RESEARCH LETTER
Metal-dependent repression of siderophore and bio¢lm formation
in Actinomyces naeslundii
Cas Moelling, Ross Oberschlacke, Price Ward, John Karijolich, Ksenia Borisova, Nikola Bjelos
& Lori Bergeron
Department of Biology, Ripon College, Ripon, WI, USA
Correspondence: Lori Bergeron, 300
Seward St, Ripon, WI 54971, USA. Tel.:
920 279 0019; e-mail: [email protected]
Received 16 May 2007; revised 9 July 2007;
accepted 9 July 2007.
First published online September 2007.
DOI:10.1111/j.1574-6968.2007.00888.x
Editor: William Wade
Keywords
DtxR; levans; AmdR; iron dependent.
Abstract
Actinomyces naeslundii is a pioneer of the oral cavity and forms a biofilm on the
tooth’s surface. Most bacteria require iron for survival and in pathogenic bacteria
iron availability regulates virulence gene expression. Metal-dependent repressors
control gene expression involved in metal transport and uptake including siderophores. Siderophores are small molecules synthesized by bacteria and fungi to
acquire iron. The A. naeslundii genome was searched for a gene encoding a metaldependent repressor. Actinomyces metal-dependent repressor or amdR was identified. The AmdR protein was examined for its ability to bind to the promoter
sequence of a gene encoding the siderophore uptake (sid gene). According to gel
shift assays, AmdR binds to the sid gene promoter sequences. In the authors’
model, when iron is available AmdR binds to the sid promoter and represses sid
gene expression. To further explore the role of AmdR, an amdR-defective strain of
A. naeslundii was constructed and biofilm formation and siderophore production
were evaluated. When iron is removed from the medium A. naeslundii increases
biofilm and siderophore production. However, amdR-defective A. naeslundii is less
sensitive to metal ion concentrations in the growth medium.
Introduction
Actinomyces naeslundii is an early colonizer of the oral cavity
(Ellen, 1976; Ellen et al., 1985; Li et al., 2004). By the age of 1
year, c. 90% of infants are colonized with Actinomyces species
(Kononen et al., 1999). Actinomyces species have been implicated in periodontal disease and root surface caries formation
(Ellen et al., 1985; Schupbach et al., 1995). To gain an insight
into the mechanisms that A. naeslundii uses to cause disease,
the role of metal ions, in particular iron, in A. naeslundii
virulence properties was examined. The role of an Actinomyces
metal-dependent repressor (AmdR) in A. naeslundii virulence
gene expression was explored.
For most pathogenic bacteria, iron is essential. In
humans, a majority of the iron is scavenged by host metalbinding proteins such as lactoferrin and transferrin. Therefore, bacteria have developed ways to acquire iron. This
low-iron environment is used as a signal to transcribe
bacterial genes involved in metal ion acquisition such as
toxins, metal transporters and siderophores. Siderophores
are metal-binding molecules produced by bacteria and fungi
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and are secreted when environmental iron concentrations
are reduced.
Mycobacterium tuberculosis and Corynebacterium diphtheriae siderophore production is repressed by metal-dependent
repressors (Qian et al., 2002; Rodriguez et al., 2002; Kunkle
& Schmitt, 2003). Corynebacterium diphtheriae produces
diphtheria toxin repressor (DtxR), which is an iron-dependent
repressor that regulates transcription from multiple promoters including the phage encoding tox gene and at least seven
other genes (Boyd et al., 1990; Kunkle & Schmitt, 2003). In
C. diphtheriae, low iron modulates the expression of surface
carbohydrates, adherence to human red blood cells and
slime production (Moreira Lde et al., 2003).
The role of metal ions in the oral cavity has been
explored in Streptococcus mutans, the primary bacterium
associated with dental caries. In S. mutans, iron-depleted
saliva resulted in increased cell aggregation (Berlutti et al.,
2004). Recent studies also support a role for the metaldependent repressor (SloR) in S. mutans biofilm formation,
antibiotic gene regulation and in glucan production
(Rolerson et al., 2006).
