Introduction

Naturally occuring, there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is pHnCBS1D which was found in Halothiobacillus neapolitanus (Cannon and Shively, 1983). We used different parts of this plasmid for the production of BioBricks. These BioBricks (BBa_K1465202-BBa_K1465223) were used for the construction of a synthetic carboxysome-encoding plasmid. It is built up of the shell proteins CsoS1ABC and CsoS4AB and the shell associated protein CsoS2. In this way, the proteins should form an anaerobic microcompartment. This bottom up approach allows verifying the essentiality of the different components. We used a translational fusion of one of the shell proteins CsoS1A and GFP (BBa_K1465222) as indicator of correct protein folding. A concentrated subcellular localisation of the gfp-fluorescence shows the positions of carboxysomes within the cells. This reporter function of GFP was identified and used for this purpose before (Waldo et al., 1999; Hsu et al., 2009).

The expression of BBa_K1465223 (pSB1C3-T7:sap-csoS4AB-csoS1CA:gfp-csoS1B) in E. coli leads to the assembly of carboxysomes. We used a translational fusion of one shell protein (CsoS1A) with the CDS of the green fluorescent protein as an indicator of correct protein folding. The observed fluorescence in our E. coli cells is concentrated at different points of the cell and indicates the presence of functional carboxysomes.

Shell protein folding

With our GFP fusion we wanted to analyse the protein folding of our constructed microcompartment, the carboxysome. On the one hand we had a GFP fusion with the shell proteins. On the other hand we analysed this GFP fusion of the microcompartiment with the additionally shell associated protein encoded by csoS2 upstream of the shell proteins on the plasmid. The exact function of this protein is still unknown (Yeates et al., 2008).

Figure 1: Fluorescence measurement of E. coli KRX wildtype (negative control) and E. coli KRX carrying one of the three different plasmids pSB1C3_p_Tet_gfp (positive control), pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B and pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B. The measurement has been done with the GloMax® Discover Multimode-Reader.

The fluorescence shown in figure 1 was quantified by photometric measurements. The signal intensity of the E. coli KRX wildtype (negative control) is compared to cells carrying a plasmid without csoS2 named T7_csoS14_GFP (BBa_K1465222). E. coli KRX cells with pSB1C3-p_Tet_gfp (BBa_I13522) serve as a positive control and lead to the highest fluorescence signal. The expression of csoS1A_gfp with all other known essential shell proteins (BBa_K1465222) does not yield any fluorescence. This indicates that the presence of the shell associated protein is needed for the correct folding of the shell proteins and the assembly of the whole carboxysome. The combined expression of csoS2 and the coding sequences of the shell proteins csoS1CAB and csoS4AB (BBa_K1465223) yields green fluorescence. From these results we deduce that the correct folding of the shell proteins is dependent of the presence of the shell associated protein CsoS2.

Carboxysome assembly

To show the successfull assembly of the carboxysome we analysed the subcellular localisation of the green fluorescent protein. Pictures taken through a fluorescence microscope are shown in figure 2.

Figure 2: Fluorescence pictures taken through a Leica DMI6000 microscope. (A) E. coli wildtype KRX (negative control), (B) Expression of E. coli KRX with plasmid pSB1C3_p_Tet_gfp (positive control), (C) Expression of E. coli KRX with plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B, (D) and (E) Expression of E. coli KRX with plasmid pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B.

The negative control with the wildytpe of E. coli in picture A does not yield any fluorescence just as well as the sample with the plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B in picture C. Regarding this we can conclude that the carboxysome cannot be folded correctly without the shell associated protein CsoS2. In picture B, the sample with the positive control, the whole cells cause fluorescence. The E. coli bacteria carrying the whole carboxysome plasmid with the GFP fusion are shown in picture D and E. As expected not the whole cells are fluorescing but only a small part of the cells. We assume that the concentrated green fluorescence results from GFP molecules which are fused to the shell proteins of a successfully assembled carboxysome.
To get a higher resolution of the carboxysomes in the cells we also used a structured illumination microscope . The pictures are shown in figure 3.

Figure 3: Fluorescence pictures taken through a structured illumination microscope microscope. (A) E. coli wildtype KRX (negative controls), (B) Expression of  coli KRX with plasmid pSB1C3_pTet_gfp (positive control), (C) Expression of E. coli KRX with plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B, (D) and (E) Expression of E. coli KRX with plasmid pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B.

With this higher resolution we could identify the carboxysomes in the cells with the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B plasmid in one specific location of the cell (picture D and E). Most of the cells seem to have only one carboxysomes but there were also some that carried two. Additionally we could detect some cells forming a filament located at the membrane of the cell (figure 4). These filaments might be formed because of a high induction of the plasmid ( Bonacchi et al., 2012). As we used the strong T7 promoter this could be the reason for this occurrence.

Figure 4: Fluorescence picture of a single E. coli carrying the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B and forming a filament. Picture taken through a structured illumination microscope microscope.

The assembly of the carboxysome was possible without the expression of csoS1D, another coding sequence which is located on pHnCBS1D. The resulting protein is probably responsible for the pore size in the carboxysome envelope (Kinney et al., 2011; Bonacchi et al., 2012). Nevertheless it seems that it has no essentiell function in protein folding.

Summary and outlook

We report the construction of a synthetic plasmid encoding a functional carboxysome. Despite the fact that it has not shown the fixation of carbon dioxide in this compartment yet, we would like to emphasise the construction of a useful microcompartment. The functional carboxysome could be used for the carbon dioxide fixation. Therefore three enzymes need to be heterologous expressed in E. coli. We already tried to combine the carboxysome encoding genes and the three required enzymes on a single plasmid. The carbonic anhydrase (csoS3) should be cloned between the shell associated protein and the shell proteins. One RubisCO (BBa_K1465202) and one phosphoribulokinase (BBa_K1465201) encoding sequence should be integrated upstream of the carboxysomal operon.
The complete carboxysome could be used for anaerobic reactions in aerobic cultures of E. coli. One possible application beside the carbon dioxide fixation could be the fixation of atmospheric nitrogen which is essential for the growth of plants.

References

• Cannon, Shively 1983. Characterisation of a Homogenous Preparation of Carboxysomes from Thiobacillus neapolitanus. Archive of Microbiology, vol. 134, pp. 52-59
• Waldo, G. S., B. M. Standish, J. Berendzen, und T. C. Terwilliger. „Rapid Protein-Folding Assay Using Green Fluorescent Protein“. Nature Biotechnology 17, Nr. 7 (Juli 1999): 691–95. doi:10.1038/10904.
• Hsu, Shang-Te Danny, Georg Blaser, und Sophie E. Jackson. „The Folding, Stability and Conformational Dynamics of Beta-Barrel Fluorescent Proteins“. Chemical Society Reviews 38, Nr. 10 (2009): 2951–65. doi:10.1039/b908170b.
• Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. Proceedings of the National Academy of Sciences 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
• Kinney, James N., Seth D. Axen, und Cheryl A. Kerfeld. „Comparative analysis of carboxysome shell proteins“. Photosynthesis Research 109, Nr. 1–3 (September 2011): 21–32. doi:10.1007/s11120-011-9624-6.
• Yeates et al., 2011. Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nature Reviews Microbiology, vol. 6, pp. 681-691