Figure 1.
Continuous culture of P. gingivalis and T. denticola mono- and co-cultures.
Cell density of P. gingivalis and T. denticola mono- and co-cultures from three independent continuous cultures in OBGM with the dilution rate of 0.044 h−1 and mean generation time of 15.8 h as determined by measuring A650 nm. The arrow shows the addition of P. gingivalis to a steady state T. denticola culture.
Figure 2.
Scanning electron micrograph of P. gingivalis and T. denticola grown in continuous culture.
Co-culture was collected, fixed on a coverslip, dehydrated, covered with colloidal silver, gold-coated and imaged using a Philips XL30 field-emission scanning electron microscope. Electron micrographs showed that P. gingivalis and T. denticola coaggregated. T. denticola is a long helical shaped spirochete with an average length of 5 to 20 µm. P. gingivalis is a coccobacillus with an average diameter of 1 µm. Putative T. denticola outer sheath vesicles and/or P. gingivalis outer membrane vesicles are indicated by arrows along the length of T. denticola.
Table 1.
T. denticola and P. gingivalis genes differentially expressed during continuous co-culture relative to mono-culture, grouped by COG category.
Table 2.
Expression of T. denticola genes encoding enzymes involved in glycine or glycine-related metabolism during co-culture with P. gingivalis.
Figure 3.
T. denticola growth and glycine.
(a) The concentration of free glycine in T. denticola culture (black square; left axis). T. denticola growth curve in the same medium (black inverted triangle; right axis). Data points are the mean and standard deviation of three biological replicates. (b) Glycine (10 mM) was added to OBGM either before inoculation with T. denticola (black square, open arrow) or at 96 h after inoculation (black triangle, filled arrow) and bacterial growth was determined by A650 nm measurement. T. denticola culture with no added glycine (white circle). Results are expressed as mean ± standard deviation obtained from eight replicates.
Figure 4.
The extracellular products of T. denticola [U-13C]glycine fermentation.
[U-13C]glycine (5.00 mM) was added to a 24 h T. denticola culture and aliquots were collected every 24 h, filtered and the identity and the quantity of the 13C-labeled compounds was determined using NMR spectroscopy. black cross, [U-13C]glycine; black circle, [U-13C]acetate; black inverted triangle, dual or uniformly-labeled lactate; black triangle, [U-13C]bicarbonate; white circle, dual or uniformly-labeled alanine.
Figure 5.
P. gingivalis cell numbers and free glycine content in different cultures.
a) The cell numbers of P. gingivalis in different media as determined by absorbance at 650 nm. b) The concentration of free glycine in different P. gingivalis cultures over time, as determined by GC-MS. Data shown are the average of three biological replicates. P. gingivalis grown in:- OBGM – black diamond; OBGM/PBS – black cross; OBGM/T. denticola conditioned medium – white square. Uninoculated OBGM/T. denticola conditioned medium – black square.
Figure 6.
Free glycine production during P. gingivalis growth.
The difference in the amount of free glycine relative to that at t = 0 h as a function of P. gingivalis cell numbers in a) OBGM/PBS, b) OBGM and c) OBGM/T. denticola conditioned medium. A regression line was fitted using a linear mixed modelling approach. The slope represents the amount of glycine produced/109 P. gingivalis cells.
Figure 7.
Excess thiamine pyrophosphate production by P. gingivalis.
The E. coli TPP auxotrophic strain JW3957-1 (black shading) and the parent strain JRG902 (white shading) were cultured in M9 growth medium (that lacks thiamine) supplemented with either (a) uninoculated P. gingivalis medium (that lacks thiamine) or (b) cell-free P. gingivalis spent medium. The bacterium was cultured with or without TPP addition (5.88 nM).
Figure 8.
Proposed T. denticola glycine catabolic pathways.
Glycine can be oxidized by the glycine cleavage system (1), producing NH3, CO2 and CH2-THF. Glycine and CH2-THF can be condensed to form serine by serine hydroxymethyltransferase (2). Serine is deaminated to produce pyruvate by serine dehydratase (3). Lactate dehydrogenase (4) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. Pyruvate can also be metabolized to acetate by pyruvate-ferredoxin oxidoreductase (5), phosphate acetyltransferase (6) and acetate kinase (7). Glycine can also be reduced to acetyl-P by the glycine reductase system (8).