FEMS Microbiol Lett 275 (2007) 214–220
215
Metal-dependent repression
While the importance of metal ions in S. mutans virulence is being explored, the role that metal ions play in the
virulence of A. naeslundii is unknown. As most Grampositive bacteria have metal-dependent repressors, it is
logical that A. naeslundii possesses a metal-dependent
repressor. However, there is little information on the effects
of iron on A. naeslundii gene expression. In this study, a
DtxR-like repressor was identified in A. naeslundii. This
AmdR is hypothesized to be a global regulator of virulence
genes in A. naeslundii regulating biofilm and siderophore
production.
Materials and methods
Bacterial strains, plasmids and growth
conditions
Actinomyces naeslundii MG1 was obtained from Sophia Piña
(University of Texas Health Science Center at San Antonio).
Actinomyces naeslundii was maintained at 5% CO2 at 37 1C
on brain heart infusion (BHI) agar. A chemically defined
medium named Actinomyces defined medium (ADM) was
used to cultivate A. naeslundii (Christie & Porteous, 1962).
ADM was modified to facilitate low-iron conditions by
omitting hemin and iron from the medium. Escherichia coli
strains M15 and DH10B were maintained on Luria broth.
Ampicillin (100 mg mL1) and kanamycin (50 mg mL1) were
added when necessary.
Isolation of AmdR
The A. naeslundii genome was scanned for putative metaldependent repressors, which revealed a 672 base pairs (bp)
ORF (Locus ANA 1929) with homology to metal-dependent
repressors. This ORF was designated AmdR gene or amdR.
Chromosomal DNA was isolated from A. naeslundii as
described by Donkersloot et al. (1985). Primers based on
the sequence of the amdR gene were designed to incorporate
a 5 0 BamHI site and a 3 0 KpnI site flanking the amdR
orf 5 0 -CACCTCAAGCAGGATCCCACCCCATGACC-3 0 and
5 0 -GCCGTCAGCGGTACCGCGAGACAG-3 0 . Utilizing PCR,
a 672-bp band was amplified corresponding to the predicted
length of amdR. The BamHI–KpnI-restricted PCR product
was ligated into BamHI–KpnI-restricted pGEM7 (Promega,
Madison, WI). The resulting plasmid, pJK9, containing the
putative metal-dependent repressor was sent for nucleotide
sequence analysis (Medical College of Wisconsin).
Isolation of siderophore uptake promoter
The A. naeslundii genome was scanned for putative siderophore
uptake proteins. Genomewide scanning revealed an 864-bp
ORF (Locus ANA 1461) with homology to siderophore uptake
proteins. This ORF was designated siderophore uptake gene or
FEMS Microbiol Lett 275 (2007) 214–220
sid. The promoter region consisting of 242 bp upstream of the
transcription start site of sid gene was isolated from chromosomal DNA via PCR using forward primer 50 -GGCCGAGGCCG
ACTGGTACCTCTACGGGGCGCAG-30 and reverse primer
5 0 -CCGGAGCGGGCACGGATCCCTTCTTCTTCGCA-3 0 .
Purification of AmdR
The gene encoding AmdR was subcloned into pQE-30 and
transformed into E. coli M15 to produce a recombinant 6 Histidine-tagged protein. The HIS-tagged AmdR was purified using nickel affinity chromatography (Qiagen Valencia,
CA). Briefly, bacterial cells grown to the mid-log phase were
induced with 1 mM isopropyl-b-D-thiogalactopyranoside
(IPTG) and allowed to grow for an additional 4 h. Cells
were lysed by and extracts were recovered by centrifugation.
Cell lysates were then passed through nickel–nitrilotriacetic
affinity spin columns. The band corresponding to AmdR
was excised from the sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). The gel slice containing
purified AmdR was used to immunize a rabbit at Lampire
Biologicals (Pipersville, PA) to obtain polyclonal antisera.
