A short story about a big magic bug

Abstract

Bacillus megaterium, the "big beast," is a Gram-positive bacterium with a size of 4 × 1.5 µm. During the last years, it became more and more popular in the field of biotechnology for its recombinant protein production capacity. For the purpose of intra- as well as extracellular protein synthesis several vectors were constructed and commercialized (MoBiTec GmbH, Germany). On the basis of two compatible vectors, a T7 RNA polymerase driven protein production system was established. Vectors for chromosomal integration enable the direct manipulation of the genome. The vitamin B(12) biosynthesis of B. megaterium served as a model for the systematic development of a production strain using these tools. For this purpose, the overexpression of chromosomal and plasmid encoded genes and operons, the synthesis of anti-sense RNA for gene silencing, the removal of inhibitory regulatory elements in combination with the utilization of strong promoters, directed protein design, and the recombinant production of B(12) binding proteins to overcome feedback inhibition were successfully employed. For further system biotechnology based optimization strategies the genome sequence will provide a closer look into genomic capacities of B. megaterium. DNA arrays are available. Proteome, fluxome and metabolome analyses are possible. All data can be integrated by using a novel bioinformatics platform. Finally, the size of the "big beast" B. megaterium invites for cell biology research projects. All these features provide a solid basis for challenging biotechnological approaches.

Keywords: Bacillus megaterium; protein secretion; recombinant protein production; vector system; vitamin B12.

Figure 1

Electron microscope image of Bacillus megaterium (yellow) and Escherichia coli (red) vegetative cells. B. megaterium and E. coli cells were aerobically cultivated separately in LB medium at 37°C. They were grown until reaching the stationary phase. The two cultures were mixed in the ration of 1:1. Aldehyde-fixed bacteria were dehydrated with a graded series of acetone, critical-point-dried with liquid CO₂, and sputter-coated with gold. Samples were examined in a field emission scanning electron microscope (FESEM) Zeiss DSM982 Gemini at an acceleration voltage of 5 kV using the Everhart-Thronley SE-detector and the SE-Inlens-detector in a 50:50 ratio. The image was colored subsequently. Magnification ×15,000. Compared to the E. coli volume of 0.5 µm³ (0.5 × 0.5 × 2), B. megaterium has at least up to 100-times higher volume.

Figure 2

Culture heterogeneity of GFP producing B. megaterium cells (A and B). B. megaterium cells were cultivated on A5 medium agarose pad at 37°C and observed using a Zeiss Axiovert 200 M microscope. Pictures were taken using an AxioCam HR under 630x total magnification (63x objective, 10x ocular). (A) fluorescent image of GFP producing cells; (B) bright image of the same cells overlaid with green-colored fluorescent image. (C) A Biostat B2 bioreactor (B. Braun, Melsungen, Germany) with 2 L working volume connected to an exhaust gas analysis unit (Maihak, Hamburg, Germany) was operated and controlled as described previously., B. megaterium carrying a plasmid coding for GFP-Strep fusion protein was grown in semi-defined minimal medium at 37°C initially in a batch phase with 4 g/L glucose. At the end of the batch phase an exponential feeding profile was started. GFP was visualized by a lamp emitting blue light and a yellow filter using a digital camera. (D) Results of flow cytometric analysis of bioreactor cultivation. Samples taken from bioreactor cultivation of B. megaterium carrying a plasmid coding for GFP before and 4.6 h after induction of the gfp gene expression were stained with propidium iodide (PI) and analyzed in a FACSCalibur (Benton Dickinson, Belgium): Living cells, no GFP: red; living cells, GFP: green; dead cells, no GFP: black; dead cells, GFP: cyan. Percentages of the subpopulation compared to all cells are given.

Figure 3

Series of expression plasmids for the (A) extra- and (B) intracellular production of recombinant proteins by B. megaterium. All expression plasmids shown allow parallel cloning of genes of interest into the identical multiple cloning sites (MCS). SP: signal peptide; Tag: Affinity purification tag; CS: protease cleavage site; Term: Terminator; P_xylA: promoter of xylA; P_T7: T7 RNA polymerase dependent promoter; TEV: tobacco etch virus protease cleavage site; X_a: factor X_a protease cleavage site; SP_lipA: signal peptide of the lipase A; SP_pac: signal peptide of the penicillin G amidase; T_T7: terminator for T7 RNA polymerase.

Figure 4

Shaking flask cultivation of B. megaterium wild type strain and mutant strain HBBm1 (integrated xylose-inducible promoter upstream of the hemA_KKXCDBL operon). Wild type bacterial cells and mutant cells were aerobically grown in LB-medium containing 0.23% xylose. Recombinant gene expression was induced with 0.5% xylose at an optical density of 0.4. The picture was taken with a digital camera 5 hours after induction. Left site: wild type cells, right site: Mutant cells. The strong formation of tetrapyrroles is indicated by the red color.