MOESM2 of The subway microbiome: seasonal dynamics and direct comparison of air and surface bacterial communities

Additional file 1: Table S1. Type of environment, latitude and longitude for all sampled stations. Table S2. Overview of all samples included in the analyses. Table S3. PCR program for 16S rRNA gene amplicon sequencing. Table S4. The best-fit models of qPCR 16S rRNA gene copies for air samples and surface samples. Table S5. Top 20 phyla, families, and genera and species in surface samples collected on kiosks, benches, and railings. Table S6. Random forest classification models of samples collected from different surface types. Figure S1. The significant predictors of qPCR 16S rRNA gene copy yields in air samples. Figure S2. The significant predictors of qPCR 16S rRNA gene copy yields in surface samples. Figure S3. Quality profile of filtered reads. Figure S4. Rarefaction curves with observed diversity and Shannon’s Diversity Index. Figure S5. A) Relative abundances of the top 15 phyla across the three surface types and seasons. B) Heatmap of most abundant families. Figure S6. Top 20 most important genera in random forest classification analysis of samples collected in different seasons. Figure S7. Top 20 most important genera in random forest classification analysis of air and surface samples. Figure S8. Interaction effect between temperature (°C) and air/surface in the linear model of Shannon’s diversity index. Figure S9. PCoA plot of Bray Curtis dissimilarity distances with the only significant predictor (surface type) from the PERMANOVA model that included only surface-specific predictors

Amino acid-induced morphogenesis requires mitochondrial oxidative phosphorylation.

A. ATP levels in macrocolonies (PMRCA18) formed 24 h after spotting cells on the indicated SXD medium (X = Asp, Arg, Orn, Pro, Am (ammonium sulfate) or Urea) incubated at 37 °C. The levels of ATP in three biological replicates normalized to total protein are plotted. The values from each biological replicate is the average of 2–3 technical replicates. Statistically significant changes in ATP levels, as compared to cells grown on Asp, are indicated (ave. ± CI; **, p value B. Uncoupling of mitochondria reduces amino acid-induced filamentation. Cells (PMRCA18) were spotted on SXD media (X = Arg, Orn or Pro) supplemented with indicated amount of methylene blue (MB); macrocolonies were grown at 37 °C and photographed after 24 h. C. Metabolic activity of C. albicans grown in the presence of inducing and non-inducing amino acids. Mitochondrial respiratory activity of macrocolonies (PMRCA18) grown on the indicated solid media was assessed using the TTC-overlay method (upper panel; representative of three independent assessments) and the reduction of the nonfluorescent resazurin dye by NADH or NADPH oxidoreductases (lower panel). Fluorescence signals plotted were from the average of four biological replicates (ave. ± CI, 95% CL).</p

Proline utilization is influenced by carbon source but not by NCR.

A. Filamentous growth is more robust at lower glucose level. Wildtype cells (PMRCA18) from overnight YPD liquid cultures were washed and then adjusted to OD600 of 8, 10 μl aliquots were spotted on media containing 10 mM of proline and the indicated levels of carbon source. Plates were incubated at 37 °C and photographed at 48 and 72 h. B. The rate of proline utilization and ATP levels are higher under glucose limited conditions. The levels of proline remaining in culture supernatants (blue bars, left axis) and ATP (red bars, right axis) of WT (PMRCA18) after 2 h of growth at 37 °C in the presence of different carbon source are indicated. Results shown are from 5 biological replicates (Ave. ± CI, 95% CL, p value PUT2/PUT2-HA) grown under identical conditions (inset). C. Respiratory growth predominates as glucose level decreases. WT cells were diluted to OD600 of 0.5 in pre-warmed synthetic proline media containing 10 mM proline (YNB+Pro+BCP) and the indicated levels of glucose with the initial pH adjusted to 6.0. Cultures were grown for 16 h under vigorous agitation at 37 °C prior to photographing the culture tubes. Glycerol was used as respiratory growth control. D. Put2 is highly expressed in cells grown in the presence of non-glucose carbon sources. Immunoblot analysis of cell extracts prepared from CFG185 (PUT2/PUT2-HA) cells grown in YPGlu (YP+2% glucose = YPD), YPGly (YP + 1% glycerol), YPLac (YP + 1% lactate), or Spider medium (with 1% mannitol) at 37 °C for the timepoints indicated. Cells were pre-grown in YPD and inoculated at an OD600 of 0.5. E. Proline utilization is insensitive to NCR. Immunoblot analysis of cell extracts prepared from CFG184 (gln3Δ/Δ gat1Δ/Δ PUT2/PUT2-HA) grown at 30 °C in SD0.2% medium, which contains 10 mM ammonium sulfate (Am) and 0.2% glucose, supplemented with 10 mM of the nitrogen sources and harvested at the timepoints as indicated. Cells were pre-grown in SD and cultured to log phase in SD0.2%.</p

Mitochondrial proline catabolism is required by <i>C</i>. <i>albicans</i> cells to escape murine macrophages.

