Journal: Applied and Environmental Microbiology

Article Title: Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate

doi: 10.1128/AEM.02920-19

Figure Lengend Snippet: Rosettes swarm in response to the EroS Pv mating factor. (A to C) Bacterial cues regulate mating and multicellularity in S. rosetta . Bars, 10 μm. (A) S. rosetta grown in the presence of the prey bacterium E. pacifica (Ctrl) proliferated as solitary cells. This culture served as the foundation for all experiments in this study. A typical S. rosetta cell has an apical collar (arrowhead) surrounding a single flagellum (arrow). (B) S. rosetta formed mating swarms within 0.5 h of treatment with the bacterially produced chondroitinase EroS Pv . (C) S. rosetta solitary cells developed into rosettes through serial rounds of cell division within 24 h of treatment with RIF-OMVs from the bacterium A. machipongonensis . (D and E) Rosettes swarm in the presence, but not in the absence, of EroS Pv . Bars, 50 μm. (D) After 24 h of treatment with a 1:1,000 dilution of RIF-OMVs and BSA (carrier control), solitary cells in an SrEpac culture developed into rosettes (arrowheads) but did not swarm. (E) Swarms of rosettes (arrows) formed after 24 h of treatment with a 1:1,000 dilution of RIF-OMVs followed by 0.5 h of treatment with 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv . (F) Scatterplot of the surface areas of cell clusters from SrEpac cultures treated with a 1:1,000 dilution of RIF-OMVs for 24 h followed by 0.5 h of incubation either with BSA (carrier control) or with 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv . According to the approach described in reference , we generated a binary mask to measure cell cluster area (the area of each cell, rosette, or swarm) (see Fig. S1 in the supplemental material). EroS Pv treatment resulted in clusters of cells, including swarms of rosettes (median, 58.7 μm 2 ; interquartile range, 21.6 to 98.0 μm 2 ), whose areas were significantly larger than those measured in the rosette-only control (median, 35.5 μm 2 ; interquartile range, 17.8 to 65.9 μm 2 ) (Kolmogorov-Smirnov test). In total, 875 cell cluster areas from 3 biological replicates were plotted for the cultures treated with RIF-OMVs, and 1,359 cell cluster areas from 3 biological replicates were plotted for the cultures treated with RIF-OMVs plus EroS Pv .

Article Snippet: SrEpac ( Salpingoeca rosetta cocultured with the prey bacterium Echinicola pacifica , ATCC PRA-390 [ ]) was cultured in 5% seawater complete medium (5% SWC [vol/vol] in ASW) at 22°C.

Techniques: Produced, Control, Incubation, Generated

Journal: Applied and Environmental Microbiology

Article Title: Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate

doi: 10.1128/AEM.02920-19

Figure Lengend Snippet: The mating inducer EroS Pv enhances rosette development. (A) EroS Pv enhances rosette induction by RIF-OMVs. Treatment of SrEpac with increasing concentrations of RIF-OMVs (circles) and BSA (carrier control) resulted in a concomitant increase in the percentage of cells in rosettes. Cotreatment of SrEpac with RIF-OMVs and 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv (triangles) resulted in rosette development at concentrations of RIF-OMVs that did not otherwise induce rosettes (e.g., at circle 1). EroS Pv also increased the maximum percentage of cells in rosettes at saturating concentrations of RIF-OMVs (e.g., at circle 2). The 1:20,000 dilution of RIF-OMVs used for the sensitized rosette induction assays in panels B to D is indicated with a vertical dotted line. (B) Cotreatment of SrEpac with 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv and a 1:20,000 dilution of RIF-OMVs leads to a dramatic increase in percentage of cells in rosettes throughout the course of rosette development relative to that in SrEpac treated only with RIF-OMVs and BSA (carrier control). After 39 h (shaded bar) of cotreatment with RIF-OMVs and EroS Pv (triangles), 88.2% ± 2.7% of S. rosetta cells were in rosettes, compared with 23.4% ± 4.9% of cells treated with RIF-OMVs alone (circles). (C) EroS Pv increased the ratio of rosettes to solitary cells in SrEpac cultures treated with RIF-OMVs. After 39 h (shaded bar) of cotreatment with a 1:20,000 dilution of RIF-OMVs and 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv (triangles), the ratio of rosettes to solitary cells was 0.96 ± 0.31 compared with 0.06 ± 0.02 after treatment with RIF-OMVs and BSA (carrier control) (circles). (D) EroS Pv increased the number of cells per rosette in RIF-OMV-treated SrEpac cultures. After 39 h (shaded bar) of cotreatment with a 1:20,000 dilution of RIF-OMVs and 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv (triangles), there were 8.9 ± 2.7 S. rosetta cells per rosette colony compared with 5.3 ± 1.7 cells per rosette colony after treatment with RIF-OMVs alone and BSA (carrier control) (circles). Means ± SDs from 3 biological replicates are plotted for each panel (A to D).

Article Snippet: SrEpac ( Salpingoeca rosetta cocultured with the prey bacterium Echinicola pacifica , ATCC PRA-390 [ ]) was cultured in 5% seawater complete medium (5% SWC [vol/vol] in ASW) at 22°C.

