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Probing phenotypic growth in expanding Bacillus subtilis biofilms

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Abstract

We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert’s law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.

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Acknowledgments

We thank Richard Losick’s group and Roberto Kolter’s group for providing triple-fluorescent-labeled Bacillus subtilis strains. This work was supported by the National Science Foundation (DMR-1310266, DMS-1411694), the Harvard Materials Research Science and Engineering Center (DMR-1420570), the National Natural Science Foundation of China (11272002), and the Beijing Higher Education Young Elite Teacher Project (YETP0363).

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Authors and Affiliations

  1. School of Mechanical Engineering, University of Science and Technology Beijing, Beijing, 100083, China

    Xiaoling Wang

  2. School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

    Xiaoling Wang, Stephan A. Koehler, James N. Wilking, Naveen N. Sinha, Siddarth Srinivasan, Shmuel Rubinstein, Michael P. Brenner & David A. Weitz

  3. Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT, 59717-3920, USA

    James N. Wilking

  4. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138, USA

    Matthew T. Cabeen

  5. CNRS, LPMC UMR 7336, Université Nice Sophia Antipolis, Parc Valrose, 06108, Nice, France

    Agnese Seminara

  6. Department of Mechanical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, China

    Qingping Sun

  7. Department of Physics, Harvard University, Cambridge, MA, 02138, USA

    David A. Weitz

Authors
  1. Xiaoling Wang
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  2. Stephan A. Koehler
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  3. James N. Wilking
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  4. Naveen N. Sinha
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  5. Matthew T. Cabeen
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  6. Siddarth Srinivasan
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  7. Agnese Seminara
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  8. Shmuel Rubinstein
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  9. Qingping Sun
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  10. Michael P. Brenner
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  11. David A. Weitz
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Corresponding authors

Correspondence to Xiaoling Wang or David A. Weitz.

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Funding

The National Science Foundation (DMR-1310266, DMS-1411694), the Harvard Materials Research Science and Engineering Center (DMR-1420570), the National Natural Science Foundation of China (11272002), and the Beijing Higher Education Young Elite Teacher Project (YETP0363).

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The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Wang, X., Koehler, S.A., Wilking, J.N. et al. Probing phenotypic growth in expanding Bacillus subtilis biofilms. Appl Microbiol Biotechnol 100, 4607–4615 (2016). https://doi.org/10.1007/s00253-016-7461-4

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  • DOI: https://doi.org/10.1007/s00253-016-7461-4

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