with primers GelEprom and SprEP2 and subsequently inserted as an EcoRI/
XhoI fragment into pAT28 cut with EcoRI/SalI. The gelE-sprE double protease
complement construct was cloned by PCR amplification of V583 genome using
primers GelEprom and GelEP4 and inserted as an EcoRI/BamHI fragment into
EcoRI/BamHI-cut pAT28 vector. Plasmid pVT08 was constructed by digesting
pVT07 with AflII and NcoI, followed by Klenow treatment to make it blunt ended,
of gelE. These constructs were transformed into the corresponding protease deletion
mutants, and phenotypic complementation was confirmed by zymography using
skim milk at a final concentration of 0.02% as a substrate (24).
Biofilm assay on polystyrene microtiter plates. Biofilm formation on polysty-
rene was quantified with crystal violet staining method as previously described
(20). Each assay was performed in octuplicate and repeated five times. Statistical
significance was calculated by using Dunnett’s test (GraphPad Software, San
Cell surface hydrophobicity assay. The cell surface hydrophobicities of E.
faecalis V583 and isogenic protease mutant strains were carried out as previously
described (43). The percentage of bacterial adhesion to hydrocarbon was calcu-
lated as follows: [1 ? (ODF/ODI)] ? 100, where ODIand ODFare the optical
densities of cells resuspended in PUM buffer (100 mM potassium phosphate [pH
7.1], 30 mM urea, 800 ?M MgSO4? H2O) determined at the beginning and the
end of the experiment, respectively. Statistical significance was computed by
using the Dunnett’s test (GraphPad Software, San Diego, CA).
Autolysis assay. Autolysis assay was carried out as previously reported (11).
Isolation of eDNA from E. faecalis planktonic culture supernatants. Superna-
tants from 24-h-old grown cultures were passed through a sterile syringe filter
(0.2-?m pore size; Nalgene) and concentrated ?20-fold using a 10-kDa cutoff
membrane (YM-10 Centricon centrifugal filter devices; Millipore) according to
the manufacturer’s instructions. The concentrated samples were loaded on a 1%
agarose gel and stained with ethidium bromide to visualize high-molecular-
weight DNA. Densitometric spot comparisons were performed by using
Alphaimager software (Alpha-Innotec, San Leandro, CA).
eDNA from culture supernatants was isolated by using the Wizard genomic
DNA purification kit according to the manufacturer’s instructions, and chromo-
somal DNA was isolated as previously described (37). For comparative PCR,
primers listed in Table 3 were designed to amplify genes from regions of the E.
faecalis V583 genome, including Ef0887, Ef1091, Ef2194, Ef2488, Ef2490, and
Laser scanning confocal microscopy. E. faecalis strains V583, VT01, VT02,
and VT03 were transformed with pMV158GFP (34) to constitutively express Gfp
for confocal imaging. The resulting strains were designated VT09, VT10, VT11,
and VT12, respectively. Confocal microscopy was performed on E. faecalis bio-
films grown on glass coverslips. Sterile glass coverslips were placed on the bottom
of six-well tissue culture plates and submerged with 5 ml of M17 broth, seeded
with a 1:100 dilution from an overnight culture (approximately 5 to 10 ? 106
CFU), and grown for 24 h at 37°C. For 2-, 3-, and 4-day-old biofilms, the culture
supernatants were replaced with fresh medium daily. Just prior to imaging,
biofilms were gently rinsed three times with sterile phosphate-buffered saline,
followed by 10 min of staining with 5 ml of propidium iodide (PI; 1 ?M). The
coverslips were mounted on a microscope slide and sealed with clear nail polish
to prevent dehydration. Slides were visualized by using a Zeiss LSM 5 Pascal
laser scanning confocal microscope. The LSM 5 system was equipped with a
Zeiss Axioplan 2 MOT research microscope, a fully motorized stage, a Plan
Apohromat objective (?63/1.4 oil) and differential contrast interference. Dual
fluorescence emission imaging of green fluorescent protein (GFP) and PI was
accomplished using a 488-nm line of 458/488/514 argon gas ion laser to excite
GFP and a 543-nm line of HeNe laser to excite PI. A secondary HFT 545
dichroic was used to split the emission signals into two signals, the shorter
wavelengths passed through a band-pass 505- to 530-nm filter to image GFP
fluorescence, and the longer wavelength passed through a long-pass 560-nm filter
to image PI fluorescence. For z-series, the Airy units of the longer and shorter
wavelengths were adjusted to give an optical slice thickness of 0.7 ?m, and this
thickness was used as the slice interval. Biofilm quantification was carried out
using the COMSTAT analysis package (21). Volumetric analysis (?m3) of rep-
resentative confocal images portraying regions within the biofilm stained by PI
were carried out using the 3D Object counter plug-in in the NIH Image J
software. For determination of statistical significance, the data were natural log
transformed, and an unpaired t test was performed using GraphPad (GraphPad
Software, San Diego, CA).
