The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm

Abstract

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

Fig. 1.

Transcription of the exoprotease-encoding bpr gene observed at the single cell level is dependent on DegU-P. Flow cytometry analysis of 3610 Pbpr–gfp (NRS2315) (black line) (a) and 3610 Pbpr–gfp degU (NRS2769) (black line) (b) using the parental 3610 strain as a negative control (grey-shaded area). Cells were grown under biofilm formation conditions for 17 h at 37 °C. A representative example is shown from three independent experiments.

Fig. 2.

An increase in transcription of the exoprotease-encoding bpr gene is observed at the single-cell level in the presence of high DegU-P. (a–e) Flow cytometry analysis. The grey-shaded area represents the parental 3610 strain as a negative control and the black lines the experimental sample. (f–o) Microscopy of Pbpr–gfp degU, amyE : : Phy-spank-degU32 hy cells (NRS2771). (f–j) Fluorescence was imaged in the FITC channel to detect GFP production. (k–o) The same cells analysed by phase-contrast fluorescence microscopy showing the overlay with the GFP expression with the cells. The cells were grown under biofilm formation conditions for 17 h at 37 °C in the presence of 0 µM IPTG (a, f, k), 2.5 µM IPTG (b, g, l), 7.5 µM IPTG (c, h, m), 10 µM IPTG (d, i, n) and 25 µM IPTG (e, j, o) prior to collection. In each case, one representative example is presented from three independent experiments.

Fig. 3.

Transcription of bpr increases over time during biofilm formation. (a) Transcription of bpr from cells extracted from complex colonies was monitored over a 72 h period using a bpr–gfp transcriptional fusion strain (NRS2315). Colonies were grown at 30 °C and collected for flow cytometry analysis (grey-shaded area, non-fluorescent control 3610 strain; black line, bpr–gfp) after 17 h (i), 24 h (ii), 48 h (iii) and 72 h (iv). (b) The same cells were then analysed by phase-contrast fluorescence microscopy after 17 h (i), 24 h (ii), 48 h (iii) and 72 h (iv). Shown are the phase-contrast (top) and FITC (GFP) channel (middle) and an overlay of both channels (bottom). A representative example is presented from three independent experiments.

Fig. 4.

An increase in transcription of the exoprotease-encoding bpr gene occurs during pellicle formation. Transcription of bpr from cells extracted from pellicle biofilms was monitored over a 96 h period using a bpr–gfp transcriptional fusion strain (NRS2315). Transcription of bpr from cells extracted from pellicles grown at 23 °C was monitored over a 96 h period using a bpr–gfp transcriptional fusion. Flow cytometry data are shown from cells extracted from pellicles at 24 h (a), 36 h (b), 48 h (c), 60 h (d), 72 h (e) and 96 h (f). The non-fluorescent NCIB3610 control line is shown as the grey-shaded area and the experimental sample as a black line. A representative example of both the expression analysis and pellicle formation is presented from three independent experiments.

Fig. 5.

Protease secretion was analysed from supernatant collected from pellicles during biofilm formation. Protease secretion was assessed using an azocasein assay from pellicle supernatants. Pellicles were grown at 25 °C for up to 96 h and samples were collected at 24 h (day 1), 48 h (day 2), 72 h (day 3) and 96 h (day 4). Enzyme activity was normalized against wet pellet weight of the pellicle. Data are presented as the mean of four independent experiments and the error bars represent sem.

Fig. 6.

Spatial analysis of bpr transcription within the mature biofilm. (a) Bright-field and FITC merged image and (b) FITC image of a 9 µm vertical cross-section of a 48 h colony biofilm harbouring the Pbpr–gfp reporter fusion (NRS3921) detected by confocal microscopy. The top (air interface) and bottom (agar interface) of the colony biofilm are indicated for reference purposes and shown as white lines. In (a), a 48 h colony biofilm is shown and the white box represents the approximate region of the biofilm that was imaged in cross-section. Bars, 10 µm. The images shown are representative of at least three independent experiments during which multiple fields of view were examined.

Fig. 7.

Co-expression of the bpr and tapA genes. (a, b) Static microscopy of NRS3378 cells harbouring the PtapA–mCherry and Pbpr–gfp transcriptional reporter fusions extracted from a pellicle biofilm after 24 h growth at 37 °C (a), where the asterisks indicate selected cells for which fluorescence was detected in both the TRITC (false coloured red) and the FITC (false coloured green) channels, for colony biofilms grown at 37 °C for the time (hours) indicated in the upper left-hand corner (b). Bars, 5 µm; the images are representative of multiple fields of view. (c) Microscopy analysis of NRS3921 harbouring PtapA–mKate2 and Pbpr–gfp transcriptional reporter constructs in real-time during microcolony development at 30 °C. Strain NRS3921 was imaged every 15 min. Images from the DIC, FITC (false coloured green) and TRITC (false coloured red) channels are shown above. The time (minutes) is indicated in the upper left-hand corner. Bars, 10 µm.

Fig. 8.

Origin of exoprotease-producing cells. Microscopy analysis of NRS3921 harbouring PtapA–mKate2 and Pbpr–gfp transcriptional reporter constructs during microcolony development at 30 °C. Strain NRS3921 was imaged every 15 min. Images from the DIC, FITC (false coloured green) and TRITC (false coloured red) channels are shown. The white and yellow arrowheads indicate cells that have transitioned directly from matrix production to exoprotease production. The green arrowheads indicate cells that have transitioned directly from a non-fluorescent state to exoprotease production. The blue arrowheads indicate cells that have transitioned from matrix-producing cells to exoprotease production and back to matrix production. The time (minutes) is indicated in the upper right-hand corners. Bars, 5 µm.