Media 1: Architecture and applications of a high resolution gated SPAD image sensor

Originally published in Optics Express on 14 July 2014 (oe-22-14-17573

SPβ genes required for prophage excision.

<p>(A) Chromosomal DNA from the vegetative (T₋₁) and the sporulating cells (T₈) of strain 168 (WT), SPRAd (<i>sprA</i>), and SPRBd (<i>sprB</i>) were digested with <i>Nde</i>I and subjected to Southern blotting. The genetic maps of SPRAd and SPRBd were shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004636#pgen.1004636.s002" target="_blank">Figure S2</a>. (B) The schematic shows construct of the SPmini strain. Thick lines indicate <i>sprA</i> and <i>ypqP</i> probes for Southern blotting. <i>Bgl</i> denotes <i>Bgl</i>II restriction sites. (C) Southern blotting. Chromosomal DNA was isolated from vegetative (left panels, T₋₁) and sporulating cells (left panels, T₈) in the DSM culture and from the SPmini vegetative cells (OD₆₀₀ = 0.25) grown in LB with or without MMC treatment (0.5 µg/ml) at 37°C for 60 min (right panels). DNA was digested with <i>Bgl</i>II and subjected to Southern blotting using the <i>sprA</i> and the <i>ypqP</i> probes.</p

Spore properties.

<p>(A) Adhesion of the mutant and wild-type spores to glass tubes. The spores purified from strain 168 (WT), SPRAd (<i>sprA</i>), YODUd (<i>yodU</i>), SPRAc (<i>sprA spsM</i>⁺), and YODUc (<i>yodU spsM</i>⁺) were resuspended in water and the final OD₆₀₀ was adjusted to 15. Each 30 µl of spore resuspension was added to a Pyrex tube (13×100 mm, Corning) and vortexed gently for 30 s. After removing the spore resuspensions, the glass tubes were briefly dried and images were acquired. (B) Adhesion of the mutant and wild-type spores to polypropylene tubes. Adhesion (%) was determined by 10 successive binding reactions of the spores to the tubes. Error bars indicate ± standard deviations based on three independent experiments. (C) The polysaccharide layer facilitates spore dispersal through water flow. Overnight cultures of <i>B. subtilis</i> cells grown in LB medium were spotted onto DSM-agar plates. The plates were incubated at 37°C for 1 week. Each colony was confirmed as containing>95% free spores using phase-contrast microscopy. The images show the spore colonies on the DSM plates before (upper panels) and after rinsing with 1 ml of DDW (lower panels). The wild-type spores on the plates were dispersed by water, whereas the mutant spores stuck to the plates.</p

Model of the phage-mediated DNA rearrangement.

<p>(A) A model of the sporulation-specific phage-mediated gene rearrangement, based on the cases of SPβ in <i>B. subtilis</i> and <i>B. amyloliquefaciens</i>. (B) Maintenance of the intervening element in the host genome. Sporulation gene (<i>spo</i> gene), black box; attachment sites, triangle; intervening element, red line; <i>sprA</i> and <i>sprB</i>, red arrow; phage-related genes, red box; host genes, open box.</p

Analysis of <i>B. subtilis</i> spore surface components.

<p>(A) Negative staining with Indian ink of the <i>B. subtilis</i> wild-type and mutants. The purified spores from strain 168 (WT), SPRAd (<i>sprA</i>), YODUd (<i>yodU</i>), SPRAc (<i>sprA spsM</i>⁺), and YODUc (<i>yodU spsM</i>⁺) were negatively stained with Indian ink and observed using phase-contrast microscopy. Untreated, native spores; boiled, heat-treated spores at 98°C 10 min in SDS buffer. Scale bars, 4 µm. (B) Electrophoresis of <i>B. subtilis</i> spore surface extracts. Spore surface extracts from strain 168 (WT), SPRAd (<i>sprA</i>), YODUd (<i>yodU</i>), SPRAc (<i>sprA spsM</i>⁺), and YODUc (<i>yodU spsM</i>⁺) were loaded onto a 5% native polyacrylamide gel. The gel was stained with Stains-All after electrophoresis. (C) Quantification of the polysaccharides in spore surface extracts. The spore surface polysaccharides from <i>B. subtilis</i> spores were ethanol-precipitated. The precipitants were dissolved in water and reacted with Stains-All. The amounts of polysaccharides were determined by measuring the OD₆₄₀ according to the method described by Hammerschmidt et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004636#pgen.1004636-Hammerschmidt1" target="_blank">[32]</a>. Error bars indicate ± standard deviations based on three independent experiments.</p

DNA rearrangement of <i>spsM</i> in <i>B. amyloliquefaciens</i>.