DNase I treatment of biofilms. To assess the significance of eDNA for E.
faecalis biofilms, 6-, 12-, and 24-h-old biofilms were treated with 100 Kunitz units
per ml of DNase I. The control contained denatured DNase I that was heated at
100°C for 15 min. The biofilms were imaged by using confocal laser scanning
Construction and complementation of E. faecalis V583 iso-
genic protease mutants. Kristich et al. recently developed a
pheS counterselectable vector system, pCJK47 to generate
markerless in-frame isogenic deletion mutations in E. faecalis
OG1RF (25). However, this vector system was unsuitable for
studies with strain V583 due to the unavailability of selectable
resistance markers, as well as difficulty associated with conjugal
mating of strains possessing multiple plasmids. In the present
TABLE 3. Oligonucleotides used in this study
Primer Sequence (5?–3?)
5692THOMAS ET AL. J. BACTERIOL.
study, we used the plasmid pLT06 (a derivative of pCJK47),
which encodes resistance to chloramphenicol and contains a
temperature-sensitive replication origin from pWV01 (26).
Extracellular protease deletion mutants VT01 (?gelE),
VT02 (?sprE), and VT03 (?gelE-sprE) (Fig. 1A) were con-
structed by using the markerless exchange vectors pVT01,
pVT02, and pVT03, respectively. The respective plasmids were
integrated into the V583 genome by homologous recombina-
tion. Subsequent plasmid excision was counterselected by plat-
ing on medium containing DL-p-chlorophenylalanine as de-
scribed previously (25). Roughly 50% of the isolates growing in
the presence of DL-p-chlorophenylalanine yielded the expected
gene deletion for each of the plasmid constructs. The proteo-
lytic phenotypes of the mutants were compared to V583 and
were consistent with previous reports (24). Strains VT01 and
VT03 lacked a zone of proteolysis on skim milk agar, whereas
strain VT02 showed a smaller zone compared to V583 (data
E. faecalis V583 isogenic protease mutants (VT01, VT02,
and VT03) were complemented with full-length genes of gelE,
sprE, and gelE-sprE in trans under the control of the native gelE
promoter. Complementation confirmed that the protease-neg-
ative phenotypes were a result of targeted protease deletions
and not due to polar effects of gene mutations elsewhere on
the chromosome (data not shown).
Biofilm formation of E. faecalis V583 isogenic protease mu-
tants. Quantitative analysis of biofilms formed by the protease
deletion mutants on polystyrene confirmed previous findings
(20). VT01 (?gelE) and the double protease deletion strain
VT03 (?gelE-sprE) were significantly reduced in biofilm bio-
mass compared to strain V583 (Dunnett’s test, P ? 0.05) (Fig.
1B). Interestingly, deletion of sprE (VT02) marginally in-
creased the biofilm biomass, although this did not appear to be
statistically significant (Dunnett’s test, P ? 0.30). Complemen-
tation of the protease-negative strains restored biofilm forma-
tion to near wild-type levels, suggesting no polar effects for the
deletion mutations (Fig. 1B).
Given the differences in biofilm biomass on polystyrene, we
sought to determine whether mutant cells exhibited any differ-
ences in primary biofilm mat formation on a glass substrate.
