Biology Reference
In-Depth Information
scheme was applied such that the total spectral counts or total intensities for all
P.
gingivalis
proteins in each condition were set equal for each comparison. This normal-
ization also had the effect of zero centering the log
2
transformed relative abundance
ratios, see Figure 2. The normalized data for each abundance ratio comparison was
tested for significance using either a global G-test or a global paired t-test for each
condition, the details of which have been published for this type of proteomics data in
which all biological replicates are compared against each other [55, 56], and are also
described in the explanatory notes. Both of these testing procedures weigh deviation
from the null hypothesis of zero abundance change and random scatter in the data
to derive a probability or
p
-value that the observed change is a random event, that is
the null hypothesis of no abundance change is true. Each hypothesis test generated a
p
-value that in turn was used to generate a
q
-value as described [24, 32], using the R
package QVALUE [26]. The
q
-value in this context is a measure of quantitative FDR
[25] that contains a correction for multiple hypothesis testing.
Ontology Analysis
An overall list of detected proteins as well as lists of proteins that showed increased
or decreased levels in the three species community were prepared using
Entrez
gene
identifiers. Ontology analyses were then conducted using the DAVID [57] functional
annotation clustering feature with the default databases. Both increased and decreased
protein level lists were analyzed using the overall list of detected proteins as the back-
ground. Potentially interesting clusters identified by DAVID were then examined
manually.
Construction of
P. gingivalis
HmuR
Mutant
A mutation in the
hmuR
gene was generated using ligation-independent cloning of
PCR mediated mutagenesis (LIC-PCR) [58]. A 2.1-kb ermF-ermAM cassette was in-
troduced into the
hmuR
gene by three steps of PCR to yield a hmuR-erm-hmuR DNA
fragment as described previously [59]. The fragment was then introduced into
P. gin-
givalis
33,277 by electroporation. The hmuR deficient mutant (ΔhmuR) was generated
via a double crossover event that replaces hmuR with the hmuR-erm-hmuR DNA frag-
ment in the 33,277 chromosome. The mutants were selected on TSB plates containing
erythromycin (5 μg/ml), and the mutation was confirmed by PCR analysis. Growth
rates of mutant and parent strains were equivalent.
Quantitative Community Development Assays
(i) Crystal violet assay. Homotypic community formation by
P. gingivalis
was quanti-
fied by a microtiter plate assay [60], as adapted for
P. gingivalis
[61]. Parental and
mutant strains in early log phase (2 × 10
8
cells) were incubated at 37°C anaerobically
for 24 hr. Wells were washed, stained with 1% crystal violet and destained with 95%
ethanol. Absorbance at 595 nm was determined in a Benchmark microplate reader. (ii)
ELISA.
F. nucleatum
was incubated at 37°C anaerobically for 36 hr in microtiter plate
wells. After washing, parental and mutant
P. gingivalis
strains (2 × 10
6
cells) were incu-
bated with the fusobacterial biofilm at 37°C anaerobically for 24 hr.
Porphyromonas
gingivalis
accumulation was detected with antibodies to whole cells (1:10,000)
