THE ADAPTIVE MORPHOLOGY OF BACILLUS SUBTILIS AS A MECHANISM FOR SURVIVAL

Sarah Gingichashvili ^1,2 Danielle Duanis-Assaf ^1,3 John Featherstone ⁴ Moshe Shemesh ³ Osnat Feuerstein ² Doron Steinberg ¹

¹Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
²Faculty of Dental Medicine, Department of Prosthodontics, The Hebrew University of Jerusalem, Jerusalem, Israel
³Department of Food Quality and Safety, Institute for Postharvest Technology and Food Sciences, Agricultural Research Organization The Volcani Center, Jerusalem, Israel
⁴School of Dentistry, University of California San Francisco, San Francisco, California, USA

Biofilms are commonly defined as accumulations of microbes, embedded in self-secreted, polysaccharide-rich extra-cellular matrix. Bacillus subtilis - a model organism in biofilm research, forms unique three-dimensional macrocolonies on solid surfaces. This study investigates the relationship between structural and functional characteristics of B. subtilis biofilms, using a specially-developed software model that quantitatively analyzes morphological features within.

B. subtilis colonies were grown to maturity on Lysogeny broth agars of varying dilute solutions of a biofilm-promoting medium. The structure of formed biofilms was subjected to software analysis that modeled the morphological changes occurring as a result of nutrient depletion – these manifest themselves as a developing network of channels that crisscross the full thickness of the biofilm. In order to assess functional characteristics of the channels, fluorescent analog of glucose, a regularly consumed carbohydrate (2-NBDG), was utilized to monitor glucose uptake by cells at different locations within the biofilm. Two-photon excitation microscopy was further utilized to reveal the detailed structure of the macrocolonies.

Our results reveal the internal organization of a mature B. subtilis colony – under minimal nutrient availability, the presence of channels hints at their possible functional role as “highways” for nutrient trafficking. Monitoring of the native uptake of glucose further demonstrates the functional role of channels with regards to nutrient absorption and distribution. Understanding the processes behind high-level bacterial organization in biofilms, pathogenic or otherwise, is important for a number of reasons, not least of which is to identify structural “weak links” that can be targeted for purposes of biofilm inhibition or eradication. Our study demonstrates how quantitative software-based models can reveal this link between structural and functional characteristics of undisrupted B. subtilis biofilms.