CLSM analysis of the structural and spatial organization of
24-h-old biofilms (Fig. 2) showed a dense and compact paren-
tal V583 biofilm (VT09). Consistent with our earlier observa-
tions, VT10 (?gelE) and VT12 (?gelE-sprE) displayed poor
biofilms (decreased by ca. 60 and 50%, respectively, compared
to VT09; Table 4) and was composed mainly of isolated and
sparse distributions of cells on the glass surface. In contrast,
biofilms of VT11 (?sprE) were more dense than those formed
by the parental strain (increased by ca. 55%; see Table 4) and
appeared to have a rugged, mountainous surface terrain con-
sistent with an early initiation of microcolony development.
Extracellular proteases do not affect the cell surface hydro-
phobicity of E. faecalis V583. To determine whether the ability
of GelE to enhance biofilm formation resulted from an in-
FIG. 1. Extracellular protease deletion mutations affect E. faecalis
V583 biofilm development. (A) Diagrammatic depiction of extracellu-
lar protease deletions. VT01, VT02, and VT03 correspond to E. fae-
calis V583 strains harboring ?gelE, ?sprE, and ?gelE-sprE protease
deletions, respectively. Solid lines indicate chromosome, boxed arrows
indicate genes, and curved arrows indicate promoter regions. The
schematic is not drawn to scale. (B) Biofilm formation of extracellular
protease mutants on polystyrene microtiter plates. The biofilm density
within microtiter plate wells was assayed as a function of crystal violet
stain retained by the biofilm biomass. Mutant strains complemented
with gelE, sprE, and gelE-sprE are designated VT04, VT05, and VT06,
respectively. Assays were performed in triplicate, and error bars indi-
cate the standard error of the mean.
FIG. 2. Confocal analysis of 1-day-old biofilms of E. faecalis wild
type and isogenic protease deletion mutants. All strains constitutively
expressed Gfp from pMV158GFP (see Materials and Methods) and
were grown on glass coverslips in M17 medium. Panels A, B, C, and D
are representative biofilm projections of VT09, VT10, VT11, and
VT12, respectively. Below each panel is the z-projection for the cor-
responding image, and the depth of the biofilm is indicated by the
height of the z-stack (see Table 4). The inset scale bar represents
VOL. 190, 2008 EXTRACELLULAR PROTEASES MODULATE BIOFILM DEVELOPMENT5693
crease in overall cell surface hydrophobicity, we tested whether
a ?gelE mutation would decrease the overall hydrophobicity of
cells. The assay was carried out by quantifying the population
of bacteria that were able to separate into an organic phase
(n-hexadecane) depending on the degree of cell surface hydro-
phobicity displayed. The presence or absence of either pro-
tease in both single- and double-deletion protease mutants did
not result in significant differences in partitioning into the
n-hexadecane phase relative to the wild-type V583 strain (Fig.
3, Dunnett’s test, P ? 0.05).
Extracellular proteases modify the rate of E. faecalis V583
autolysis. Given the ability of enterococcal proteases to modify
autolysins (48) and based on the observations seen using con-
focal imaging of biofilms, we hypothesized that GelE and SprE
may differentially regulate the autolysis rates of E. faecalis. We
observed that VT01 (?gelE) and VT03 (?gelE-sprE) exhibited
a decrease in the rate of autolysis compared to V583 (Fig. 4A),
a finding consistent with observations reported by Waters et al.
(54). In contrast, VT02 (?sprE) displayed a significant increase
in the rate of autolysis compared to V583 (Fig. 4A, Student t
test, P ? 0.05).
eDNA in E. faecalis V583 culture supernatants. Based on the
altered rates of autolysis, we hypothesized that eDNA resulting
from cell lysis would be more abundant in culture supernatants
of E. faecalis V583 than mutants deficient in GelE production.
Concentrated (20-fold) supernatant fractions were assessed for
the presence of eDNA by agarose gel electrophoresis. High-
molecular-weight DNA was detected in V583 and VT02
(?sprE) fractions (Fig. 4B, lanes 1 and 3), but not in mutants
VT01 and VT03 (Fig. 4B, lanes 2 and 4), a finding consistent
with a decreased rate of autolysis in strains lacking GelE.
Densitometric determination of band intensity between DNA
present in V583 and VT02 culture supernatants indicated a
?2-fold increase in the amount of eDNA from an SprE?
mutant, a finding consistent with a role for SprE as a negative
regulator of autolysis. Initiation of DNA release in V583 cul-
ture supernatants followed expression of GelE in the transition
to stationary phase (data not shown), a finding consistent with
the earlier observation that GelE initiates autolysis. Finally,
comparative PCR using eDNA and chromosomal DNA as
templates confirmed that eDNA was indeed chromosomal in
nature since amplification with primer pairs targeted to ran-
domly distributed regions of the V583 genome could be am-
plified from both templates (data not shown).
