JOURNAL OF BACTERIOLOGY, July 2004, p. 4246–4253 Vol. 186, No.

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0021-9193/04/$08.00⫹0 DOI: 10.1128/JB.186.13.4246–4253.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Involvement of Streptococcus gordonii Beta-Glucoside Metabolism

Systems in Adhesion, Biofilm Formation, and In Vivo
Gene Expression
Ali O. Kiliç,1 Lin Tao,1* Yongshu Zhang,2 Yu Lei,2 Ali Khammanivong,2 and Mark C. Herzberg2,3
Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612,1 and
Department of Oral Sciences, School of Dentistry,2 and Mucosal and Vaccine Research Center,3
University of Minnesota, Minneapolis, Minnesota 55455
Received 4 November 2003/Accepted 15 March 2004

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart
valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite
(sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these
induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene
inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metab-
olism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and
a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides.
Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochi-
tosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci.
Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic
surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism
may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

Streptococcus gordonii is a pioneer colonizer on the surface
of human teeth, initiating the formation of dental plaque (25).
Although not directly associated with dental or gingival dis-
eases in the oral cavity, S. gordonii colonizes damaged heart
valves once it enters the bloodstream in the experimental rab-
bit (15) and is considered to cause endocarditis in humans (1,
8, 12). Thus, the virulence of S. gordonii is reflected in its ability
to adhere, colonize, and survive in the heart, where it is a
pathogen, and on teeth in the oral cavity, where it is not. Its
virulence in a specific environment may reflect expression of
genes uniquely in that environment.
Using an in vivo expression technology (IVET) library con-
structed in S. gordonii V288, at least 13 genes were shown to be
expressed in vivo on infected heart valves during experimental
endocarditis, but they were unexpressed in vitro under labora-
tory conditions (15). Since expression occurred only on heart
valves, these genes were suggested to contribute to virulence
and perhaps pathogenicity. Similarly, S. gordonii genes ex-
pressed during in vitro colonization of saliva-coated hydroxy-
apatite (sHA) were identified using the IVET library (17).
Among the genes expressed during colonization of sHA and
damaged heart valves in the rabbit were beta-glucoside metab-
olism-encoding genes. We therefore initiated in vitro studies to
understand how the beta-glucoside metabolism genes might
contribute to colonization of heart valves in infective endocar-
ditis and on sHA.
Bacteria ferment beta-glucoside sugar substrates, including
cellobiose, arbutin, salicin, and esculin. While common in
* Corresponding author. Mailing address: Department of Oral Bi-
ology, College of Dentistry, University of Illinois at Chicago, 801 South
Paulina St., Chicago, IL 60612. Phone: (312) 355-4077. Fax: (312)
996-6044. E-mail: Ltao@uic.edu.
plants and useful in establishing phenotypic fermentation
patterns of bacteria, the aryl-beta-disaccharides are not found
in mammalian tissues and fluids. Mammalian extracellular
matrix, however, is rich in glycosaminoglycans (GAGs),
which contain beta-linked disaccharide repeating units (22,
33). Structural analogues of cellobiose or N,N⬘-diacetylchito-
biose [(GlcNAc)2], beta-linked disaccharides are released
when GAGs degrade. In the rabbit model of endocarditis,
S. gordonii may have metabolized GAGs from the injured
heart valves. Metabolism of beta-glucosides by other species
involves known genes. In Escherichia coli, the cel (cellobiose)
operon is induced by (GlcNAc)2 and has been renamed the chb
(N,N⬘-diacetylchitobiose) operon (14). Listeria monocytogenes
expresses beta-glucoside permease in vivo (10). Hence, metab-
olism of beta-glucosides, perhaps in the form of GAG-derived
oligosaccharides, may be an important adaptive response to
survival in vivo.
In the present study, we compared the gene sequences of the
IVET clones with the S. gordonii genomic data (www.tigr.org)
to identify the proximal genes in each locus. Five beta-glu-
coside-associated genes expressed in vivo during experimental
endocarditis were shown to localize with other beta-glucoside
metabolism genes comprising two different novel phospho-
enolpyruvate-dependent phosphotransferase systems (PTS)
and a binding protein-dependent sugar transport and metab-
olism. We also used this IVET system to identify S. gordonii
genes critical to and perhaps induced during biofilm formation
under in vitro conditions. Among the newly identified genes,
many are associated with the metabolism of beta-glucosides
through yet another PTS. Therefore, four novel systems for
beta-glucoside metabolism have been identified in S. gordonii
which may contribute to virulence during colonization in mam-
malian environments.

4246
VOL. 186, 2004 S. GORDONII BETA-GLUCOSIDE METABOLISM 4247

TABLE 1. S. gordonii strains used in the study The absorbency at 568 nm was determined with a plate reader. Bacteria with
weak CV staining were picked from the duplicate plates as putative biofilm
Relevant Isolation Source or formation-defective mutants.
Strain
characteristics method reference
Selecting mutants defective in beta-glucoside fermentation. Single colonies of
V288 Wild type Gary Dunny the S. gordonii V288 reporter gene-fusion library were plated onto modified
KG212 bglC-amy-cat Tcr IVET 15 Jordan agar supplemented with esculin and ferrous salt (4). The clones that did
KG206 escB1-amy-cat Tcr IVET 15 not show black pigmentation were tested again for their abilities to ferment
KG207 escR-amy-cat Tcr IVET 15 cellobiose, arbutin, and salicin.
