TU BRAUNSCHWEIG

Phenotypic heterogeneity during heterologous protein production in Bacillus megaterium

During the last decade Bacillus megaterium was systemically developed for the gram per liter production of recombinant proteins. In contrast to the usual bacterial gene expression scenario, recombinant protein production systems often rely on multicopy plasmid encoded promoters, repressors and target genes. During production of the green fluorescent model protein (GFP) as a model culture heterogeneity was observed at the single cell level using time-lapse microscopy. The two subpopulations (producing versus low-producing) were separated by cell sorting and initially analyzed for limiting components of the expression system via quantitative RT-PCR. Further cultivation of both subpopulations revealed a systematic reajustment of their subpopulation distributions, indicationg bistability. An initial model was deduced as working hypothesis to identify the molecular basis for the observed phenotype and to improve our mathematical model.
Fluorescence microscopic image of a growing B. megaterium microcolony and colony heterogeneity of plated cells. The same image is shown as fluorescent image (A) and bright image overlaid with green-colored fluorescent image (B). Arrows indicate low-producing cells. Colony heterogeneity is shown in C) with small green-colored high GFP producing colonies and large white low GFP producing colonies. In the enlarged detail of image (C) three large white colonies are marked indicating low-producing cells.

References:

Contact details:
Prof Dr. Dieter Jahn
Technische Universität Braunschweig
Institute of Microbiology
Spielmannstraße 7
D-38106 Braunschweig
Tel.: +49-(0)531-391-5800
Fax: +49-(0)531-391-5854
 d.jahn@tu-bs.de
Homepage Link
Prof. Dr. Johannes Müller
Technische Universität München
Centre for Mathematical Sciences
Unit M12, Room 02.06.033
Boltzmannstr. 3
D-85747 Garching / Munich
Tel.: +49-(0)89 289 18392
Fax.: +49-(0)89 31873029
 johannes.mueller@mytum.de
Homepage Link
Dr. Rebekka Biedendieck
Technische Universität Braunschweig
Institute of Microbiology
AG Biotechnology
Spielmannstraße 7
D-38106 Braunschweig
Tel.: +49-(0)531-391-5812
Fax: +49-(0)531-391-5854
 r.biedendieck@tu-bs.de
Homepage Link
Dr. Richard Münch
Technische Universität Braunschweig
Institute of Microbiology
AG Computational Microbiology (CoMic)
Spielmannstraße 7
D-38106 Braunschweig
Tel.: +49-(0)531-391-5847
Fax: +49-(0)531-391-5854
 r.muench@tu-bs.de
Homepage Link
Co-workers: Collaborations:


References:

Münch, K. M., Müller, J., Wienecke, S., Bergmann, S., Heyber, S., Biedendieck, R., Münch, R. and Jahn, D. (2015) Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity. Appl. Environ. Microbiol., 81, 5976-5986.

Roselius, L., Langemann, D., Müller, J., Hense, B. A., Filges, S., Jahn, D. and Münch, R. (2014) Modelling and analysis of a gene-regulatory feed-forward loop with basal expression of the second regulator. J. Theor. Biol., 363C, 290-299.

Rulands, S., Jahn, D. and Frey, E. (2014) Specialization and bet hedging in heterogeneous populations. Phys. Rev. Lett., 113, 108102.

Klein, J., Leupold, S., Biegler, I., Biedendieck, R., Münch, R. and Jahn, D. (2012) TLM-Tracker: Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28, 2276-2277.

Münch, R., Klein, J. and Jahn, D. (2011) Prediction and analysis of gene regulatory networks in prokaryotic genomes. In Systems and Computational Biology - Molecular and Cellular Experimental SystemsInTech, Rijeka, Croatia, pp. 149-162.

Eppinger, M., Bunk, B., Johns, M. A., Edirisinghe, J. N., Kutumbaka, K. K., Koenig, S. S., Creasy, H. H., Rosovitz, M. J., Riley, D. R., Daugherty, S., Martin, M., Elbourne, L. D., Paulsen, I., Biedendieck, R., Braun, C., Grayburn, S., Dhingra, S., Lukyanchuk, V., Ball, B., Ul-Qamar, R., Seibel, J., Bremer, E., Jahn, D., Ravel, J. and Vary, P. S. (2011) Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J. Bacteriol., 193, 4199-4213.

Stammen, S., Müller, B. K., Korneli, C., Biedendieck, R., Gamer, M., Franco-Lara, E. and Jahn, D. (2010) High-yield intra- and extracellular protein production using Bacillus megaterium. Appl. Environ. Microbiol., 76, 4037-4046.

Bunk, B., Schulz, A., Stammen, S., Münch, R., Warren, M. J., Rohde, M., Jahn, D. and Biedendieck, R. (2010) A short story about a big magic bug. Bioeng Bugs, 1, 85-91.