Tracking cell death in enterococcal biofilms. Because autol-
ysis and biofilm formation of E. faecalis was directly dependent
on the presence of gelatinase, we questioned whether biofilms
formed by the parental strain would contain foci of lysed cells
compared to VT01 (?gelE). To test this, biofilms of V583 and
VT01 expressing Gfp (VT09 and VT10, respectively) were
grown over a period of 3 days and were stained for the pres-
ence of DNA and dead cells with PI. Regions within the bio-
film of VT09 contained concentrated foci of DNA (as detected
by PI staining) in contrast to the few random dead cells in
VT10 biofilms (Fig. 5). ImageJ analysis software was used to
quantify the amount of PI-stained volumes within the biofilm
as a measure of eDNA present in the biofilm. From this anal-
ysis, it is apparent that a common feature shared by both
GelE?mutant and wild-type cell populations is the presence of
damaged cells capable of taking up PI, and this cell population
is accounted for in our analysis. A property unique to the
wild-type cells compared to the GelE?mutant is the presence
of larger volumes of PI staining associated with lysed cells. The
mean values for PI-stained volumes is ?4.4-fold higher in the
TABLE 4. COMSTAT analysis of wild-type and isogenic protease
mutant biofilm images
Mean ? SD
6.7 ? 0.93
2.6 ? 0.37
10.5 ? 0.33
3.0 ? 0.48
6.3 ? 0.98
2.4 ? 0.41
10.1 ? 0.48
2.4 ? 0.49
6.3 ? 0.98
2.45 ? 0.49
10.15 ? 0.49
2.45 ? 0.5
5.6 ? 0.38
0.035 ? 0.04
11.61 ? 0.46
0.194 ? 0.04
7.9 ? 1.04
0.022 ? 0.027
19.9 ? 0.01
0.28 ? 0.03
11.2 ? 0.0
8 ? 2.26
20.8 ? 0.0
7.2 ? 2.26
FIG. 3. Cell surface hydrophobicity of E. faecalis V583 and extra-
cellular protease mutants. The overall measure of hydrophobicity of
wild-type and mutant populations were calculated as the percent bac-
teria that adhered to hydrocarbon (BATH). Assays were performed in
triplicate, and error bars represent the standard error of the mean.
FIG. 4. Extracellular proteases influence autolysis rates and eDNA
release. (A) Differences in autolysis rates of V583 (F) and extracellular
protease mutants VT01 (f), VT02 (Œ), and VT03 (}) are exhibited as
percent values of the initial optical density at 600 nm (OD600). Assays
were performed in quadruplicate, and error bars denote the standard
error of the mean calculated from three independent assays. (B) High-
molecular-weight bacterial chromosomal DNA was detected by
ethidium bromide staining, after 20-fold concentration of 24-h-old
culture supernatants. Lanes: 1, V583; 2, VT01; 3, VT02; 4, VT03; and
4, 1-kb DNA ladder showing the 12-, 10-, and 8-kb bands (the 12-kb
band is labeled in lane 5).