Biofilm assay
Biofilm formation was detected according to a modification
of the procedure described by Huber et al. (2001). Briefly,
A. naeslundii grown overnight at 37 1C, 5% CO2 was inoculated in 2.0 mL deep six-well plates, containing BHI supplemented with dipyridyl for 24 h without shaking. Nonadhering
cells were removed and 1 mL of aqueous solution of crystal
violet (1% w/v) was added to each well. After 20 min of roomtemperature incubation, the dye was removed and the wells
were washed three times with dH2O. The plate was incubated
at 37 1C and was allowed to dry for 3 h. The biofilm was
quantified by solubilizing the crystal violet with a 95%
ethanol solution and determining the OD470 nm.
Levan production
Actinomyces naeslundii were grown in BHI with 1% sucrose.
Divalent metal ions were removed from the medium by
adding dipyridyl. Actinomyces naeslundii grown in the above
medium was examined for fructan metabolism utilizing a
levan enzyme-linked immunosorbent assay (ELISA) (Bergeron & Burne, 2001). To detect levans bacteria were grown
overnight in the medium indicated above in a six-well plate.
Plates were inverted to remove all nonadhering cells. Plates
were washed with phosphate-buffered saline (PBS) and
blocked with 5% dry milk. Levans were detected using a
mouse monoclonal antibody (mAb) UPC-10 that detects
b2,6 linkages (MP Biomedicals Aurora, OH). Plates were
washed with 0.05% Tween 80 PBS and subsequently incubated with a peroxidase-conjugated rabbit anti-mouse IgG
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216
C. Moelling et al.
antibody. Unbound antibody was removed and the ELISA
was developed by 3,3 0 ,5,5 0 -tetramethylbenzidine, citrate
buffer, peroxide. The reaction was stopped by addition
of 0.5 M H2S04 and the OD450 nm was determined spectrophotometrically. Cells grown without sucrose and wells
without UPC-10 were used as negative controls.
the supernatant was retained for siderophore assays.
Five hundred microliters of the supernatant was combined
with 500 mL of CAS reagent and the OD was recorded at
590 nm. The levels of siderophore production were normalized for cell growth by factoring in OD of the culture
OD600 nm.
Generation of amdR -defective A. naeslundii
Electrophoretic mobility shift assay
An Actinomyces naeslundii mutant containing an insertion in
the amdR gene was constructed as follows: a 672-bp fragment
containing the amdR ORF was subcloned into pGEM-7,
which does not replicate in A. naeslundii (Yeung et al., 1998).
A kanamycin-resistant cassette KmR was released from
pJRD215 by XhoI–DraI digestion and blunt ends were
generated by T4 DNA polymerase (New England Biolabs).
The KmR gene was introduced into a unique StuI site of the
amdR gene leaving 300 bp upstream and 370 downstream of
the insertion site. The resulting plasmid pKB was electroporated into A. naeslundii MGI as described previously (Yeung
& Kozelsky, 1994). Chromosomal DNA was isolated from
KmR colonies and was screened for the presence of the
kanamycin-resistant marker via PCR. Primers 5 0 -CTCAGAA
GAACTCGTCAAGA-3 0 and 5 0 -CTGCTATTGGGCGAAGTG
CCGGG-30 corresponding to sequences of the KmR gene were
used for PCR amplification.
Sid promoter sequence was amplified by PCR using biotinlabeled primers. The AmdR was purified under native conditions using Qiagen nickel affinity columns. Five micrograms
of AmdR protein was added to 2 pmol of biotin-labeled sid
promoter. Samples were loaded onto a 6% nondenaturing
acrylamide gel. The gel was electrophoresed at 100 V for
60 min and blotted onto a Biodyne B membrane (Pierce
Biologicals, Rockford, IL). The membrane was UV crosslinked and developed as indicated by Pierce Biologicals.
Siderophore detection
Siderophore production was detected by a modification of
the chrome azurol S (CAS) assay (Schwyn & Neilands,
1987). Briefly, 100 mL of A. naeslundii grown overnight in
1 ADM were inoculated into tubes containing 5 mL of
0.5 ADM alone or supplemented with FeCl3. Actinomyces
naeslundii strains were grown in ADM and their growth
was monitored spectrophotometrically. Cells were collected
at early, mid- and late-log and after 48 h, 600 mL of cells
were collected and centrifuged at 10 000 g for 2 min and
Results
Identification of amdR
Sequence data support the existence of a DtxR-like repressor
in A. naeslundii, which the authors have coined AmdR.