KG210 escA-amy-cat Tcr IVET 15 PCR procedures. Arbitrary PCR was used to determine the DNA sequence
KG202 gomA-amy-cat Tcr IVET 15 flanking the inserted IVET vector (2, 28). In the first-round PCR, a primer
KG205 iviE-amy-cat IVET 15 unique to the ori sequence of pACYC184 (oriExt, 5⬘-AGCTCAGAGAACCTT
KG208 iviH-amy-cat IVET 15 CGAAAAAACC-3⬘) and an arbitrary primer (ARB1, 5⬘-GGCCACGCGTCGA
KG211 msrA-amy-cat IVET 15 CTAGTACNNNNNNNNNNGATAT-3⬘) were used in 50-␮l PCR mixtures (1⫻
MG1006 bfbF1-amy-cat Tcr Biofilm This study Vent polymerase buffer, MgSO4 [1 mM], deoxynucleoside triphosphates [0.25
MG1007 bfbB-amy-cat Tcr Biofilm This study mM], and Vent [exo⫹] DNA polymerase [1 U] with 5 ␮l of an overnight THB-
MG1021 escB2-amy-cat Tcr Biofilm This study grown culture as the DNA template). The first-round PCR conditions were 95°C
MG1015 bfrA-amy-cat Tcr Biofilm This study for 5 min; 6 cycles of 94°C for 30 s, 30°C for 30 s, and 72°C for 1 min; 30 cycles
MG2025 bfbF2-amy-cat Tcr Biofilm on sHA This study of 94°C for 30 s, 43°C for 30 s, and 72°C for 1 min; and a final step at 72°C for
MG2026 bfbR-amy-cat Tcr Biofilm on sHA This study 10 min. The second-round PCR was performed with the same conditions as the
CG420 escA Emr Insertion This study first round, except 5 ␮l of the first-round PCR product was used as DNA
CG425 escA Kmr Insertion This study template with primers ARB2 (5⬘-GGCCACGCGTCGACTAGTAC-3⬘) and ori-
CG421 escP Emr Insertion This study Int (5⬘-CAAGAGATTACGCGCAGACC-3⬘). The ARB2 sequence was identi-
CG426 escP Kmr Insertion This study cal to the 5⬘ end of the ARB1 primer. The oriInt primer was derived from a
CG422 gomG Emr Insertion This study sequence of pACYC184 located closer than the oriExt sequence to the junction
CG423 bglJ Emr Insertion This study between the inserted pAK36 and the S. gordonii chromosome.
CG427 gomF Emr Insertion This study Nucleotide sequence determination and genetic loci identification. The PCR
CG480 fucA Emr Insertion This study products were purified using the QIAquick PCR purification kit (QIAGEN). The
CG482 manB Emr Insertion This study purified PCR products were sequenced using the oriInt primer at the Advanced
CG484 gomH Emr Insertion This study Genetic Analysis Center, University of Minnesota. The sequences were com-
CG486 gomR Emr Insertion This study pared with the GenBank database using the BLAST program. The DNA se-
CG495 ⌬gom Emr Deletion This study quence data that matched genes encoding the utilization of beta-glucoside sugars
CG532 merA-amy-cat Tcr Esculin-negative This study from our previous IVET study (15) and those from the present study were used
for BLAST analysis against the partially completed S. gordonii genome (The
Institute for Genomic Research [TIGR]; www.tigr.org). In case complete se-
quence data were not available for a certain locus, the inverse PCR technique
MATERIALS AND METHODS (26) was used to complete the sequence data.
Phylogenetic analysis. From their nucleotide sequences, genes in S. gordonii
Bacterial strains, media, and chemicals. Bacterial strains used in this study are
beta-glucoside regulons were translated into putative proteins. Protein sequences
listed in Table 1. S. gordonii V288 and its reporter gene-fusion strain library
of other bacterial species were obtained from GenBank via a BLAST search. The
based on the streptococcal IVET vector pAK36 (15) were grown in Todd-Hewitt
sequences were aligned using the CLUSTAL X program (version 1.81; http://inn
broth (THB; Difco) or FMC medium (40) at 37°C under CO2-rich conditions.
-prot.weizmann.ac.il/software/ClustalX.html) (41), and phylogenetic trees were
Tetracycline (TET) was added at 15 ␮g/ml to maintain the selective pressure for
constructed with the TreeExplorer program (http://evolgen.biol.metro-u.ac.jp
growth of the strain library. For sugar fermentation studies, purple broth (Difco)
/TE/TE_man.html). Bootstrap values were obtained with the CLUSTAL X pro-
supplemented with appropriate carbohydrate was used. To detect esculin hydro-
gram.
lysis, a ferrous medium was used (4). For the amylase reporter assay, modified
Mutant construction and analysis. Mutants were constructed by two methods.
Jordan medium (13) supplemented with 0.5% starch (wt/vol) and 1.5% agar
All mutants except CG495 were constructed by insertion duplication using a
(wt/vol) plus 0.1% glucose (wt/vol) with or without 0.1% esculin (wt/vol) was
streptococcal integration plasmid, pVA891 or pSF151 (38). Strain CG495 was
used.