5694 THOMAS ET AL.J. BACTERIOL.
wild-type strain (113.5 ? 59.88) than in the GelE?mutant
(25.80 ? 5.09), and this was shown to be statistically significant
(P ? 0.0004) by using an unpaired t test, after data transfor-
mation, to account for the fact that stained volumes present in
the V583 biofilms were not normally distributed compared to
VT01 biofilms. A graph of this analysis is shown in Fig. 5C, and
the z-stack image comparing V583 and VT01 biofilms stained
with PI is also shown in Fig. 5. Collectively, these results sug-
gest that GelE enhances biofilm formation by inducing lysis in
discrete pockets of cells that appear to initiate biofilm devel-
Since the expression of GelE and SprE may only be opti-
mally activated after the establishment of a quorum of bacteria
on a surface, we hypothesized that we would see more prom-
inent defects in the differentiation of the biofilm at later stages
of development rather than the initial stages of attachment and
proliferation. Consistent with this hypothesis, we observed that
VT10 (?gelE) and VT12 (?gelE-sprE) were able to form a
primary biofilm matt on a glass surface within 48 to 72 h of
growth (data not shown). However, unlike the parental VT09
or VT11 (?sprE), even after 96 h of growth these two strains
were not able to differentiate into microcolonies (Fig. 6). PI
staining of the dead bacteria and eDNA in 4-day-old biofilms
revealed clusters of dead bacteria around the base and stalk of
a microcolony, whereas live bacteria interspersed with DNA
frequently occupied the top of microcolonies within biofilms
(Fig. 6). This suggested that pockets or clusters of dead cells
that are dependent on the expression of GelE visualized at an
earlier phase of biofilm development (Fig. 5) may actually be
sites of initial microcolony development. Consistent with a role
for SprE in negatively regulating GelE activity, we observed
significantly more biofilm biomass (107% increase compared
to the wild type) in an SprE?mutant after 96 h of growth than
in the parental strain (Fig. 6 and Table 4).
Functional role of eDNA in enterococcal biofilms. To deter-
mine whether eDNA of E. faecalis played a structural role in
biofilm development, we analyzed the affect of DNase I on
biofilm formation. Static biofilms grown on glass substrates
were treated with DNase I after 6, 12, and 24 h of growth.
Biofilm defects were most pronounced after early treatments
of DNase I at 6 and 12 h of biofilm growth (Fig. 7). The affect
of DNase I treatment at later stages of development was less
significant, as exhibited by the 24-h treatment.
The importance of extracellular proteases of E. faecalis in
pathogenesis has been well demonstrated in a number of bio-
logical models (14, 15, 49, 50). Components of the host innate
immune response are known to be cleaved by the proteolytic
activity of gelatinase and include LL37 (45), ?-defensin (46),
and the complement components C3a and C3b (35), providing
a mechanism for host immune evasion. GelE has also been
shown to cleave fibrin, possibly enhancing efficient dissemina-
FIG. 5. Bacterial cell death and eDNA release in 3-day-old bio-
films. E. faecalis biofilms grown in M17 medium over a period of 3 days
were stained with PI (1 ?M) before being visualized by CLSM.
(A) Top-down view of VT09 biofilm displaying discrete foci of lysed
bacteria, along with dead bacterial cells. (B) View of isolated dead
bacterial cells within the VT10 biofilm. Below each panel is the z-
projection for the corresponding image, and the depth of the biofilm is
indicated by the height of the z-stack. The inset scale bar represents 20
?m. (C) Volumetric analysis of PI-stained foci for VT09 and VT10
biofilms. Vertical scatter plots with each of the values of stained foci
(cubic microns) are shown along with the mean and standard error of
FIG. 6. Comparison of biofilm architectures and relative eDNA
localization. Four-day-old Gfp-expressing strains of E. faecalis V583
and isogenic protease mutants were grown in M17 and stained for the
presence of eDNA with PI (1 ?M) as indicated in Materials and
Methods. Live bacteria are green, and eDNA and dead cells are
visualized in red. High concentrations of eDNA laced among live
bacteria present on each raised microcolony and surroundings appear
in shades of yellow. Panels A, B, C, and D are representative biofilm
projections of VT09, VT10, VT11, and VT12, respectively. Below each
panel is the z-projection for the corresponding image, and the depth of
the biofilm is indicated by the height of the z-stack (see Table 4). The
inset scale bar represents 20 ?m.
VOL. 190, 2008 EXTRACELLULAR PROTEASES MODULATE BIOFILM DEVELOPMENT5695
tion of the organism in vivo (54). Aside from its proteolytic
affects on host factors, gelatinase has also been shown to have
a positive role in E. faecalis biofilm development (20, 25).
Hence, our present focus was to elucidate the mechanism
behind GelE-dependent biofilm development and to further
examine the role of SprE in that process.
A speculative role for GelE in biofilm development included
its potential ability to increase cell surface hydrophobicity by
cleaving surface polypeptides at hydrophobic residues (6, 27).