A. Confocal immunofluorescence microscopy of C. albicans cells expressing Put2-HA (CFG185) in the phagosomes of RAW264.7 macrophages. Primary antibodies (rat anti-HA and rabbit anti-Lamp1) and secondary antibodies (Alexa Fluor 555 conjugated goat anti-rat antibody and Alexa Fluor 488 conjugated goat anti-rabbit) were used to visualize Put2 and the lysosomal compartment, respectively. Orthogonal view of merged channels is shown in the lower right panel. Scale bar = 10 μ. B. Proline catabolism is required for hyphal growth of C. albicans in macrophages. Wildtype (WT; SC5314), heat killed WT, put1-/- (CFG139), put2-/- (CFG207), put3-/- (CFG146), put1-/- put2-/- (CFG159) and CRISPR/Cas9 control strains CFG181 (pV1093) and CFG182 (pV1524) pre-grown in YPD and stained with FITC were co-cultured with RAW264.7 macrophages at a MOI of 3:1 (C:M). After 30 min, external non-phagocytosed cells were removed by washing, and the co-cultures were incubated an additional 4 h. External (escaping) hyphal cells were stained with calcofluor white (CFW). Scale bar = 10 μ. C. Macrophage killing of C. albicans. Strains as in B (not stained) were co-cultured with RAW264.7 at a MOI of 3:1 (C:M) for 3 h. After lysing macrophages, viability of C. albicans was assessed by quantitating the number of CFU. The percent killing was determined by comparison to the viability of cells grown in the absence of macrophages. D. put1-/- mutant is unable to escape the phagosome of primary BMDM. The growth and morphogenesis of wildtype (Green, ADH1/PADH1-GFP) and put1-/- (Red, ADH1/PADH1-RFP) cells phagocytosed in the same primary BMDM was monitored by time-lapse microscopy for 5 h.</p

Amino acid-induced morphogenesis is dependent on amino acid uptake.

A. Macrocolonies of wildtype C. albicans (PMRCA18) grown on SXD medium containing 10 mM of the indicated amino acids (X = Pro, Arg, Orn or Asp) and 2% glucose after 48 h of growth at 37 °C (upper panels). Cells scraped from macrocolonies stained with calcofluor white (lower panels); scale bars = 30 μ. B. Amino acid-induced SPS-sensor signaling. Cells expressing Stp2-6XHA (PMRCA48) were grown to log phase in SD medium and induced with 50 μM or 5 mM of the indicated amino acids for 5 min at 30 °C. The levels of latent and processed Stp2 in extracts were analyzed by immunoblotting (upper panels). Similarly, reporter strain (CFG001) carrying an integrated PCAN1-NanoLuc-PEST construct was grown to log phase in SD medium and induced with 50 μM of the indicated amino acids for 2 h at 30 °C. The average luciferase signal (ave. ± CI, 95% CL) are plotted; threshold for significance ≥ 1.5X fold change). C. Macrocolonies of wildtype (WT; PMRCA18) and strains carrying mutations inactivating SPS-sensing pathway components ssy1Δ/Δ (YJA64), ssy5Δ/Δ (YJA53), stp1Δ/Δ (PMRCA59), stp2Δ/Δ (PMRCA57), stp1Δ/Δ stp2Δ/Δ (PMRCA94) and csh3Δ/Δ (PMRCA12) grown on the indicated SXD media. D. Constitutively active Stp2* but not Stp1* bypasses the filamentous growth defect of a ssy1 null mutant in the presence of ornithine. Macrocolonies of WT (PMRCA18), STP1* (PMRCA23), STP2* (PMRCA44), ssy1-/- STP1* (CFG078), and ssy1-/- STP2* (CFG073) grown on SOD with ornithine (O) as sole nitrogen source. Images in C and D were obtained after 24 h of incubation 37 °C.</p

A bifurcated pathway for arginine-induced morphogenesis.

A. Scheme of arginine catabolic pathway. B. Growth-based assays. Five microliters (5 μl) of serially diluted cells were spotted onto the surface of SXD (X = Am (Ammonium sulfate), Arg, Orn, Pro or Urea) and then grown for 48 h at 30°C. Strains used: wildtype (WT; PMRCA18), car1-/- (CFG077), dur1,2-/- (CFG091), put1-/- (A) (CFG122), put1-/- (B) (CFG155), and put1-/- dur1,2 -/- (CFG158). C. Arginine rapidly derepresses proline catabolic genes. PUT1, PUT2, PUT3 and DUR1,2 expression in wildtype (SC5314) cells 1 h at 37 °C after shifting from YPD (t = 0) to YNB+Arg (pH = 6.0). Gene expression was determined by qRT-PCR using the levels of RIP1 to normalize expression. D. Proline catabolic pathway is required for growth in arginine as a sole nitrogen and carbon source. Cells with the indicated genotypes were harvested from log phase YPD cultures and diluted in YNB+Arg+BCP medium (pH = 4.0) to OD600 ≈ 0.01, cultures were incubated for 16 h at 37 °C. Alkalization (shift to purple) correlates with growth. Strains used: WT (SC5314), dur1,2-/- (CFG246), put1-/- (CFG149), and put2-/- (CFG143). Identical results were obtained using PMRCA18-derived mutants. E. Rapid activation of proline catabolism in the presence of arginine, ornithine, and proline. Immunoblot analysis of Put2-HA in whole cell lysates prepared from CFG185 (PUT2/PUT2-HA) cells grown at 37 °C in the specified SXD media for the indicated times. Cells were pre-grown in YPD and inoculated at an OD600 of 0.5. Levels of α-tubulin were used as loading control. F. Pharmacological inhibition of proline dehydrogenase (Put1) reduced filamentous growth of C. albicans in the presence of arginine. Macrocolonies of wildtype (WT; PMRCA18) grown at 37 °C for 72 h on SRD medium supplemented with L-THFA as indicated. G. Filamentous growth of strains on SXD media (X = Arg, Urea, or Arg + Urea as sole nitrogen sources). Macrocolonies were grown at 37 °C and photographed at 24 and 48 h. Strains used: wildtype (WT; PMRCA18), car1-/- (CFG077), dur1,2-/- (CFG091), and put1-/- (CFG122).</p

