Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: Optimization of formamide concentration and probe specificity. ( A–C ) C. acetobutylicum (Cac), C. ljungdahlii (Clj), and C. kluyveri (Ckl) were hybridized with each probe at formamide concentrations between 10% and 50% to determine ideal stringency. The median population fluorescence intensity (in arbitrary units, a.u.) was plotted for each sample on a linear axis indexed at 0. ( A ) ClosLjun selectively binds to C. ljungdahlii ’s rRNA between 10 and 40% formamide, with optimal fluorescence at 30% formamide. ( B ) ClosAcet selectively binds to C. acetobutylicum ’s rRNA between 10% and 20% formamide, with optimal fluorescence at 20% formamide. ( C ) ClosKluy selectively binds to C. kluyveri ’s rRNA between 10% and 30% formamide, with optimal fluorescence at 10% formamide. ( D ) C. acetobutylicum was hybridized with ClosAcet, ClosKluy, and ClosLjun simultaneously at 20% formamide concentration and interrogated via flow cytometry on the channels corresponding to each probe’s fluorescent marker. The x -axis is fluorescent intensity, and the y -axis is the number of events at that intensity. “Labeled” samples were compared to “unlabeled” samples which underwent the same hybridization procedure but without any probes. Panel E shows anexperiment analogous to panel D performed in C. ljungdahlii . Panel F shows an experiment analogous to panel D performed in C. kluyveri . For each species, only the corresponding probe induced a shift in the population’s fluorescence. The gating strategy established from this set of experiments was maintained throughout the work.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Concentration Assay, Fluorescence, Flow Cytometry, Marker, Labeling, Hybridization

Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: rRNA is compatible with common fluorescent protein labeling techniques. ( A ) C. ljungdahlii -p100ptaHalo ( Clj -ptaHALO, top row) is labeled with ClosLjun and Janelia 549, a yellow-emitting ligand for HaloTag. ( B ) C. acetobutylicum -p100ptaHalo ( Cac -ptaHALO, bottom row) is labeled with ClosAcet (pseudo-colored magenta) and Janelia 646, a far-red ligand for HaloTag. ( C ) C. kluyveri ’s proteins were labeled with CellTracker Deep Red since there have been no reports of successful exogenous gene expression in the organism. This dye was also found to be compatible with in-solution rRNA-FISH.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Labeling, Expressing

Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: rRNA-FISH as an indicator of culture health/activity. OD 600 and fraction of the population which was labeled by rRNA-FISH during batch cultivation of C. acetobutylicum in triplicate. Cells were labeled with ClosAcet. Cells with a fluorescent signal brighter than background were deemed “labeled.” Error bars represent a single standard deviation above and below the average.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Activity Assay, Labeling, Standard Deviation

Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: rRNA localization and “chaining” in exponentially growing C. ljungdahlii . ( A ) The OD 600 and ( B ) flow cytometric data demonstrating a steep drop-off in fluorescence once the cells enter stationary phase. ( C ) DIC images of a typical C. ljungdahlii at early stationary phase (t3), which clearly show distinct cells bodies which have not undergone cleavage. Trace and fluorescent images show localization at the cleavage furrow and at the mid-cell. The fluorescent profile plot of the agglomerate’s major axis is indicated by the yellow line in panel C . Fluorescent peaks are extremely prominent during exponential phase. ( D ) Very few cells imaged microscopically from stationary phase (t5) displayed fluorescence (Fig. S13). The brightest cell from these images cell was selected and analyzed as cells in panel C but had decreased peak prominence compared to exponential phase.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Fluorescence

Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: rRNA localization in C. kluyveri . ( A ) The OD 600 and ( B ) flow cytometric data demonstrating a modest decrease in fluorescence once the cells leave exponential phase. ( C ) Cells exhibited ribosomal localization during exponential growth (t3) to varying extents epitomized by the four cells in this grouping. Cells 1, 2, and 4 show very little localization patterns, but cell 3 has distinct puncta at its poles, suggesting ribosomal localization is a transient phenomenon in C. kluyveri. ( D ) In late stationary phase (t7), the cells remained highly fluorescent, unlike in C. ljungdahlii, but mostly lacked fluorescent peaks.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Fluorescence

Journal: mSystems

Article Title: Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

doi: 10.1128/msystems.00572-24

Figure Lengend Snippet: rRNA localization in C. acetobutylicum . An inoculum grown from a colony to an OD 600 of ~4. Our rRNA-FISH method was used to analyze the inoculum (t1), exponential (t2), and stationary phase (t5) of the culture. ( A ) The OD 600 . ( B ) Flow cytometry histograms of fluorescence in the population show a decrease in overall fluorescence during early exponential phase, but sustained fluorescence in early stationary phase. ( C ) Microscopy images and fluorescent profile plots of typical inoculum cells show no localization. ( D ) Microscopy images and fluorescent profiles plots of typical exponential-phase cells indicate strong ribosomal localization. ( E ) Microscopy images and fluorescent profile plots of C. acetobutylicum in early stationary phase.

Article Snippet: Orthogonality was checked with the fluorescent spectrum tool from AAT BioQuest ( https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer ).

Techniques: Flow Cytometry, Fluorescence, Microscopy