Biedendieck, R., Yang, Y., Deckwer, W.-D., Malten, M. and Jahn, D. (2007) Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium. Biotechnol. Bioeng., 96, 525-537.

Münch, K. M., Müller, J., Wienecke, S., Bergmann, S., Heyber, S., Biedendieck, R., Münch, R. and Jahn, D. (2015) Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity. Appl. Environ. Microbiol., 81, 5976-5986.

Grote, J., Krysciak, D., Schorn, A., Dahlke, R. I., Soonvald, L., Müller, J., Hense, B. A., Schwarzfischer, M., Sauter, M., Schmeisser, C. and Streit, W. R. (2014) Evidence of autoinducer-dependent and autoinducer-independent heterogeneous gene expression in Sinorhizobium fredii NGR234. Appl. Environ. Microbiol.

Roselius, L., Langemann, D., Müller, J., Hense, B. A., Filges, S., Jahn, D. and Münch, R. (2014) Modelling and analysis of a gene-regulatory feed-forward loop with basal expression of the second regulator. J. Theor. Biol., 363C, 290-299.

Hense, B. A., Müller, J., Kuttler, C. and Hartmann, A. (2012) Spatial heterogeneity of autoinducer regulation systems. Sensors (Basel), 12, 4156-4171.

Fekete, A., Kuttler, C., Rothballer, M., Hense, B. A., Fischer, D., Buddrus-Schiemann, K., Lucio, M., Müller, J., Schmitt-Kopplin, P. and Hartmann, A. (2010) Dynamic regulation of N-acyl-homoserine lactone production and degradation in Pseudomonas putida IsoF. FEMS Microbiol. Ecol., 72, 22-34.

Hense, B. A., Kuttler, C., Müller, J., Rothballer, M., Hartmann, A. and Kreft, J.-U. (2007) Does efficiency sensing unify diffusion and quorum sensing? Nat. Rev. Microbiol., 5, 230-239.

Münch, K. M., Müller, J., Wienecke, S., Bergmann, S., Heyber, S., Biedendieck, R., Münch, R. and Jahn, D. (2015) Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity. Appl. Environ. Microbiol., 81, 5976-5986.

Klein, J., Leupold, S., Biegler, I., Biedendieck, R., Münch, R. and Jahn, D. (2012) TLM-Tracker: Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28, 2276-2277.

Eppinger, M., Bunk, B., Johns, M. A., Edirisinghe, J. N., Kutumbaka, K. K., Koenig, S. S., Creasy, H. H., Rosovitz, M. J., Riley, D. R., Daugherty, S., Martin, M., Elbourne, L. D., Paulsen, I., Biedendieck, R., Braun, C., Grayburn, S., Dhingra, S., Lukyanchuk, V., Ball, B., Ul-Qamar, R., Seibel, J., Bremer, E., Jahn, D., Ravel, J. and Vary, P. S. (2011) Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J. Bacteriol., 193, 4199-4213.

Stammen, S., Müller, B. K., Korneli, C., Biedendieck, R., Gamer, M., Franco-Lara, E. and Jahn, D. (2010) High-yield intra- and extracellular protein production using Bacillus megaterium. Appl. Environ. Microbiol., 76, 4037-4046.

Bunk, B., Schulz, A., Stammen, S., Münch, R., Warren, M. J., Rohde, M., Jahn, D. and Biedendieck, R. (2010) A short story about a big magic bug. Bioeng Bugs, 1, 85-91.

Biedendieck, R., Yang, Y., Deckwer, W.-D., Malten, M. and Jahn, D. (2007) Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium. Biotechnol. Bioeng., 96, 525-537.

Münch, K. M., Müller, J., Wienecke, S., Bergmann, S., Heyber, S., Biedendieck, R., Münch, R. and Jahn, D. (2015) Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity. Appl. Environ. Microbiol., 81, 5976-5986.

Roselius, L., Langemann, D., Müller, J., Hense, B. A., Filges, S., Jahn, D. and Münch, R. (2014) Modelling and analysis of a gene-regulatory feed-forward loop with basal expression of the second regulator. J. Theor. Biol., 363C, 290-299.

Klein, J., Leupold, S., Biegler, I., Biedendieck, R., Münch, R. and Jahn, D. (2012) TLM-Tracker: Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28, 2276-2277.

Münch, R., Klein, J. and Jahn, D. (2011) Prediction and analysis of gene regulatory networks in prokaryotic genomes. In Systems and Computational Biology - Molecular and Cellular Experimental SystemsInTech, Rijeka, Croatia, pp. 149-162.

Bunk, B., Schulz, A., Stammen, S., Münch, R., Warren, M. J., Rohde, M., Jahn, D. and Biedendieck, R. (2010) A short story about a big magic bug. Bioeng Bugs, 1, 85-91.


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