Although cell surface hydrophobicity has previously been pro-
posed to be a key factor in the initial attachment of bacteria to
a substratum (12), our analysis of the different protease mu-
tants does not support a role for GelE or SprE in altering cell
surface hydrophobicity since the deletion of either protease
singly or in tandem resulted in minimal changes. A second
hypothesis centered on the ability of GelE to alter rates of
autolysis, based on observations by Shockman and Cheney (48)
and Waters et al. (54). Our data appear to confirm the impor-
tance of autolysis in driving the development of E. faecalis
biofilms, since we observed altered rates of autolysis, changes
in eDNA release, and differences in biofilm development in
mutants defective in extracellular protease production. The
contributions of both proteases to the process of biofilm de-
velopment was readily observed only after confocal analysis.
We did not initially observe a contribution for SprE in the
microtiter plate biofilm assay. The apparent discrepancy be-
tween the two assays is consistent with observations reported
by Tendolkar et al. (51) in which the plate assay significantly
underestimated biofilm biomass compared to confocal imaging
and COMSTAT analysis.
The major autolysin, AtlE of Staphylococcus epidermidis was
recently shown to contribute to biofilm development through
the generation of eDNA upon autolytic activation (40). A role
for muramidase 2, a major autolysin of E. faecalis, in biofilm
formation was reported by Mohamed et al. (30), and these
authors concluded that it played a major role in the initial
adherence phase of biofilm development. The findings re-
ported by Qin et al. (40) that eDNA is an integral component
of the biofilm matrix in S. epidermidis biofilms may warrant a
reevaluation of the role of autolytic processes in biofilm devel-
opment in E. faecalis. The observed alterations in eDNA re-
lease that are dependent on protease activity and appear to
mediate the ability of E. faecalis to develop microcolonies
within biofilms suggest that autolytic processes may govern not
only initial attachment but also the subsequent development of
the biofilm. Our findings have not only confirmed the role for
GelE in activating autolysis since its deletion resulted in au-
tolysis and biofilm defects but also provide direct evidence that
SprE is involved in negatively regulating autolysis, eDNA re-
lease, and biofilm maturation.
Previous reports have identified and characterized SprE as a
virulence factor whose activity is altered in the presence of
GelE (24). This activity is similar in nature to that reported for
the corresponding homologous extracellular proteases of S.
aureus, where the metalloprotease aureolysin processes the
cotranscribed SspA (V8 protease) (41). In S. aureus, SspA is
known to alter the autolytic profile (41), which is consistent
with our observations for the role of GelE and SprE in regu-
lating autolysis. Because our data suggest that SprE prevents
early maturation of biofilms by negatively regulating GelE
activity, we postulated whether there would be a fitness cost
associated with the bacterial cell in the absence of SprE. Our
observations suggest that the quick biofilm maturation pheno-
type of VT02 is associated with cell surface perturbations that
may be disadvantageous at a planktonic level of existence. For
instance, the SprE?mutant is at least fourfold more sensitive
to vancomycin compared to wild-type V583 (data not shown).
Hence, it would seem that the trade-off for rapid biofilm de-
velopment is costly and, in an evolutionary sense, unstable.
It has been observed in several model systems that eDNA
serves as an important matrix component of microbial biofilms
(2, 40, 42, 57). Consistent with a role for eDNA as a matrix
component, we observed that treating a developing biofilm
with DNase I at 6 and 12 h postinoculation resulted in dimin-
ished biofilm accumulation compared to a heat-inactivated
DNase I control. In contrast, the addition of DNase I at 24 h
showed only a marginal reduction in biofilm accumulation,
suggesting that changes in the matrix composition may take
place at later stages of development. Consistent with our find-
ings, the observation that disrupting biofilms with DNase I
treatment works better at earlier stages of development has
been reported for Pseudomonas aeruginosa (57) and S. aureus
Although the factors regulating the spatial death of a sub-
population of bacterial cells in a biofilm are not clear, the
extracellular nature of the proteases and their opposing phe-
FIG. 7. DNase I inhibits biofilm formation at early stages of devel-
opment. V583 biofilms grown on glass coverslips were treated with
DNase I after 6, 12, and 24 h of growth (represented in panels B, C,
and D, respectively) and analyzed after 26 h by CLSM. The biofilm
micrograph on the far left (panel A) shows a control experiment with
heat-inactivated DNase I introduced after 6 h of biofilm development.
