Display of Cellulase A from C. thermocellum on the B. subtilis cell surface

The first task of this part was creation of a cell-based cellulase chip using the cell display system which was constructed by Nguyen and Schumann (Nguyen and Schumann, 2006). For anchoring the protein on the B. subtilis surface, the sortase A from L. monocytogenes was employed for the catalysis of the covalent link between the cell wall and the polypeptides containing the sorting sequence. The sorting sequence using in here was from S. aureus FnbB with the sorting motif LPETG.

3.3.1.1 Permanent expression of sortase A (SrtA) from L. monocytogenes in B. subtilis

In the original cell surface display system, the SrtA enzyme was controllably produced by addition of IPTG. Therefore, the protein anchoring process needed the use of this costly inducer.

Because of this, I constructed a new B. subtilis strain which can permanently produce SrtA from L. monocytogenes in the cytoplasm.

The srtA gene of L. monocytogenes was fused to the PdnaK promoter and integrated at the thrC locus of B. subtilis strain WB800N resulting in strain QAC04. To demonstrate expression of the srtA gene, B. subtilis strains WB800N (control, no srtA gene), NHD03 (control, IPTG-inducible construct) and QAC04 were grown to the mid-logarithmic growth phase and NDH03 culture was submitted to IPTG-induction. The cultures were further grown for 2 h. Aliquots were

fractionated in SDS-PAGE and subjected to immunoblot probed with polyclonal antibodies against sortase A. The anti-DnaK antibodies were used for loading control. Analysis of the extraction from strain QAC04 showed a clear signal appeared at the molecular mass of sortase A (estimated 30 kDa) (Fig 3.22, lane 3). This signal was comparable with the one from the extract from strain NDH03 (Fig 3.22, lane 1). However, due to the lower amount of loading sample from NDH03 extraction (see Fig 3.22, lane 1-probed with α-DnaK), the production of SrtA in QAC04 was reduced. No unexpected band was detected with the negative SrtA strain (Fig 3.22, lane 2).

It can be concluded that strain QAC04 is able to produce SrtA in the cytoplasm constitutively.

The amount of expressed SrtA in QAC04 was lower when compared to IPTG-inducible NDH03 strain. However, because SrtA is an enzyme needed for anchoring the main target protein, even a small amount of it would be sufficient for this purpose. Therefore, the QAC04 strain was used for further studies.

Fig 3.22 Detection of SrtA of L. monocytogenes in extracts of B. subtilis. B. subtilis strains NHD03, WB800N and QAC04 were grown in LB medium at 37 °C to the mid-exponential growth phase (OD578 ∼ 0.8), 0.5 mM IPTG was added into the culture of strain NDH03 to induce expression of SrtA, while the other two cultures were further grown without induction for 2 h. Samples were taken and treated as described in 2.2.2.1, then processed for immunoblotting using rabbit anti L. monocytogenes SrtA antibodies. The anti-DnaK antibodies were used for loading control.

3.3.1.2 Less CelA can be anchored on the B. subtilis cell surface

To display cellulase A (CelA) on the cell surface, the celA gene was inserted into the pNDH11 plasmid resulting in pQAC07, so that a fusion of Secretion Signal-CelA-Sorting Signal would be produced upon induction using xylose.

Fig 3.23 Visualization of CelA on the B. subtilis cell surface. The B. subtilis strains QAC04 (thrC::PdnaK-srtA) and QAC07 (PxylA-SamyQ-celA-fnbB) were grown in LB medium at 37 °C to the mid-exponential growth phase (OD578 ∼ 0.8), 0.5% xylose was added to induce the expression of celA. Further growth for 2 h was performed. Then, aliquots were collected;

the cells were separated from the growth medium by centrifugation and processed for immunofluorescence microscopy as described in 2.2.4.1. The immobilization of CelA on the cell wall of B. subtilis was visualized by transmission and fluorescence Leica SP2 microscopy.

The strain QAC04 harboring the plasmid pQAC07 (PxylA-SamyQ-celA-fnbB – strain QAC07) was grown in LB medium to the mid-logarithmic growth phase, then 0.5% xylose was added. Further growth for 2 h was performed. The cells were withdrawn, gently washed twice with AP buffer and used directly for immunofluorescence staining using primary antibodies against CelA and Alexa-conjugated secondary antibodies (see 2.2.4.1). The QAC04 strain itself was grown under similar conditions, then treated with the same antibodies and used as a negative

control. The microscopic analysis showed the presence of CelA on the cell surface with the QAC07 cells. Little unspecific fluorescence was detected with the negative control (Fig 3.23).

Fig 3.24 Detection of celA expression on the cell wall by cellulase activity (A) and immunoblotting (B). The B. subtilis strains QAC07 and WB800N harboring plasmid pQAC07 and QAC04 were grown in LB medium and equal amounts of cells were washed as described in the legend to Fig 3.23. Cellulase activities were determined with whole cells and within the supernatant and presented in units per OD578 (A). The cell wall fractions from an identical number of cells from each strain were prepared as described in 2.2.2.2, loaded on 12% SDS-PAGE and subjected to immunoblot using rabbit anti CelA antibodies (B). The anti-DnaK antibodies were used for controlling the leakiness of the cytoplasmic membrane per sample.

To obtain a more precise confirmation for anchoring of CelA on the cell wall by SrtA transpeptidase, plasmid pQAC07 was transformed into the strain WB800N (does not contain SrtA). This strain was expected to secrete the fusion CelA-sorting signal into the medium. Next, this strain and strain QAC07 were grown in LB and induced by xylose. Whole cells were separated from the growth medium by centrifugation and analyzed separately for cellulase activity. The QAC04 was used in this experiment as a negative control. While a background level in the absence of any CelA was minor, the activities were increased with the cells from strain WB800N carrying plasmid pQAC07. The strongest activities from cells were measured with strain QAC07, about 65 milli units (Fig 3.24A). The cellulase activities measured from

supernatant of the two strains QAC07 and WB800N/pQAC07 were equivalent. However, as compared to activities from cells, they were significantly higher (Fig 3.24A). This indicated that more CelA was secreted to supernatant than anchored on cell wall.

In addition, the cell wall fractions from the three strains were prepared as described in 2.2.2.2 and subjected to immunoblot analysis using primary antibodies against CelA. The polyclonal antibodies against DnaK were used for controlling the leakiness of the cytoplasmic membrane in each sample. As anticipated, no CelA band was detected with the QAC04 sample (Fig 3.24B, lane 1). The signals from the WB800N/pQAC07 and QAC07 samples were detected with comparable amount (Fig 3.24B, lane 2 and 3). The result made it difficult to demonstrate the anchoring of CelA on the cell wall.

In summary, the CelA could not be effectively anchored on the B. subtilis cell wall using the L. monocytogenes SrtA transpeptidase.