constructed by gene replacement to delete the target gene(s). To delete the
Genes induced during biofilm formation on sHA. The adhesion assay de-
target genes, the flanking genes, fucA and bglJ, of the target S. gordonii gom locus
scribed by Gong et al. (11) was modified to study biofilm formation by using the
IVET library for S. gordonii. An overnight culture of the S. gordonii V288 were amplified by PCR and cloned jointly into PCR cloning vector pGEMT-Easy
reporter gene (amy-cat) fusion library in THB with TET was diluted 1,000-fold (Promega). Next, an erythromycin resistance cassette derived from the strepto-
in FMC medium. Chloramphenicol was added to a concentration of 15 ␮g/ml to coccal shuttle plasmid pVA838 (21) was inserted into the fucA-bglJ joint gene
provide selective pressure for biofilm-induced genes. Bacteria were mixed with fragment, replacing parts of both genes. The recombinant gene cassette was then
sHA and incubated at 37°C for 24 h. Adherent surviving S. gordonii cells were excised by restriction enzyme digestion. Following gel purification, the excised
removed and spread on THB plates containing TET. Single colonies were replica recombinant gene cassette was used to transform S. gordonii V288 to erythro-
plated onto three new THB plates. One contained 5 ␮g of chloramphenicol/ml; mycin resistance. The gene insertion or deletion in each mutant was confirmed
the second contained 0.5% starch (wt/vol); and the third was THB without by PCR with appropriate primers.
antibiotic supplement. After incubation for 24 h at 37°C, the colonies on the Sugar fermentation. Mid-exponential-phase cells growing in THB with appro-
starch agar plate were flooded with a solution of 0.5% I2 and 1% KI to detect priate antibiotics were harvested by centrifugation, washed once, and then re-
amylase activity (starch hydrolysis). Colonies that were sensitive to chloramphen- suspended in purple broth supplemented with a sugar and antibiotics. Sugars (all
icol and negative for amylase activity were selected from the third plate. from Sigma or as noted) tested for fermentation included glucose, fructose,
Selecting mutants defective in biofilm formation on plastic. Biofilms were galactose, lactose, maltose, mannose, cellobiose, arbutin, salicin, and esculin and
formed in 96-well polystyrene microtiter plates (18, 27). The S. gordonii V288 chitin oligosaccharides [(GlcNAc)n; n ⫽ 2 to 8], N, N, N⬙-triacetyl chitotriose,
reporter gene-fusion library was spread on THB plates containing TET and ␣1-2, 1-3, or 1-6 D-mannobiose, ␣1-3-␣1-6-D-mannopentose, Lewis-X oligosac-
incubated at 37°C for 24 h. Individual colonies were picked and transferred into charide, GlcNAc-␤1-2 mannose (Dextra), heparin, chondroitin sulfate, and li-
wells on microtiter plates (Costar 3799) containing 200 ␮l of THB with TET. chenan. The cultures were incubated at 37°C, and color changes were monitored
Duplicates were made by transferring 100 ␮l of each culture to another well on hourly. To assay for esculin hydrolysis, mid-exponential-phase cultures of the
a new microtiter plate. Plates were incubated at 37°C for 24 h, and then 25 ␮l of wild-type strain and mutants were spotted onto ferrous salt-containing agar and
a 1% crystal violet (CV) solution was added into each well. The plates were incubated for 24 h. A black color forms from the hydrolysis product of esculin,
incubated at room temperature for 15 min and rinsed thoroughly with water. 6,7-dihydroxycoumarin, which reacts with the ferrous ion. To analyze for glucose-
Biofilm formation was quantified by the addition of 200 ␮l of 95% ethanol to mediated catabolite repression, the ferrous medium was supplemented with
each CV-stained well, of which 125 ␮l was transferred to a new microtiter plate. glucose.
4248 KILIÇ ET AL. J. BACTERIOL.

FIG. 1. Genetic loci encoding beta-glucoside metabolism in S. gordonii. Shown are genes induced specifically in the rabbit heart during
experimental endocarditis (15), genes induced during biofilm formation on sHA, and genes required for in vitro biofilm formation on plastic,
indicated by diamonds, triangles, and circles, respectively. The star indicates the gene induced by shifting S. gordonii from mildly acidic (oral) to
neutral (blood) pH (42). The small bending arrows represent promoter-like sequences. U, overlapping reading frames.

Biofilm and sHA adhesion assays. Mutants and previously isolated IVET
clones were analyzed for their abilities to form biofilms on microtiter plates as
described above. For the sHA adhesion assay, bacteria were cultured overnight
(16 to 18 h) in 3 ml of FMC with [3H]thymidine (10 ␮Ci/ml). Cells were
centrifuged and resuspended to a final optical density at 620 nm of 0.32 (109
cells/ml). Suspensions (1 ml) were added to tubes with sHA and rotated for 1 h
at room temperature. The sHA with bound bacteria was allowed to settle,
washed three times, and transferred to scintillation vials. Radioactivity associated
with sHA was counted with a scintillation counter. Triplicates of each sample
were analyzed. Statistical analysis was performed by using the two-tailed, un-
equal variance t test with comparisons between the values for the wild type and
each mutant.
Sugar induction assay. To analyze whether the expression of the genes en-
coding beta-glucoside metabolism and/or other genes induced during endocar-
ditis can be induced by any beta-glucoside sugars, strains with the pAK36 plasmid
reporter gene fusion were tested on Jordan agar plates supplemented with 0.5%
starch and 0.5% cellobiose, arbutin, salicin, esculin, or oligochitosaccharide.
Mid-exponential-phase cultures in THB were diluted 10-fold with THB and
spotted (5 ␮l) onto the starch agar plate. After incubation for 16 h, the plates
were flooded with the iodine solution to detect the expression of amylase. The
wild-type strain V288 was used as a control.
