The Myxococcus xanthus spore cuticula protein C is a fragment of FibA, an extracellular metalloprotease produced exclusively in aggregated cells

Abstract

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns.

Figure 1. Protein C accumulation is heterogeneous.

A. Anti-Protein C immunoblot analysis of wild type (M. xanthus strain DZ2) cells grown on the surface of a Petri plate in vegetative (CYE) media. Cells were harvested, and cells in aggregates were pelleted at 50 x g for 5 min. Each lane contains lysate from 4.3×10⁷ cells harvested from the supernatant (S) or aggregated cell pellet (P) fractions. FB: Control demonstrating the Protein C accumulation in 4.3×10⁷ cells isolated from fruiting bodies formed after 48 hours of development. B. Anti-PilA (top panel) and anti-PilC (bottom panel) immunoblot of the supernatant (S) or pellet (P) fractions from A. Protein C was detected exclusively in the pellet cell fraction whereas PilA and PilC were equally represented in both cell fractions.

Figure 2. Mass spectrometry maps Protein C to FibA.

Domain architecture of the 744 amino acid (aa) FibA preprometalloprotease as predicted by SMART (http://smart.embl-heidelberg.de) . ss: signal sequence (aa 1–24); FTP: Fungalysin/Thermolysin Propeptide PFAM domain (aa 100–149); Peptidase _M4/M4_C: peptidase family M4 and M4-Cterminal PFAM domains (aa 218–518); PPC: Bacterial pre-peptidase C-terminal PFAM domain (aa 544–614 and 638–724). Black lines correspond to the length and sequence position of the peptides identified from mass spectrometry analysis of Protein C isolated from the spore coat (above schematic) or by immune-precipitation of the aggregated cell fraction using anti-Protein C sera (below schematic).

Figure 3. Protein C is the 31 kDa fragment of FibA.

A and B) Immunoblot analysis using anti-Protein C polyclonal antibodies (pAb) (left) and anti-FibA 2105 monoclonal antibodies (mAb) (right). Black arrows: ∼66 kDa band previously assigned to FibA . Grey arrows: ∼31 kDa band previously assigned to FibA and to Protein C . A) Total cell lysates prepared from equal numbers of cells from vegetative cultures (0 hours development) of dsp (DK3470) and wild type (DZ2) cells, and dsp cultures developing for 24 hours on nutrient-limited CF agar plates. B) FibA is present only in the aggregated cell fraction. Wild type (DZ2) and fibA (PH1018) cells were developed under submerged culture for 24 hours. Cell lysates were prepared from aggregated cell fractions (P) and supernatant cell fractions (S) as described in Fig. 1.

Figure 4. The fibA promoter region is active in both cell fractions.

A) Coomassie stain (left), and anti-Protein C (aka FibA) (middle), or anti-mCherry (right) immunoblot analysis of aggregated (P) and supernatant (S) cell fractions harvested from strain PH1019 (DZ2 attB::P_fibA-mCherry) developed for 24 hours under submerged culture. Supernatant and aggregated cell lysates were prepared from equal numbers of cells. Black and grey arrows indicate the ∼66 kDa and ∼31 kDa bands previously attributed to FibA and FibA/Protein C, respectively. B. Distribution of individual cell mCherry fluorescence intensities recorded from the samples above. Background subtracted intensity measurements of ≥250 cells from each fraction were recorded. The distribution of intensity measurements (bin size 50 relative intensity values) is displayed as a histogram for the aggregated (pelleted fraction) and supernatant cell fractions as indicated. Histograms were generated using Origin (ver. 6.1) data analysis and graphing software (Northampton, MA, USA). A and B. Results from one assay are shown, but triplicate biological repetitions produced identical results.