Amino acid-induced morphogenesis is dependent on catabolism and Ras1 activated Efg1-dependent transcription.

A. Scheme of possible signaling pathways controlling amino acid-induced morphogenesis. B. Amino acid-induced morphogenesis requires a functional Ras1/cAMP/PKA pathway (Efg1-dependent) but not on the MAPK signaling pathway (Cph1-dependent). Wildtype (WT; PMRCA18) and strains lacking Ras1 (CDH107), Cph1 (JKC19), Efg1 (HLC52), and both Cph1 and Efg1 (HLC54) were spotted onto the indicated SXD media (X = Pro, Arg, Orn or Asp) and incubated at 37 °C for 48 h. C. Levels of active GTP bound form of Ras1 (Ras1-GTP) increase upon amino acid induction. Extracts were prepared from pooled WT (PMRCA18) macrocolonies grown for 24 h at 37 °C on the specified SXD medium. The levels of total Ras1 and the activated forms (Ras1-GTP) were determined by immunoprecipitation. D. Arginine catabolism is required for arginine-induced morphogenesis. Cells were spotted on SRD (Arg) supplemented with nor-NOHA, a competitive inhibitor of arginase. E. Proline catabolism is required for proline-induced morphogenesis. Cells were spotted on SPD (Pro) supplemented with L-THFA, a competitive inhibitor of proline dehydrogenase. For D and E, macrocolonies (PMRCA18) were grown at 37 °C and photographed after 72 h. Lower images are magnified 2X in comparison to upper images.</p

Arginine induces morphogenesis in <i>C</i>. <i>albicans</i> through mitochondria-dependent activation of Ras1/cAMP/PKA pathway.

The presence of extracellular arginine enhances arginine uptake by binding to the SPS-sensor, leading to the endoproteolytic activation of transcription factor Stp2. The active form of Stp2 efficiently targets to the nucleus and binds the UASaa in the promoter of genes encoding amino acid permeases (AAP). Amino acid permeases are cotranslationally inserted into the membrane of the ER, and transported to the plasma membrane (PM, arrow) via the secretory pathway, a process that requires the ER membrane-localized chaperone Csh3. The increased functional expression of amino acid permeases (Aap) lead to an enhanced capacity to take up arginine. Intracellular arginine is catabolized by arginase (Car1) yielding ornithine and urea. Urea is further degraded by the urea amidolyase (Dur1,2) generating CO2 and NH3. Ornithine is further catabolized to proline in the cytoplasm in a series of enzymatic reactions starting with the ornithine aminotransferase (Car2). Proline is transported into the mitochondria where it is catabolized by Put1 and Put2 forming glutamate. These reactions generate the reduced electron carriers FADH2 and NADH,H+, respectively. Glutamate is oxidized by glutamate dehydrogenase (Gdh2) forming α-ketoglutarate in a reaction that liberates NH3 and generates NADH,H+. α-ketoglutarate feeds directly into the mitochondrial-localized TCA cycle. The reduced electron carriers generated by proline, glutamate and TCA cycle metabolic events are oxidized in reactions coupled to the generation ATP by mitochondrial oxidative phosphorylation. The elevated levels of ATP in the cytoplasm activate the adenyl cyclase (Cyr1) in a Ras1-dependent manner, which activates the downstream protein kinase A (PKA) signaling pathway and the effector transcription factor Efg1. The active phosphorylated form of Efg1 binds the UASEfg1 in the promoter of hyphal specific genes (HSG), thereby inducing yeast-to-hyphal morphogenesis. The catabolism of arginine via the proline pathway induces hyphal growth more rapidly (FAST) than the Dur1,2 generated CO2 (slow). Mitochondrial activity is repressed in the presence of high glucose.</p

Analysis of multivariate homogeneity of group dispersions.

Analysis of multivariate homogeneity of group dispersions.</p

Differential abundance analysis of gut bacteria in <i>Daphnia magna</i> exposed to Ciprofloxacin.

Bacterial genera significantly associated with (a) exposure to Ciprofloxacin; (b) high somatic growth and fecundity of the host observed during the experiment. The fold change (log2FC) and the associated statistics were determined using the edgeR package.</p