RESULTS
Identification of genes and loci associated with the utiliza-
tion of beta-glucosides. By homology analysis, DNA sequences
of 5 (iviL [new name, bglC], iviJ [escA], iviF [escB], iviG [escR],
and iviB [gomA]) of the 13 unique S. gordonii clones induced in
vivo during experimental endocarditis were found to be asso-
ciated with three separate genetic loci encoding the utilization
of beta-glucosides. Additional sequences of these loci were
obtained by searching the partially completed S. gordonii ge-
nome at the TIGR website. The sequence of the gom locus was
not completed by TIGR. Therefore, the inverse PCR tech-
nique was used to synthesize flanking DNA and obtain addi-
tional sequences (accession number AY526569). Among 44
S. gordonii IVET clones with genes induced during biofilm
formation on sHA, 11 had reporter gene insertions in two
genes of a new beta-glucoside locus (bfbF and bfbR). Among
35 mutants with genes required for biofilm formation on plas-
tic, 9 clones with six unique insertion sites were related to
beta-glucoside metabolism. Four of the unique insertion sites
were within the same locus as the sHA biofilm-associated
genes. The fifth insertion site matched one of the three loci
identified from the IVET studies on heart valves, and the sixth
site was associated with an isolated beta-glucosidase gene.
Therefore, biofilm formation by S. gordonii in different envi-
ronments appeared to involve as many as four putative regu-
lons for the utilization of beta-glucoside-like sugars (Fig. 1).
A mutant, CG532, defective in esculin, arbutin, and salicin
fermentation was isolated from the S. gordonii pAK36 plasmid
insertion strain library. Its target gene was sequenced using the
Amy-R primer. The genetic locus was identified from the S.
VOL. 186, 2004
gordonii genome and showed high homology to the mercury
resistance gene (merA) of many bacteria.
Nucleotide sequence analysis. (i) The bgl locus. The bgl locus
includes five open reading frames (ORFs). The first two ORFs,
separated by 14 nucleotides, encode two putative transcrip-
tional regulators, BglD (249 amino acids [aa]) and BglE
(322 aa). The remaining three ORFs are highly homologous
to genes encoding three components, BglA (107 aa), BglB
(109 aa), and BglC (433 aa), of a PTS permease (enzyme II)
for transporting beta-glucosides in Streptococcus pyogenes
(Q8NZ66) and Streptococcus pneumoniae (Q97SS4). bglA is
223 nucleotides downstream of bglE and has its own putative
promoter. The two downstream ORFs, bglB and bglC, are
separated by only 11 and 2 nucleotides, respectively, from their
immediate upstream ORFs. However, a gene encoding a phos-
pho-beta-glucosidase is not found in this locus.
(ii) The esc locus. The esc locus includes six ORFs. The first
ORF encodes the PTS permease (enzyme II), EscP (629 aa),
for transporting beta-glucosides. The second ORF encodes a
hypothetical protein, EscB (420 aa), which is homologous to
the CapA protein associated with capsule synthesis of Strepto-
coccus agalactiae (Q8E4G0). The third and fourth ORFs en-
code two conserved hypothetical proteins, EscC (75 aa) and
EscD (55 aa). The fifth ORF, transcribed in the opposite
direction, encodes a protein EscR (186 aa) homologous to the
Streptococcus mutans regulator BglC (Q9KJ78). The last ORF,
escA, encodes a protein (478 aa) homologous to S. mutans
phospho-beta-glucosidase BglA (Q9KJ76). These ORFs, ex-
cept ORF4, have their own putative promoters. A putative
RNA antiterminator (RAT) sequence, GGATTGTTACTGA
TCACGCAGGCAAAAC CTA, is located 125 bases upstream
of the initiation codon for the escP gene. It matches the con-
sensus sequence of RATs (5).
(iii) The bfb locus. The bfb locus includes seven ORFs. The
first ORF, bfbF, encodes a protein (488 aa) homologous to
S. pneumoniae phospho-beta-glucosidase BglA (Q8DR89).
The second ORF encodes a putative protein, BfbG (79 aa).
The third ORF, bfbB, encodes the B subunit of a beta-glu-
coside permease (106 aa; PTS enzyme IIB). The fourth ORF,
bfbR, encodes a BglG-like antiterminator (656 aa). The fifth
ORF, bfbA, encodes the A subunit of a PTS enzyme II (105
aa). The sixth ORF, bfbD, encodes a hypothetical protein (161
aa), possibly the D subunit of the PTS enzyme II. The seventh
ORF, bfbC, encodes the C subunit of the PTS permease en-
zyme II (451 aa) for transporting beta-glucosides.
(iv) The gom locus. The gom locus has 15 ORFs, including
fucA (␣-1,3/4 fucosidase; 576 aa), manA (␣-1,2-mannosidase;
697 aa, in opposite orientation), gomA (hypothetical protein;
435 aa), manB (␣-1,6 mannosidase; 877 aa), gomR (regulator;
292 aa), bhsA (N-acetyl-beta-hexosaminidase; 627 aa), gomF
and gomG (binding protein-dependent sugar transporter mem-
brane components; 311 aa each), gomE (sugar-binding protein;
515 aa), gomB (hypothetical protein; 204 aa), gomH (two-
component sensor histidine kinase; 529 aa), gomD (two-com-
ponent response regulator; 427 aa), bglJ (beta-glucosidase; 461
aa), gomL (VirJ-like sensor histidine kinase; 176 aa), and glmS
(glucosamine-6-P synthase; 603 aa). An ATP-binding protein
for the ABC sugar transport cassette is not found in this locus.
(v) The bfr locus. Although the bgl locus does not have a
beta-glucosidase, a gene encoding 6-phospho-beta-glucosidase
S. GORDONII BETA-GLUCOSIDE METABOLISM 4249
(BglF; 479 aa) was identified in a separate locus, the bfr locus.
Immediately downstream of bglF are two genes, bfrA and bfrB,
encoding a biofilm-related two-component system (44).