Techniques: Control

Journal: Applied and Environmental Microbiology

Article Title: Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate

doi: 10.1128/AEM.02920-19

Figure Lengend Snippet: Purified RIFs and EroS are sufficient for enhancement of rosette induction. (A) Cotreatment of SrEpac with 10 μg/ml (16.7 μM) HPLC-purified RIFs and 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS Pv for 24 h resulted in an increase in the percentage of S. rosetta cells in rosettes compared to that after treatment with HPLC-purified RIFs and BSA (carrier control). Rosettes do not form in the absence of RIFs. Means ± SDs from 2 biological replicates are plotted (unpaired t test). (B) Cotreatment of SrEpac with a 1:20,000 dilution of RIF-OMVs and either 0.1% EroS from V. fischeri (EroS Vf ) or 0.05 U/ml (0.2 to 1 μg/ml, ∼2 to 8 nM) EroS from P. vulgaris (EroS Pv ) for 24 h resulted in an increase in the percentage of rosette colonies compared to that after treatment with RIF-OMVs and BSA (carrier control). Means ± SDs from 5 biological replicates (RIF-OMVs alone, RIF-OMVs plus EroS Pv ) or 4 biological replicates (RIF-OMVs plus EroS Vf ) are plotted (unpaired t test).

Article Snippet: SrEpac ( Salpingoeca rosetta cocultured with the prey bacterium Echinicola pacifica , ATCC PRA-390 [ ]) was cultured in 5% seawater complete medium (5% SWC [vol/vol] in ASW) at 22°C.

Techniques: Purification, Control

Journal: Applied and Environmental Microbiology

Article Title: Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate

doi: 10.1128/AEM.02920-19

Figure Lengend Snippet: S. rosetta integration of bacterial cues. S. rosetta phenotypes induced over time by EroS Pv , RIF-OMVs, and the synergistic effect of both cues. (A) Untreated SrEpac proliferates as solitary cells. (B) Treatment with EroS Pv induces swarming of unrelated cells within 0.5 h. Cells with different genotypes (depicted as cells of different colors) can gather together to form nonclonal swarms. (C) Treatment with RIF-OMVs induces rosette development through cell division within 11 to 24 h. Cells in rosettes arise through serial rounds of cell division and share the same genotype (depicted as cells of the same color). (D) Cotreatment with RIF-OMVs and EroS Pv for 0.5 h results in swarming, showing that RIF-OMVs do not interfere with or enhance the activity of EroS Pv . (E) After 11 to 24 h of cotreatment with RIF-OMVs and EroS Pv , rosettes develop and swarm. Compared to that after treatment with RIF-OMVs alone, cotreatment with RIF-OMVs and EroS Pv induces the development of more rosettes and rosettes containing more cells.

Article Snippet: SrEpac ( Salpingoeca rosetta cocultured with the prey bacterium Echinicola pacifica , ATCC PRA-390 [ ]) was cultured in 5% seawater complete medium (5% SWC [vol/vol] in ASW) at 22°C.

Techniques: Activity Assay

Journal: ACS Omega

Article Title: Characterization of γ-Cadinene Enzymes in Ganoderma lucidum and Ganoderma sinensis from Basidiomycetes Provides Insight into the Identification of Terpenoid Synthases

doi: 10.1021/acsomega.1c06792

Figure Lengend Snippet: Characterization of γ-CSs from G. lucidum and G. sinensis in E. coli . (A) Expression of various γ-CSs from G. lucidum and G. sinensis . Lane M, molecular weight marker protein; S, soluble fraction of total cell extracts; and I, insoluble fraction of total cell extracts. (B) GC–MS chromatograms of cultural supernatants of strains GlSTS6, GsSTS43, and GsSTS45b and the control strains pET32a _ctrl and the positive STC9_posi. (C) Prediction and cloning of a gene in the G. sinensis STS family. The predicted gene was GsSTS45. The cloned and reannotated genes were GsSTS45a and GsSTS45b, and only GsSTS45b revealed to be functional.

Article Snippet: The successfully constructed plasmids were transformed into E. coli Rosetta (DE3) (TIANGEN) hosts to heterologously express proteins.

Techniques: Expressing, Molecular Weight, Marker, Gas Chromatography-Mass Spectrometry, Control, Cloning, Clone Assay, Functional Assay

Journal: bioRxiv

Article Title: An accessible transfection protocol for choanoflagellates

doi: 10.64898/2026.03.10.710884

Figure Lengend Snippet: Preliminary trials identify Chicabuffers 1M and 3H as candidates in-house electroporation buffers for further optimization. ( A ) Schematic overview of the post-electroporation workflow used to identify candidate buffers. ( B ) Chicabuffers and AmaxaV buffers were evaluated across five independent trials, with three transfection reactions per buffer. Filled circles indicate Salpingoeca rosetta survival in one of three transfections after 5 days of antibiotic selection.

Article Snippet: Salpingoeca rosetta was maintained in monoxenic co-culture with the bacterium Echinocola pacifica (co-culture “SrEpac”; American Type Culture Collection [ATCC], Manassas, VA; Cat No. PRA-390).

Techniques: Electroporation, Transfection, Selection

Journal: bioRxiv

Article Title: An accessible transfection protocol for choanoflagellates

doi: 10.64898/2026.03.10.710884

Figure Lengend Snippet: Chicabuffer 1M with pulse DS-137 yields the optimal combination of in-house buffer and pulse program for further fine-tuning of transfection efficiency. Plasmid-encoded nanoluciferase was transfected into S. rosetta using ( A ) Chicabuffer 1M and ( B ) Chicabuffer 3H across a range of pulse programs varying in efficiency and viability in triplicate. Nanoluciferase activity was measured 24 hours post-nucleofection and reported as luminescence relative to the average of three no-pulse controls.

Article Snippet: Salpingoeca rosetta was maintained in monoxenic co-culture with the bacterium Echinocola pacifica (co-culture “SrEpac”; American Type Culture Collection [ATCC], Manassas, VA; Cat No. PRA-390).

Techniques: Transfection, Plasmid Preparation, Activity Assay