Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jul;17(7):1116-1127.
doi: 10.1038/s41396-023-01426-9. Epub 2023 May 11.

Glycerol metabolism supports oral commensal interactions

Puthayalai Treerat  1 , David Anderson  2 , Rodrigo A Giacaman  3 , Justin Merritt  2   4 , Jens Kreth  5   6
Affiliations

Glycerol metabolism supports oral commensal interactions

Puthayalai Treerat et al. ISME J. 2023 Jul.

Abstract

During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Interspecies interaction of S. sanguinis and C. durum on the phenotypic and genotypic level.
Representative immunofluorescence microphotographs of SK36 (GFP) cocultured with (A) DAPI or (B) CellTracker Red CMTPX fluorescent dye-labeled C. durum (Cd) in saliva supplemented with 100 mM glucose. Interspecies coaggregation was shown in the merged images by white arrows. Scale bars indicate A 20 μm and B 10 μm. C Volcano plot demonstrates the overall transcriptional changes across Cd supernatant-treated SK36 (red) in comparison to the medium control (blue) condition. Each data point represents one gene. The X-axis represents the log2 fold change of each gene and the Y-axis represents the log10 of its adjusted p value. Genes with an adjusted p < 0.05 and log2 fold change > 1 (indicated in red dots) show upregulation. Genes with p < 0.05 and log2 fold change < −1 (indicated in blue dots) show downregulation. Hierarchical clustering analysis and heatmap of SK36 genes transcriptomic profiles responses to Cd supernatant in comparison to the medium control (n = 3). The top genes involved in D lipid and glycerol metabolism and E pilus biosynthesis were upregulated and downregulated, respectively, in the presence of Cd supernatants. The color gradient in D and E indicates the range of gene expression levels within a given sample. Red represents low expression levels, yellow intermediate, and green represents high expression levels.
Fig. 2
Fig. 2. Glycerol consumption of S. sanguinis.
A Total glycerol production of Cd and the unique ability of S. sanguinis SK36, SK408, SK1056, and VMC66 strains to utilize the glycerol being produced by Cd. Cd was cultured in BHI overnight and its supernatant was collected and filtered through 0.45 μm membrane. SK36 was then cultured in the Cd supernatant overnight. Both samples were subsequently measured for total glycerol concentration. Results shown are the average of biological triplicates. Error bars represent the standard deviation. B Ribbon diagram of SK36 glycerol kinase (GlpK) with the β-strands shown as arrowed ribbons, α-helices, and the connecting loops. Colors indicate the N-terminus (blue) to the C-terminus (red). The histidine 232 (H232) phosphorylation site is marked in magenta.
Fig. 3
Fig. 3. Effect of glycerol on SK36 chain morphological alteration via the glpK metabolic pathway.
SK36 wild-type (SK36), ΔglpK, the complemented mutant (ΔglpKc), and its single point mutant H232E (glpKHE) strains were treated with the–Glycerol and + Glycerol conditions. A Chain morphologies of the SK36, ΔglpK, glpKHE, and ΔglpKc were then examined and imaged using an Olympus IX73 inverted microscope. The pictures are representative of three independent experiments. Scale bars indicate 10 μm. B For quantification, bacterial chain length was measured from 100 bacterial chains per biological replicate using ImageJ software. Results shown are the average of biological triplicates. Error bars represent the standard deviation.
Fig. 4
Fig. 4. Total glycerol production under the influence of glucose.
SK36, ΔglpK, and the complemented mutant (ΔglpKc) were cultured overnight in BHI with glucose (+ Glucose) compared to BHI without glucose (− Glucose). The supernatants were collected, filtered through a 0.45 μm membrane, and then measured for total glycerol concentration (mM). Results shown are the average of biological triplicates. Error bars represent the standard deviation.
Fig. 5
Fig. 5. Effect of glpK in bacteria-phagocytic interactions.
Bacterial uptake was determined by challenging murine macrophage-like RAW 264.7 cells with the wild-type SK36, ΔglpK, and the complemented ΔglpK strains with (+ Cd) and without (− Cd) Cd. The number of internalized bacteria (CFU/ml) in RAW 264.7 cells then enumerated. Data are presented as the means of biological triplicates. Error bars denote standard deviations.
Fig. 6
Fig. 6. Differences in surface twitching/swarming motility phenotypes of S. sanguinis SK strains under the influence of Cd.
All S. sanguinis SK strains were prepared and spotted on 1% agar plates containing (B) Cd supernatant (+ Cd) in comparison to (A) BHI control (− Cd) plates. C Two key SK strains (SK36 and SK408) were further investigated twitching/swarming motility phenotypes on 1% agar plates supplemented with 5% defibrinated sheep blood. The plates were then incubated anaerobically. Images are representative of three independent experiments. White asterisks indicate a visible halo or hazy zone of bacteria that have twitched/swarmed across the plate. Full images of the plates are exhibited in Supplementary Fig. S5.
Fig. 7
Fig. 7. Comparison of type IV pilus gene expression in S. sanguinis SK strains ± Cd.
A Single-nucleotide polymorphism (SNP)-based neighbor-joining phylogram illustrating variations within core gene clusters that lack paralogs (modified from [54]). Core pilus genes of the selected SK strains (**) with their gene IDs and sequence identities (%) were listed in Supplementary Table 4. Relative core type IV pilus gene expression (pilE2, pilE1, pilT, and pilB) in B SK36, C SK408, D SK1056, and E VMC66 strains in the presence (+ Cd) versus the medium control (− Cd). Gene expression data are presented relative to the medium control (− Cd), which was arbitrarily assigned a value of 1. Data represent the means of biological triplicates. Error bars denote standard deviations.
Fig. 8
Fig. 8. Scanning electron micrographs (SEM) of type IV pilus morphology in the SK strains.
(Top left) SK36 and (Top right) SK408 single species cultures (− Cd). (Bottom left) SK36 and (Bottom right) SK408 dual species cocultures with Cd (+ Cd). Top left scale bar indicates 1 µm. Top right scale bar indicates 2 µm. Bottom left and right scale bars indicate 1 µm. White arrows indicate putative pili. The pictures are representative of three independent experiments. Full images are exhibited in Supplementary Fig. S6A.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Colwell RK, Rangel TF. Hutchinson’s duality: the once and future niche. Proc Natl Acad Sci USA. 2009;106:19651–58. doi: 10.1073/pnas.0901650106. - DOI - PMC - PubMed
    1. Kreth J, Merritt J, Qi F, Dong X, Shi W. Antagonistic, synergistic, and counteroffensive strategies for streptococcal interspecies interactions. In: Kolenbrander PE, editor. Oral microbial communities: genomic inquiry and interspecies communication. Washington, DC: ASM Press; 2011. p. 331–44.
    1. Kreth J, Merritt J, Shi W, Qi F. Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species. Mol Microbiol. 2005;57:392–404. doi: 10.1111/j.1365-2958.2005.04695.x. - DOI - PMC - PubMed
    1. Roberts AP, Kreth J. The impact of horizontal gene transfer on the adaptive ability of the human oral microbiome. Front Cell Infect Microbiol. 2014;4:124. doi: 10.3389/fcimb.2014.00124. - DOI - PMC - PubMed
    1. Ona L, Giri S, Avermann N, Kreienbaum M, Thormann KM, Kost C. Obligate cross-feeding expands the metabolic niche of bacteria. Nat Ecol Evol. 2021;5:1224–32. doi: 10.1038/s41559-021-01505-0. - DOI - PubMed

Publication types