Phylogenetic analysis. S. gordonii may use four different
systems to metabolize beta-glucosides during biofilm forma-
tion and/or in vivo growth. To determine their genetic similar-
ities and relationships with related proteins of other bacterial
species, phylogenetic trees were constructed (data not shown).
S. gordonii BglJ is more homologous to beta-glucosidases of
S. pneumoniae (BglA2; Q8DRA9) and S. pyogenes (BglA2;
Q8K735) than its three other beta-glucosidases, BglF, BfbF,
and EscA. Likewise, S. gordonii EscP is more similar to BglP of
S. mutans than to its two other beta-glucoside permeases, BglC
and BfbC, which contain three or four subunits. S. gordonii
EscB is highly homologous to capsule synthases of a range of
species and genera, including CapA of S. agalactiae (Q8E4G0).
Phenotypes of various strains. In addition to the reporter
gene-fusion strains (some also created mutations by splitting
target genes), insertion or deletion mutants were constructed
to assign phenotypes to the genes located in these loci. When
sugar fermentation patterns were compared, the mutants were
similar to the wild-type except for hydrolysis of two beta-glu-
cosides, arbutin and salicin. Most mutants defective in one
gene involved in beta-glucoside metabolism appeared to fer-
ment these two sugars more slowly. The mutants and wild-type
strains, in contrast, fermented two other beta-glucoside sugars,
esculin and cellobiose, similarly. Only two mutants, MG1015
(bfr amy cat) and CG532 (merA amy cat), were defective in
arbutin, salicin, and esculin fermentation, suggesting suppres-
sion of multiple beta-glucoside regulons in these mutants. Re-
gardless of insertion sites, all 13 strains with pAK36 insertions
isolated by in vivo selections and about one-third of random
clones of the pAK36 library displayed glucose inhibition of
esculin hydrolysis, suggesting a possible artifact introduced by
the plasmid. Therefore, strains (all CG strains except CG532)
constructed with pSF151 or pVA891 were used to test for
esculin hydrolysis. The wild-type and the tested mutant strains,
except CG423 (bglJ) and CG495 (⌬gom), fermented esculin in
the presence of glucose.
The wild-type strain did not ferment ␣1-2- or ␣1-3-D-man-
nobiose, ␣1-3-␣1-6-D-mannopentose, heparin, chondroitin sul-
fate, or lichenan, but it fermented Lewis-X oligosaccharide,
N,N,⬘,N⬙-triacetylchitotriose (chitin oligosaccharides), ␣-1,6-D-
mannobiose, and GlcNAc-␤1-2 mannose. All mutants ferment-
ed Lewis-X oligosaccharide and chitin oligosaccharides. Mu-
tants defective in manB, gomH, or gomR showed reduced
fermentation of GlcNAc-␤1-2-D-mannose and no fermentation
of ␣1-6-D-mannobiose.
To determine if selected genes were induced by any ␤-glu-
coside sugars, ␣-amylase activities of the reporter gene-fused
clones were detected in agar containing starch (Fig. 2). Based
on amylase reporter activities, cellobiose, arbutin, and salicin
did not induce expression of any genes tested. Esculin and
oligochitosaccharides induced genes in the esc system but not
any of the other three systems for ␤-glucoside metabolism.
Also, by testing other genes induced during endocarditis (15)
or required for biofilm formation (44), four additional genetic
loci were induced by the same two sugars. These were iviE
(located upstream of dnaX), iviH (an amino acid transporter),
iviK (msrA, peptide methionine sulfoxide reductase), and
4250 KILIÇ ET AL. J. BACTERIOL.

FIG. 2. Esculin induction of reporter gene-fused S. gordonii strains. The reporter amylase activity was disclosed on starch agar plates
supplemented with the sugars indicated after incubation with an iodine solution. (A) Esculin only; (B) esculin plus glucose; (C) glucose only. The
identities and locations of these strains are as follows: top, KG202; second row (from left to right) KG206, KG207, KG210, and KG212; third row,
MG1006, MG1007, MG2025, and MG2026. Note the esculin induction and glucose inhibition of amylase activities in the three mutant strains of
the esc regulon, KG206, KG207, and KG210. Interestingly, these three strains presented diminishing amylase activities, as indicated by a dark color
on agar in the presence of glucose, suggesting the accumulation of IPS (35). These plates contained TET to maintain the inserted plasmid. The
wild-type strain V288 was on three separate plates without TET. Its results were the same as those for KG202 (data not shown).

bfrAB. Interestingly, mutants defective in these esculin-induc-
ible genes showed iodophilic polysaccharide (IPS), as detected
on plates containing glucose and starch. IPS was not seen in
the wild type or strains with plasmid insertions in other beta-
glucoside regulons. The escA (CG420 and CG425) and escP
(CG421 and CG426) mutants constructed with plasmids
pSF151 or pVA891 without the amy-cat reporter gene also
showed IPS on agar containing glucose and starch (data not
shown). This ruled out the possibility that IPS accumulation in
these strains was an artifact introduced by the amy reporter
gene.
To ascertain if genes in these loci were associated with func-
tions in vitro, adhesion to sHA, biofilm formation on plastic,
and growth rates of these strains were analyzed in vitro (Table
2). Mutation of many genes in these four presumptive regulons
resulted in decreased biofilm formation and/or growth rate.
Remarkably, mutation in escA, escR, or gomH and deletion of
the gom regulon resulted in cells with defective abilities to
adhere to sHA in buffer. In some cases, these mutations were
accompanied by defects in growth rate or in vitro biofilm for-
mation (bglC and escA). Other genes associated with defective
adhesion did not manifest defects in biofilm formation or
growth rate (gomH and ⌬gom). While defective in adhesion,
the escR mutant formed biofilm and showed a growth rate
similar to that of wild type. Mutation in escB2 showed no
adhesion defect, but biofilm formation was substantially re-
duced.