<p>(A) Schematic representation of the gene organization of SPβ-like elements at the <i>spsM</i> locus of <i>B. amyloliquefaciens</i> strains. Eight <i>B. amyloliquefaciens</i> strains with genome sequences deposited in KEGG are shown here as representative examples. The <i>yodU</i> and <i>ypqP</i> ORFs are located at the left and right ends, respectively. The red arrows indicate <i>sprA</i> and <i>sprB</i>, which are required for SPβ excision. The size (kb) of the element and number of genes in the element are shown above the diagram. The red and green boxes indicate SPβ-related and non-SPβ-related genes, respectively. The conserved SPβ genes in <i>B. amyloliquefaciens</i> strains are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004636#pgen.1004636.s007" target="_blank">Table S1</a>. (B) Diagram of SPβ-like element excision in <i>B. amyloliquefaciens</i> FZB42. The thick lines indicate the DIG-labeled probes used for Southern blotting. <i>Eco</i> indicates <i>Eco</i>RV sites. Triangles point to the attachment sites for SPβ. (C) DNA rearrangement of <i>spsM</i> in <i>B. amyloliquefaciens</i> FZB42. <i>B. amyloliquefaciens</i> FZB42 cells were cultured at 37°C in DSM medium. Chromosomal DNA samples from the cells in the vegetative (T₋₁) and sporulation phases (T₈) were digested with <i>Eco</i>RV and subjected to Southern blotting. The <i>sprA_Bam</i> and <i>ypqP_Bam</i> probes were specific to <i>B. amyloliquefaciens sprA</i> and <i>ypqP</i>, respectively.</p

DNA rearrangement at the <i>spsM</i> locus.

<p>(A) Diagram showing SPβ excision in <i>Bacillus subtilis</i> 168. The thick lines indicate the location of the digoxigenin (DIG)-labeled probes used for Southern blotting. <i>Nde</i> indicates <i>Nde</i>I sites. Triangles point to the attachment sites for SPβ. (B) SPβ excision upon mitomycin C (MMC) treatment and during sporulation. Left panel shows induction of SPβ excision by MMC treatment. <i>B. subtilis</i> 168 cells were grown in LB medium. Vegetative cells in the early log phase (OD₆₀₀ = 0.25) were treated with 0.5 µg/ml MMC. Time 0 indicates the time point immediately after MMC addition. Right panels show SPβ excision (top panel) and <i>spsM</i> reconstitution (bottom panel) during sporulation. <i>B. subtili</i>s 168 cells were grown in DSM, and samples were taken at the indicated times (in h) after the onset of sporulation (T₀). The DNA samples were digested with <i>Nde</i>I and subjected to Southern blotting. (C) Mother cell-specific SPβ excision. Chromosomal DNA from the vegetative cells (V) at T₋₁, whole sporangia (W) at T₈, and the forespores (FS) at T₈ were isolated, digested with <i>Nde</i>I, and subjected to Southern blotting. (D) Lytic activity of SPβ phages. SPβ phage lysate, which was prepared by treating the <i>B. subtilis</i> 168 vegetative cells with MMC, was spotted on the plate (MMC). The DSM culture of <i>B. subtilis</i> 168 at T₆, T₁₂, T₂₄, and T₄₈ was centrifuged and the supernatant was filtrated with 0.44 µm Millex filter (Millipore). The filtrate was spotted on the lawn of a SPβ sensitive strain CU1050 (DSM T₂₄ and DSM T₄₈). (E) Horizontal transfer of <i>spsM</i> rearrangement system. A new SPβ-lysogen, CU1050 (SPβ) was obtained by infecting CU1050 cells with the SPβ phage lysate. The CU1050 and CU1050 (SPβ) cells were induced to sporulate on DSM-agar plates at 37°C for 3 (Vegetative cells, Veg) and 12 hours (Sporulating cells, Spo). Chromosomal DNA of the CU1050 and CU1050 (SPβ) cells was subjected to Southern blotting.</p

Mother cell-specific expression of <i>sprB</i> during sporulation.