The amdR gene encoding the putative metal-dependent
repressor is c. 672 bp and the deduced amino acid sequence
shares the highest degree of similarity to Streptomyces
coelicolor iron repressor (Table 1a). AmdR was isolated
from A. naeslundii MG1 genomic DNA via PCR. The gene
was subcloned into pGEM7 (Promega, Madison, WI) and
pQE-30 (Qiagen, Valencia, CA).
Identification of sid
The A. naeslundii MG1 genome was searched for genes that
encode proteins involved in metal uptake. A gene with
homology to a siderophore transport gene was identified
using BLAST search of the A. naeslundii genome on TIGR. The
Table 1. Homology of the proteins encoded by (a) amdR genes and (b) sid gene of Actinomyces naeslundii and those of other bacteria
A. naeslundii
Protein
AmdR % identity/Similarity
GenBank ID
(a)
Streptomyces coelicolor A3(2)
Streptomyces avermitilis
Mycobacterium tuberculosis CDC1551
Corynebacterium diphtheriae NCTC13129
Iron repressor
Iron repressor
IdeR
DtxR
58%/70%
57%/69%
53%/69%
53%/66%
CAB95905.1
BAC71567.1
AAK47100.1
CAE49945.1
A. naeslundii
Protein
Sid % identity/Similarity
GenBank ID
FepC
ATPase protein ironsiderophore
FepC
Iron siderophore uptake
64%/79%
62%/78%
AAG07545.1
AAL46004.1
60%/77%
58%/75%
AAY92888.1
CAB52849.1
(b)
Pseudomonas aeruginosa PAO1
Agrobacterium tumefaciens
Pseudomonas fluorescens Pf-5
Streptomyces coelicolor A3(2)
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FEMS Microbiol Lett 275 (2007) 214–220
217
Metal-dependent repression
1.6
1.4
1.2
OD450 nm
deduced amino acid of the A. naeslundii gene has 64%
identity to Pseudomonas aeruginosa FepC (Table 1b). The
promoter sequence upstream of the sid gene was isolated for
gel shift studies. Few characterizations of A. naeslundii
promoters exist. No significant homologous sequences
could be identified in the sid promoter when compared with
the promoters of urease and fructosyltransferase genes
(Morou-Bermudez & Burne, 1999; Bergeron et al., 2000).
1
0.8
0.6
0.4
Biofilm formation of A. naeslundii MG1 and
amdR- mutant
0.2
0
No cells
Biofilm studies were conducted to see whether metal ions
influence A. naeslundii biofilm formation. In BHI A. naeslundii MG1 biofilm production is barely detectable; however, the amdR-mutant forms a biofilm in BHI (Fig. 1).
When divalent metal ions are removed from the BHI
(addition of 5 mm dipyridyl), both A. naeslundii and amdRstrains form biofilms (Fig. 1). At dipyridyl concentrations
4 5 mm, biofilm production of the amdR mutant and wild
type decreases sharply (Fig. 1).
Levan production is independent of metal ion
availability
Levan ELISAs were conducted to see whether metal
ion availability influences levan production. Wild-type
A. naeslundii was examined for levan (b2,6 linked fructans)
production using a mAb that detects b2,6 linkages in an
ELISA. Actinomyces naeslundii were grown for 48 h in the
presence of sucrose and dipyridyl. No levans were detected
1.4
AN MG1
1.2
AmdR –
OD470 nm
1
0.8
0.6
0.4
BHI
5 µm DP
10 µm DP 15 µm DP
Fig. 2. Levan ELISA. Actinomyces naeslundii MG1 wild type overnight in
six-well polystyrene plates in BHI. Dipyridyl concentrations reflect the
final concentration of dipyridyl in micromolar. In the no-cells column, just
BHI was incubated as a negative control. The values presented here are
the means of three separate experiments performed in triplicate, and the
error bars show the SDs.
in 24 h (data not shown). However, at 48 h levans were
detected but levan production appeared to be independent
of iron availability (Fig. 2) and no statistically significant
differences of levan production were found (P o 0.05) by a
t test. Interestingly, levans were detected in BHI supplemented with 10 mm dipyridyl, which yielded little biofilm
formation in studies without sucrose supplementation.