DISCUSSION
Several S. gordonii surface components probably mediate
adhesion to the tooth surface, including sialic acid-binding
adhesin (37), amylase-binding protein (30), CshA/B (23),
SspA/B (6), and ScaA (16). While genes for these adhesin
proteins may contribute to colonization in vivo, none are well
established at this time. Recently described genes associated
with S. gordonii biofilm formation encode putative proteins
involved in signal transduction, peptidoglycan biosynthesis,
manganese homeostasis, and fructose PTS (18–20). Further-
more, S. gordonii cells forming biofilms in vivo on heart valves
(15) or in vitro on sHA (17) induced genes associated with
beta-glucoside metabolism at unexpected frequencies.
To survive in vivo and colonize as a biofilm, S. gordonii must
seek a carbon source for growth. Available nutrients, especially
sugars, will differ from those found in laboratory growth media.
Due to variations in available sugars in nature, bacteria have
developed highly controlled sugar metabolism systems. If mul-
tiple sugars exist in the environment, bacteria would first con-
sume primary sugars, such as glucose. When primary sugars
are exhausted or unavailable, bacteria induce genes for metab-
olizing secondary sugars (29), including beta-glucosides. Ex-
pression of these genes is often subject to carbon catabolite
repression by primary sugars (32). Exceptions exist. For exam-
ple, S. mutans has two systems to metabolize beta-glucoside
sugars (3–5). One system is inhibited by glucose, while the
other is not.
Induced in vivo during experimental endocarditis (15) and in
vitro during biofilm formation on sHA (17), and also required
for biofilm formation on plastic microtiter plates (44), four
novel transport and metabolism systems for beta-glucosides
have been identified. Three are PTS, and the fourth is a bind-
ing protein-dependent sugar uptake system for metabolizing
multiple sugars including beta-glucosides. Because each of
these systems consists of multiple transcriptional units, they
appear to organize and function as regulons, rather than oper-
ons.
The first putative regulon, bgl, has five genes in two tran-
scriptional groups. The first group encodes two putative tran-
scriptional regulators. The second group encodes three sub-
units of a PTS enzyme II permease. Since the bgl locus does
not have a beta-glucosidase gene, this gene may be at a sepa-
rate locus. The bfr locus has a gene encoding a beta-glucosi-
dase (BglF). Whether this gene is related to the bgl regulon is
VOL. 186, 2004 S. GORDONII BETA-GLUCOSIDE METABOLISM 4251

TABLE 2. Phenotypes of various S. gordonii beta-glucoside metabolism-related mutants

Physiological functiona Carbohydrate fermentationd Esculin hydrolysise Induction f
Strain (genotype)
Adhesion Biofilm Growth rate Arbutin Salicin Man2 GlcN-Man ⫺Glu ⫹Glu Esculin Chito

V288 (wild type) 100 100 100 ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
KG212 (bglC-amy-cat) 72b 70 65 ⫹ ⫹ ⫹⫹⫹ ⫺ ⫺ ⫺
KG206 (escB1-amy-cat) 91 82 65 ⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹
MG1021 (escB2-amy-cat) 95 47 107 ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹
KG207 (escR-amy-cat) 50b 98 112 ⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹
KG210 (escA-amy-cat) 79b 78 109 ⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹
MG1006 (bfbF1) 113 23 95 ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺
MG1007 (bfbB) 84 30 49 ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺
MG2025 (bfbF2-amy-cat) 70 59 55 ⫹ ⫹ ⫹⫹⫹ ⫺ ⫺ ⫺
MG2026 (bfbR-amy-cat) 75 69 103 ⫹⫹⫹ ⫺ ⫺ ⫺
CG422 (gomG) 88 101 103 ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹
CG423 (bglJ) 88 103 119 ⫹ ⫹ ⫹⫹⫹ ⫹
CG427 (gomF) 97 101 97 ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹
CG480 (fucA) 97 99 99 ⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
CG482 (manB) 81 99 104 ⫹ ⫹ ⫺ ⫹ ⫹⫹⫹ ⫹⫹⫹
CG484 (gomH) 60b 98 100 ⫹ ⫹ ⫺ ⫹ ⫹⫹⫹ ⫹⫹⫹
CG486 (gomR) 108 96 102 ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
CG495 (⌬gom) 67c 101 114 ⫹ ⫹ ⫹⫹ ⫺ ⫺
CG532 (merR-amy-cat) 58 97 116 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
MG1015 (bfrA-amy-cat) 36 65 87 ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹
a
As percentage of the wild-type activity. Each value represents the average of three sample measurements. Note: several strains showed significant growth reduction,
which may have proportionally affected their adhesion and/or biofilm formation. The phenotypes of three mutants, MG2026 (bfbR), CG427 (gomF), and CG486 (gomR),
might have polar effects on their downstream genes (Fig. 1).
b
P ⬍ 0.05.
c
P ⬍ 0.001.
d
Fermentation of all other sugars tested, including cellobiose, was not different from that of the wild type. Fermentation results of the four listed sugars are shown.
