Biology Reference
In-Depth Information
Only two stress proteins showed increased abundance, and then only 30% increas-
es, the molecular chaperone DnaK (PGN1208) and a PhoH family protein possibly
involved in oxidation protection (PGN0090).
Role of the Differentially Regulated
P. gingivalis
Protein HmuR
To begin to test the functional relevance of proteins identified as differentially regu-
lated in the three species community, we undertook a mutational analysis. For this
purpose it was important to target a protein that directly effectuates a biological func-
tion and lacks homologs in the genome. The hmuR, a major hemin uptake protein,
and potential adhesin [42], was selected. As shown in Figure 7A, while wild type
P.
gingivalis
cells are abundant within a
S. gordonii
-
F. nucleatum-P. gingivalis
commu-
nity,
P. gingivalis
cells lacking hmuR are deficient in community formation. Biovol-
ume analysis showed a 70% reduction in community formation by the hmuR mutant
(Figure 7C). Furthermore, this effect was specific for the three species community as
a decrease in accumulation by the hmuR deficient mutant was not observed in mono-
species biofilms, or in two species communities of
P. gingivalis
with either
S. gordonii
or
F. nucleatum
(Figure 7B, D-G). Hence loss of hmuR, that is up-regulated by
P.
gingivalis
when the organism is associated with
S. gordonii
and
F. nucleatum
, results
in a phenotype that is restricted to three species community formation.
Porphyromonas
gingivalis
cells were first cultured in hemin excess, under which conditions the hmu
operon is expressed at a basal level [42]. As the three species model system involves
metabolically quiescent
P. gingivalis
cells in buffer, it is unlikely that the role of hmuR
is related to its hemin uptake capacity. However, TonB dependent receptors can exhibit
functions distinct from transport across the outer membrane. For example, in
E. coli
the TonB dependent catecholate siderophore receptor Iha confers an adhesin function
and contributes to colonization and virulence in the mouse urinary tract [43]. Hence,
hmuR may have a cohesive function in community formation by
P. gingivalis
although
further studies are necessary to resolve this issue.
MATERIALS AND METHODS
Bacteria and Culture Conditions
Fusobacterium nucleatum
subsp. nucleatum American type culture collection (ATCC)
25586 and
Porphyromonas gingivalis
ATCC 33277 were grown anaerobically (85%
N
2
, 10% H
2
, 5% CO
2
) at 37°C in trypticase soy broth supplemented with 1 mg/ml yeast
extract, 1 μg/ml menadione, and 5 μg/ml hemin (TSB).
Streptococcus gordonii
DL1
was grown anaerobically at 37°C in Todd-Hewitt broth (THB).
CHEMICALS
The HPLC grade acetonitrile was from Burdick & Jackson (Muskegon, MI, USA);
high purity acetic acid (99.99%), and ammonium acetate (99.99%), from Aldrich
(Milwaukee, WI, USA). High purity water was generated with a NANOpure UV sys-
tem (Barnstead, Dubuque, IA, USA).