Detection of siderophores in A. naeslundii
Siderophore studies were conducted to see whether
A. naeslundii produces siderophores. AmdR-mutant and wildtype A. naeslundii siderophore production was examined at
early, log and stationary phases of growth. Siderophore
production is barely detectable at the early and log phase
of growth (data not shown). However, in the stationary
phase siderophores were detected in A. naeslundii and
the amdR-defective A. naeslundii culture supernatants
(Fig. 3). In wild-type A. naeslundii, siderophore production
decreased when the iron was added to the medium. The
amdR-defective strain showed a similar repression when
iron was supplemented to the growth medium. However,
siderophore production in the amdR-defective strain is
roughly twice that of the wild-type strain (Fig. 3).
0.2
0
bacteria
5 µm DP
10 µm DP 15 µm DP
Fig. 1. Biofilm formation of Actinomyces naeslundii MG1 and AmdRmutant. Cells were grown overnight in six-well polystyrene plates in BHI.
Dipyridyl concentrations reflect the final concentration of dipyridyl in
micromolar. AN MG1 is A. naeslundii MG1. AmdR- is the amdR-defective
strain. The values presented here are the means of three separate
experiments performed in triplicate, and the error bars show the SDs.
FEMS Microbiol Lett 275 (2007) 214–220
AmdR binds to the promoter region of
siderophore uptake gene
Gel shift studies were conducted to see whether AmdR binds
to the siderophore uptake gene (sid) promoter region. The
sid promoter region was shifted in the presence of AmdR
(Fig. 4). When polyclonal sera containing anti-AmdR antibodies were added to the sid promoter and AmdR, a supershift was observed (Fig. 4 lane 4). Increasing the iron
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218
C. Moelling et al.
Discussion
6
AN MG1
5
AmdR –
CAS units
4
3
2
1
0
20 µm Fe
0 Fe
40 µm Fe
Fig. 3. Siderophore production in Actinomyces naeslundii and amdR
mutant. Bacteria were grown in Actinomyces-defined medium containing no additional iron or supplemented with ferric chloride at the
concentrations indicated. The cell suspension was centrifuged and
culture supernatants were analyzed for siderophore production. The
values presented here are the means of three separate experiments
performed in triplicate, and the error bars show the SDs. In all cases of
measurements of supernatant activities, the mutant and wild-type
strains are statistically different (P o 0.05) by t test.
1
2
3
4
5
Fig. 4. Gel shift assay confirms that AmdR targets the siderophore
promoter region. The Sid promoter (psid) was labeled with biotin. Lane 1
psid promoter. Lane 2 psid promoter, AmdR and 1 mm FeCl3. Lane 3 psid
promoter, AmdR and 5 mm FeCl3. Lane 4 psid promoter, AmdR, 5 mm FeCl3
and anti-AmdR antibody. Lane 5 psid promoter, AmdR and 50 mM EDTA.
Gel mobility shifts were performed with a HIS-tagged Native purification
of AmdR protein. This gel is representative of four separate experiments.
concentration of the reaction from 1 to 5 mm did not result
in a significant increase in the shift of the promoter sequence
(Fig. 4 lanes 2 and 3). To show metal-dependent binding of
AmdR to promoter sequences, the metal ion chelator EDTA
(to a final concentration of 50 mM) was added. However,
EDTA did not entirely diminish the AmdR-dependent shift
of the promoter region (Fig. 4 lane 5).
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The ability to of A. naeslundii to form biofilms and to
coaggregate with oral bacteria influences plaque ecology
(Palmer et al., 2003; Shen et al., 2005). This study is the first
to examine the effects of metal scarcity on A. naeslundii
biofilm formation. The results indicate that A. naeslundii
increases biofilm formation when environmental iron concentrations decrease.