Man2, ␣-1,6-D-mannobiose; GlcN-Man, GlcNAc-␤1-2 mannose. ⫹⫹⫹, ⫹⫹, and ⫹ indicate strong, medium, and weak positive results; ⫺ indicates a negative result.
e
Esculin hydrolysis was detected on the esculin ferrous salt agar with or without glucose (Glu).
f
Induction was performed on agar plates with 0.5% starch for detection of expression of the in-frame fused amy gene. Only strains constructed with the reporter
gene-fusion plasmid, pAK36, were tested. In addition to the five strains induced by esculin and chitin oligosaccharides, three additional strains from our previous study
(15), KG205 (iviE-amy-cat), KG208 (iviH-amy-cat), and KG211 (msrA-amy-cat), were also induced by the same two sugars.

unknown. The two different regulators may be responsible for
controlling the two separate loci. bglC is induced in vivo during
experimental endocarditis (15), but it is not induced by any of
the commercially available beta-glucoside sugars in vitro. Yet,
inactivation of bglC reduced the S. gordonii growth rate (Table
2) and its ability to hydrolyze arbutin and salicin. Nonetheless,
the reduction in adhesion to sHA and biofilm formation on
plastic might be associated with its defective growth.
The second putative regulon, esc, which is induced by the
beta-glucoside esculin and oligochitosaccharides, includes six
genes. Three genes encode proteins for transport (EscP), me-
tabolism (EscA), and regulation (EscR) of a PTS. Although
the functions of the remaining three genes (escB, escC, and
escD) are unknown, escB may encode a capsule (polygluta-
mate) synthase, because it is homologous to the capA gene of
other bacteria, especially group B streptococci. Three genes of
the regulon, escA, escR, and escB, are induced in vivo in the
heart during experimental endocarditis (15) and also were
induced in vitro by esculin and oligochitosaccharides (Fig. 2A).
Although repressed by glucose (Fig. 2B), induction was attrib-
utable to the specific presence of esculin rather than the ab-
sence of glucose in the esculin medium, because other beta-
glucosides, cellobiose, salicin, and arbutin did not induce genes
in this regulon. Hence, the sensor system shows considerable
specificity for esculin and mammalian cognate sugars.
When the wild-type S. gordonii strain grew in a glucose-
containing medium, IPS accumulated (data not shown). En-
dogenous amylase was induced when the cells were grown in a
medium containing both glucose and starch, and IPS was not
seen (Fig. 2C). When mutants defective in esculin-inducible
genes were grown in the presence of glucose and starch, how-
ever, intracellular ␣-amylase appeared to be repressed (35)
and IPS accumulated in the cells (Fig. 2B and C). How these
genes affect the accumulation of IPS in S. gordonii is unknown.
In S. mutans, the accumulation of IPS is regulated by the cell
wall synthesis-associated dlt operon (36). Similarly, esculin-
inducible genes may play a role in S. gordonii cell wall and/or
surface polysaccharide synthesis, because mutation in these
genes caused defects in in vitro adhesion to sHA, biofilm for-
mation on plastic, and/or the growth rate (Table 2).
The esc regulon may be highly conserved, because esc-like
regulons exist in many bacteria, including S. mutans (3). In
comparison with the S. mutans bgl (5), the S. gordonii esc also
has a RAT site upstream of escP, but it does not have a
licT-like gene nearby. Moreover, escB, the gene immediately
downstream of escP, is not homologous to S. mutans bglB.
Although both S. gordonii esc and S. mutans bgl are induced by
esculin, the former is repressed by glucose while the latter is
not (4). It is unknown why esculin and oligochitosaccharides
also induce several other genes, including msrA. Interestingly,
when S. gordonii is shifted from the oral pH (slightly acidic) to
blood pH (neutral), both msrA and escR (previous name,
SGp1224) are induced (42).
The third putative regulon, bfb, encodes proteins apparently
involved in biofilm-associated beta-glucoside metabolism. It
includes seven ORFs, which encode four subunits (BfbA, -B,
-C, and -D) of a PTS enzyme II permease, a phospho-beta-
glucosidase, and a bglG-like antiterminator. Although unin-
4252 KILIÇ ET AL.
duced in vivo during experimental endocarditis, this regulon
was induced during in vitro S. gordonii biofilm formation on
sHA. As expected, mutants with mutations in several bfb genes
showed reduced biofilm formation on plastic in vitro. Likewise,
a fructose PTS operon has been recently linked to S. gordonii
biofilm formation (20).
The fourth putative regulon, gom, is for glycoprotein-derived
oligosaccharide metabolism. It includes 15 genes encoding a
binding protein-dependent sugar transport module, a two-com-
ponent system, a regulator, and five sugar-degrading enzymes
(a fucosidase, two mannosidases, an N-acetyl-beta-hexosamini-
dase, and a beta-glucosidase). The regulon may encode a bind-
ing protein-dependent system for transport and the metabo-
lism of multiple sugars, like the msm system in S. mutans (31,
39). However, an msmK-like gene encoding an ATP-binding
protein is absent in the gom locus. It may be located elsewhere,
as is the case in Streptococcus equisimilis (24). Since the sub-
strate sugars, such as fucosides, mannosides, N-acetyl-beta-
hexosamines, and beta-glucosides, are commonly found in
mammalian glycoprotein-derived oligosaccharides, it is not
surprising that this regulon was induced only in vivo in the
rabbit and not under in vitro growth conditions. Due to the
presence of a two-component regulatory system, gomH and
gomI, expression of the gom regulon may also be more sensi-
tive to environmental changes than other binding protein-de-
pendent sugar metabolism systems, such as the S. mutans msm
operon (39). The gomA gene was expressed in vivo and was an
insertion site of the IVET plasmid pAK36, but its function is
currently unknown. Mutants defective in manB, gomH, or gomR
showed reduced or negative fermentation of GlcNAc-␤1-2-
mannose and ␣1-6-D-mannobiose, but the wild-type strain fer-
mented both disaccharides. Hence GlcNAc-␤1-2-mannose and
␣1-6-D-mannobiose may be substrates of gom, which may be
the sole system for hydrolysis of the latter.