Below each panel is the z-projection for the corresponding image,
indicating the depth of the biofilm. The inset scale bar represents
5696 THOMAS ET AL.J. BACTERIOL.
notypes may play a role in this process. Our current model
(Fig. 8) proposes two possible means by which these proteases
may exert their regulatory affects on biofilm development. The
first mechanism involves an autolytic pathway, wherein GelE
localizes to the cell wall of the producing cell to activate au-
tolysis. If insufficient levels of SprE are present to control the
autolytic activation induced by GelE, then that cell will likely
undergo autolysis. The second mechanism would involve an
allolytic or fratricidal event, wherein GelE freely diffuses from
the producer cell to a target sibling cell to activate autolysins
present on the sibling cell wall. A delay in responding to the
quorum signal by siblings would render them susceptible to the
action of autolysins activated by GelE secreted from another
cell. SprE would also likely be present in this extracellular
environment, but differences in diffusion and affinity for the
cell wall may likely give rise to regions in the biofilm where
GelE could act independently of SprE activity. In the rare
instances in which GelE would function independently of
SprE, a sibling cell would lyse providing the necessary eDNA
scaffold on which a developing biofilm could form. Consistent
with the above model is the fact that only a few pockets within
the observed biofilms give rise to cell lysis, which is indicative
of the fact that the process is highly regulated.
In recent times, bacterial death in biofilms has been com-
pared to programmed cell death in eukaryotes (4). Often such
comparisons propagate the idea that defective cells within a
biofilm population are eliminated in response to environmen-
tal challenges due to their altruistic suicidal acts (4). Our
model adds to this complexity by proposing that GelE-medi-
ated lysis appears to be an important aspect of biofilm devel-
opment by E. faecalis. The cytotoxic activity of GelE toward
the producer cell (autolysis) or sibling cells (allolysis) by the
activation of autolysins may result in the release of eDNA
crucial for the early development of biofilms. Although in this
case a subpopulation of cells may not be defective per se, their
inability to produce the immunity factor (SprE) would result in
their death. Allolysis has also recently been referred to as
microbial fratricide (“sibling killing sibling”), and this term has
been applied to the competence developmental program in
Streptococcus pneumoniae (18), and a model was proposed on
how this process might contribute to the development of bio-
films (16). Allolysis (18) and cannibalism (17) regulate the
differentiation of competent cells in S. pneumoniae and sporu-
lation in Bacillus subtilis, respectively. Consistent with these
fratricidal systems, a model for how fratricide in E. faecalis
regulates the development of biofilms is also proposed (Fig. 8).
Interestingly, all three processes of differentiation may be con-
sidered attributes of multicellularity resulting from cell-cell
communication and involve quorum sensing, killing factors,
and immunity proteins (8, 13). In E. faecalis biofilm develop-
ment, quorum sensing is mediated through a peptide lactone
(FsrD) originally characterized as the gelatinase biosynthesis-
activating pheromone (31). The extracellular accumulation of
the peptide triggers expression of both GelE (the effector) and
SprE (the regulator). Both proteases are cotranscribed, sug-
gesting an equal number of both molecules in the extracellular
milieu. For this reason, we anticipate that most cells would be
protected from autolysis or allolysis. However, within discrete
foci, the balance of these two proteases may not be the same,
giving rise to effector-mediated processes in the absence of
regulatory control. Ongoing studies will better clarify which of
the two mechanisms (autolysis versus fratricide) plays the dom-
inant role in E. faecalis biofilm development.
We thank Gary Dunny and Chris Kristich (University of Minnesota)
for plasmid pCJK47 and Arne Heydorn (Technical University of Den-
mark, Kongens Lyngby) for the COMSTAT software. We also thank
Helmut Hirt for a critical review of the manuscript and helpful com-
ments in the preparation of the manuscript.
This study was supported by a Heartland Affiliate Beginning Grant-
in-Aid 0660072Z from the American Heart Association (to L.E.H.)
and a grant-in-aid from the Terry C. Johnson Cancer Center at Kansas
State University (V.C.T.).
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FIG. 8. Model of GelE-mediated lysis in E. faecalis biofilm devel-
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mediate lytic activity. The first mechanism is referred to as autolysis
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