In the absence of iron, bacteria may transcribe a variety of
genes in order to acquire iron from their host or environment. Some of these gene products, such as toxins, cause
destruction to host tissues. Actinomyces naeslundii virulence
properties are influenced by the availability of environmental metal ions, presumably iron. Like other pathogenic
bacteria, it was hypothesized that A. naeslundii regulates
gene expression in response to metal ions via a metaldependent repressor. In support of this theory, searches of
the A. naeslundii genome revealed an ORF with homology
to a metal-dependent repressor (AmdR). In order to characterize AmdR, an amdR-defective strain of A. naeslundii
was constructed. The working hypothesis is that in the
presence of iron AmdR binds to operator regions of genes
involved in adherence and siderophore uptake. Supporting
this hypothesis, the results indicate that AmdR binds to the
promoter region of the siderophore uptake gene. In addition, A. naeslundii biofilms are reduced in rich media
containing excess iron. Once the concentration of metal
ions in the growth medium is decreased, biofilm and siderophore production increases in A. naeslundii. However,
when the gene governing metal dependence (amdR) is
mutated, the amdR-strain is less sensitive to metal ion
concentrations. Thus, the amdR-mutant forms biofilms in
enriched media with increased concentration of metal ions
whereas the wild-type A. naeslundii does not. However,
small amounts of metal ions are required for the growth of
most bacteria because these ions are necessary for electron
transport chain function. Therefore, when the concentrations of metal ions become too limited (dipyridyl concentrations 4 5 mm), the growth of mutant and wild-type
A. naeslundii drops off steeply.
In other microorganisms, metal ion concentrations influence the production of polysaccharides. In the oral cavity, it
is known that polysaccharides such as levans and glucans
play a role in the formation of dental plaque. In S. mutans
and C. diphtheriae, metal ions have been shown to influence
the production of polysaccharides (Moreira Lde et al., 2003;
Rolerson et al., 2006). Glucan production in S. mutans is
influenced by the metal-dependent repressor SloR (Rolerson
et al., 2006). As polysaccharides are components of biofilms,
the upregulation of polysaccharide production in decreasing
iron concentrations may explain the increased A. naeslundii
biofilm formation under these conditions. As A. naeslundii
FEMS Microbiol Lett 275 (2007) 214–220
219
Metal-dependent repression
is known to produce levans (Bergeron & Burne, 2001), a
change in levan production in response to decreasing iron
availability was checked for. It was found that the iron
concentration does not influence levan production. Levans
are produced independent of environmental metal ion
concentration; most likely, levans are regulated by the
availability of carbon sources (Bergeron & Burne, 2001).
The purpose of this study was to determine which
A. naeslundii genes are controlled by the availability of
environmental iron. In most microorganisms, siderophore
production and uptake is governed by metal-dependent
repressors. However, there have been no studies to date
establishing whether or not A. naeslundii produces siderophores. The present experiments indicate that A. naeslundii
MG1 produces siderophores in a chemically defined medium without iron supplementation. The amdR-defective
strain of A. naeslundii produces twice the amount of
siderophores as wild-type A. naeslundii. Interestingly, siderophore production in the amdR mutant is still dependent
on iron availability. In the absence of the repressor, one
might expect a constitutive production of siderophores,
because the genes responsible for the synthesis of siderophores should no longer be repressed. There may be
multiple mechanisms governing the regulation of iron
uptake systems in A. naeslundii.
In the oral cavity, microorganisms become starved of
metal ions and other nutrients between meals. During these
times of iron starvation, A. naeslundii produces siderophores. In order to use the iron scavenged by siderophores,
bacteria must possess siderophore uptake systems to transport siderophores inside the bacterium. Some bacteria that
do not produce siderophores have the ability to take up
exogenous siderophores (West & Buckling, 2003). These
bacteria can utilize the siderophores produced by other
bacteria that share the same environmental niche. Therefore,
A. naeslundii siderophore production may not only be
important to A. naeslundii survival but may also contribute
to the overall biofilm ecology. The siderophores produced by
A. naeslundii may be utilized by the several hundred species
of bacteria in the mouth.
Acknowledgements
Funding for this work was made possible by the American
Association of University Women (AAUW) Educational
Foundation.
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