Controlling expression of the gom regulon, gomR may en-
code a positive transcriptional regulator, and gomH and gomI
may encode a two-component system. Both gomR and gomH
are downstream of manB (␣1-6 mannosidase), and the gomR
and gomH mutants were defective in fermentation of ␣1-6-
D-mannobiose. This suggested that the gom regulon for the
metabolism of ␣1-6-D-mannobiose was under the control of
the two-component system. Moreover, the gom regulon may
also control, in part, cell wall biosynthesis, since it contains
glmS, which encodes glucosamine-6-phosphate synthase (9). It
catalyzes the first step of N-acetylglucosamine biosynthesis,
providing a critical constituent for cell wall peptidoglycan. A
glmS-related gene, glmM (phosphoglucosamine mutase), in
S. gordonii has been associated with the biofilm phenotype
(18). Since the gom locus deletion, gomH, and manB mutants
showed reduced adhesion to sHA (Table 2), the putative gom
regulon may contribute to the integrity of the cell wall and the
fidelity of expression of surface adhesin molecules.
Except for the gom regulon, pathways for beta-glucoside
metabolism in S. gordonii may be inhibited by the presence of
the catabolite, glucose. In the presence of glucose, esculin hy-
drolysis was repressed in the gom deletion mutant (data not
shown). Therefore, other pathways for beta-glucoside metab-
olism appear to be sensitive to catabolite repression by glucose.
A post hoc analysis, however, shows that about one-third of the
reporter gene-fusion strains constructed by random-site pAK36
J. BACTERIOL.
insertion showed complete glucose-mediated catabolite re-
pression. The reporter amy gene fused in frame with any gene
may enable the expressed amylase to hydrolyze intracellular
glycogen to release glucose (35). The internally released glu-
cose may in turn repress beta-glucoside regulons. This explains
why all amy-cat report gene-fusion strains isolated by induction
(KG201 to -213) displayed glucose-mediated inhibition of es-
culin hydrolysis, while strains isolated due to defects in biofilm
formation (MG1006 and MG1007) did not, probably because
the reporter amy gene in these mutants was inserted off frame
and thus was not expressed (Table 2). Since wild-type S. gor-
donii showed esculin hydrolysis in the presence of glucose, at
least one regulon, gom, is not subject to catabolite repression
by glucose. Internally released glucose also appears to be a
potent catabolite repressor. It can repress multiple regulons
for beta-glucoside metabolism. Arbutin and esculin were not
fermented by the amy-cat reporter gene-fusion strains,
MG1015 (bfr) and CG532 (merA), in the presence or absence
of glucose. In wild-type cells, therefore, conditions favoring
hydrolysis of stored polysaccharides may promote strong ca-
tabolite repression of several beta-glucoside regulons as an
important mechanism for survival during sugar starvation.
Based on currently available genomic data published by
TIGR, S. gordonii has four different beta-glucosidases. Phylo-
genetic analysis showed that the S. gordonii beta-glucosidases
of the three PTS are related, but the one in the gom regulon,
bglJ, is relatively different. The four different beta-glucosidases
do differ more in amino acid sequences from one another,
however, than from their respective homologues in other spe-
cies, reflecting different conserved functions and substrate
specificities. Because mammalian-derived beta-glucosides are
unknown, we tested multiple commercially available beta-glu-
coside sugars, cellobiose, arbutin, salicin, esculin, and oligochi-
tosaccharides, to identify possible preferred substrates for each
system. Only the esc regulon was induced by esculin and oli-
gochitosaccharides. The several beta-glucoside-related genes
expressed specifically in vivo may require natural substrates
that are embedded in the mammalian oligosaccharides on the
heart valve. Interestingly, the bfb genes were not induced in
vivo but were induced during biofilm formation on sHA. This
suggested that bfb may have a different function from the three
other regulons. Since Streptococcus exopolysaccharide has be-
ta-linked repeating disaccharides (7, 43) and its capsule resem-
bles a typical GAG, hyaluronan (34), the expression of these
genes in S. gordonii may be needed for the synthesis, remod-
eling, and/or recycling of cell surface adhesins (glycoproteins)
and/or capsule-like polysaccharides.
The amy gene as a reporter might have introduced artifact
due to possible amylase degradation of intracellular glycogen.
There might be still-other-undetected artifacts, but the isola-
tion of beta-glucoside genes could not be attributed to one of
them because both the in vivo and in vitro selections were done
by the induction of cat gene expression (chloramphenicol re-
sistance). Moreover, Gahan and Hill (10), using listerolysin as
an IVET reporter, have also identified an in vivo-induced gene
encoding beta-glucoside permease in Listeria.
In summary, four different beta-glucoside metabolic systems
have been identified in S. gordonii. These systems are induced
in vivo and/or contribute to in vitro adhesion and biofilm for-
mation. Not only could they metabolize sugars to provide en-
VOL. 186, 2004
ergy and a carbon source for S. gordonii survival in the host, but
also they may be involved in other physiological processes,
such as the synthesis of cell surface glycoproteins and/or poly-
saccharides involved in adhesion and biofilm formation. To
understand their preferred substrates and potential roles in
streptococcal virulence, further studies of these systems will be
necessary.
ACKNOWLEDGMENTS
This work was supported by U.S. Public Health Service grants
DE08590 and DE11400.
We thank TIGR for its publication of the partially completed
S. gordonii genomic sequence.
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