Detection of putative RuBisCO proteins by western blot

The presence of the large subunit of RuBisCO was checked in pure anoxic autotrophic and mixotrophic cultures of Candidatus “T. syntrophicum” strain Cad16^T. These cultures were grown with a photoperiod of 12 h of light (7:00 – 19:00) fallowed by 12 h of dark (19:00 – 7:00) until they reached the starting concentration of approximately 10⁷ cell ml^-1 (see Material and Methods of Chapter 2). The total proteins were then extracted every 4 h during 24 h (T1-6), with an extra sample at 9:00 in the period of maximal CO₂ fixation activity (see Results of Chapter 2) and checked with polyclonal antibodies against the large subunit of RuBisCO (CbbL-CbbM).

Unfortunately, these antibodies are not specific for CbbL or CbbM, but both large subunits of strain Cad16^T are recognized (Figure A1).

During both autotrophic (Figure A1, left panel) and mixotrophic (Figure A1, right panel) conditions of growth, the concentration of CbbL-CbbM was very low at the sampling time T1 after 12 h of dark, with values of 0.254 and 0.161 CbbL-CbbM per µl^-1, respectively. The maximal quantity of RuBisCO was recorded at T3 after 8 h of light for both autotrophic and mixotrophic growing conditions, with 1.265 CbbL-CbbM per µl^-1 and 1.047 CbbL-CbbM per µl

-1, respectively. During the light period (T1.2 to T4), the quantity of CbbL-CbbM in the autotrophic cultures was up to 5-fold higher than in mixotrophic cultures. However, during the dark period (T5, T6 and T1) no relevant differences in the concentration of CbbL-CbbM were measured between the 2 growing conditions. In the autotrophic growing condition, the concentration of the protein appears to correlate with the ¹⁴CO2 assimilation results of the Chapter 3, indicating higher activity during the light phase compared with the dark phase.

Moreover, the maximal concentration of the large subunit of the RuBisCO enzyme occurs in T3 exactly during one of the maximal peaks of expression of the cbbL gene (see Results of Chapter 2).

Figure A1. Detection of large subunit of the RuBisCO

Total proteins extracted after autotrophic (left) and mixotrophic growth (right). The purified large subunit of RuBisCO was used as positive control (RbcL = 0.6 ug). The protein samples were taken every 4 h over 24 h (12 h of light followed by 12 h of dark): T1 = 7:00, T1.2 = 9:00, T2 = 11:00, T3 = 15:00, T4 = 19:00, T5 = 23:00 and T6 = 3:00.

With the aim of identifying the large subunit of the form I and the form II of RuBisCO, we decided to use the antibody described above in a 2-dimensional gel (2D-gel). Approximately 10 ng of total proteins extracted from autotrophic cultures at T2 (after 4 h of light 11:00) were loaded on a specific IPG strip (3-10 NL of 7 cm) and divided by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Figure A2 (right) shows the presence of different protein spots recognized by antibody. Three independent spots were visualized at the level of the hypothetical RuBisCO large subunit, which has a size of approximately 52 KDa in a pI range between 6.0 and 7.5, and these spots were also visible in the silver-stained gel. These protein spots were identified by MALDI-TOF mass spectrometry as the large subunit CbbM of the form II RuBisCO (see Material and Methods of Chapter 4).

Moreover, a line of several spots of approximately 45 KDA were visualized along the same pI range of the 3 independent spots mentioned above. These protein spots were not visible in the silver-stained gel, most likely because of their low quantity. Unfortunately, the low concentration of these proteins also prevented their identification by MALDI-TOF mass spectrometry.

The presence of the enzyme RuBisCO activase (RA) was as also determined (Figure A2 left) using another large-spectrum polyclonal antibody. The presence of different protein spots was demonstrated, and three of these were in the same place as the three spots identified as form II

RuBisCO (CbbM). Unfortunately, we did not have the annotation of the RA gene in our draft genome, so we could not estimate a hypothetical size or pI. The lack of specificity of this second antibody most likely resulted in recognition of protein spots only than the target RA.

Figure A2. Western blot against the large subunit from the RuBisCO in a 2D-GEL of proteins extracted during

the light exposure

(center) 2D-PAGE with 10 µg of proteins extracted at the beginning of the light phase (T2 = 11:00) from pure autotrophic cultures of strain Cad16^T. The samples were run on a linear isoelectric focusing (IEF) strip (pH 3–10 non-linear) at approximately 85 000 Vh and then separated by 12.5% SDS-PAGE of 7 cm width.

(right) Blot of a same twin gel (12.5% 7 cm 3-10 NL) shown in the center. The presence of the large subunit of RuBisCO was checked with an antibody tagging a key functional region conserved across all known plant, algal and (cyano)bacterial RbcL protein sequences (form I L₈S₈ and form II L₂) with 100% homology in the amino acid sequence with both Cad16^T proteins.

(left) Blot of another 7 cm 2D-PAGE (12.5% 7 cm 3-10 NL) gel run with the same conditions as the gel in the center. The presence of RuBisCO activase was checked with an antibody tagging a key functional region conserved across all known plant, algal and (cyano)bacterial RA protein sequences with 100% homology in the amino acid sequence with this present in strain Cad16^T.

Material and Methods

Cell lysis, protein sample preparation, quantification and gel separation. Liquid bacterial cultures (100 ml) of a concentration up to 10⁷ cell ml^-1 were washed 3 times with PBS 1x and resuspended in 1 ml of lysis buffer (Tris 10 mM, 60 mg; EDTA 1 mM, 18.6 mg; NADP 0.5 mM, 19.7 mg, deionized water 50 mL, pH adjusted to 6.8 with HCl). The cells were lysed by sonication (SONOPLUS HD 2070, Bandelin electronics, Berlin, Germany) with 5 cycles of 15 sec at approximately 20% of the maximal power with a pause of 2-3 min in ice between each cycle. After cell lysis, the sample was centrifuged for 15 min at 14000 rpm at 4°C, and the protein concentration of the supernatant as measured by Bradford assay (Bio-Rad, cat. no. 500-0201), according to the manufacturer’s instructions. A quantity of 1 µg of total proteins extracted for each sample was loaded on a SDS-PAGE according to Laemmli (Laemmli 1970).

The total proteins isolated for the 2D-PAGE analysis needed some extra steps and another lysis buffer (see Material and Methods of Chapter 5).

Immunoblot analysis. After SDS-PAGE or 2D-PAGE, the protein samples were transferred to a nitrocellulose membrane as described by Towbin (Towbin et al. 1979). After the transfer, the membrane was blocked for 1 hr at ambient temperature using 10% of DifcoTM Skim Milk (Becton, Dickinson and company, cat. no. 232100) in TBS containing 0.1% Tween 20 (TBS-T).

Subsequently, the membrane was incubated for 1 hr at room temperature with TBS-T containing a 1:5000 dilution of the primary rabbit polyclonal antibody anti-Rubisco large subunit (RbcL) or anti- RA/Rubisco activase (Agrisera, cat. no. AS03 037 and AS10 700, respectively). After washing with TBS-T (3 times for 15 min), the membrane was incubated for 30 min at room temperature with a HRP conjugated anti-Rabbit IgG (whole molecule) secondary antibody (Abcam^®, cat. no. ab6721) diluted 1:5000 in TBS-T. The antibody detection was conducted by enhanced chemiluminescence substrate detection as recommended by the manufacturer (Western Lightning^TM Plus-ECL; PerkinElmer, cat. no. NEL103001EA).

References

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences 76, 4350.