<p>(A) Genetic organization of the <i>sprB</i> region. The black and red promoter symbols indicate the promoter upstream of <i>yosX</i> and the mother cell-specific promoter directly upstream of <i>sprB</i>, respectively. The thick black line indicates the <i>sprB</i> probe for Northern blotting. The wavy lines indicate the <i>sprB</i> transcripts with their respective lengths (kb). The red and black arrows indicate the <i>sprB</i>-specific primer for reverse transcription (RT primer) and the <i>yosX</i>-, <i>yotB</i>C<i>D</i>-, and <i>sprB</i>-specific primers for the PCR reactions, respectively. The gray lines show the products of RT followed by PCR amplification. (B) Nucleotide sequence of the <i>sprB</i> promoter region. The transcriptional start site (TSS) of <i>sprB</i> is indicated by the red arrow. Boxes indicate −35 and −10 elements of the <i>sprB</i> promoter. The consensus sequences for σ^E and σ^K binding are shown below (K = G or T; N = A, T, G, or T). (C) Northern blotting. Total RNA was isolated from <i>B. subtilis</i> 168 vegetative cells treated with (+) or without (−) 0.5 µg/ml MMC at 37°C for 60 min and from sporulating cells 4 hours after onset of the sporulation (T₄). The RNA samples were subjected to Northern blotting using the <i>sprB</i> probe. The bottom panel shows methylene blue-stained 16S rRNA as a loading control. (D) RT-PCR. The <i>sprB</i> cDNA was synthesized using the <i>sprB</i>-specific primer (Figure 5A, the red arrow RT primer) and total RNA from the <i>B. subtilis</i> 168 vegetative cells treated with (+) or without (−) MMC and from sporulating cells (T₄). Internal regions of the cDNA were amplified with the <i>yosX</i>-, <i>yotB</i>C<i>D</i>-, and <i>sprB</i>-specific primer sets. The PCR product was analyzed by 2% agarose gel electrophoresis. (E) Compartmentalization of SprB–GFP expression. BsSPRBG, carrying the <i>sprB</i>–<i>gfp</i> fusion gene under the control of the mother cell specific <i>sprB</i> promoter, was cultured at 37°C in liquid DSM containing FM4-64 (0.25 µg/ml) and kanamycin (10 µg/ml). Sporulating cells at T₄ were observed using fluorescence microscopy. PC, phase contrast; Membrane, cell membranes stained with FM4-64; GFP, GFP fluorescence; Merge, merged images of Membrane and GFP. Scale bar, 2 µm.</p

Table_1_Integrated analysis of the oral and intestinal microbiome and metabolome of elderly people with more than 26 original teeth: a pilot study.XLSX

Elderly subjects with more than 20 natural teeth have a higher healthy life expectancy than those with few or no teeth. The oral microbiome and its metabolome are associated with oral health, and they are also associated with systemic health via the oral-gut axis. Here, we analyzed the oral and gut microbiome and metabolome profiles of elderly subjects with more than 26 natural teeth. Salivary samples collected as mouth-rinsed water and fecal samples were obtained from 22 healthy individuals, 10 elderly individuals with more than 26 natural teeth and 24 subjects with periodontal disease. The oral microbiome and metabolome profiles of elderly individuals resembled those of subjects with periodontal disease, with the metabolome showing a more substantial differential abundance of components. Despite the distinct oral metabolome profiles, there was no differential abundance of components in the gut microbiome and metabolomes, except for enrichment of short-chain fatty acids in elderly subjects. Finally, to investigate the relationship between the oral and gut microbiome and metabolome, we analyzed bacterial coexistence in the oral cavity and gut and analyzed the correlation of metabolite levels between the oral cavity and gut. However, there were few associations between oral and gut for bacteria and metabolites in either elderly or healthy subjects. Overall, these results indicate distinct oral microbiome and metabolome profiles, as well as the lack of an oral-gut axis in elderly subjects with a high number of natural teeth.</p