Abstract
Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or “wrinkles” that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels’ interiors which supports their possible role in the transport of molecules throughout biofilm structures.
Highlights
Bacteria, in a natural setting, routinely encounter a wide variety of stress types such as temperature related, oxidative, nitrosative, mechanical, and reductive
B. subtilis biofilm formation on solid lysogeny broth (LB) medium was shown by Shemesh et al [14] to occur when the growth medium is supplemented with glycerol and manganese; the biofilm-promoting effect was demonstrated by the appearance of a robust biofilm phenotype alongside increased extracellular matrix production and biofilm-associated sporulation
The plates were incubated at 30 ◦C for a period of three days during which the green fluorescent protein (GFP)-labelled colony-type biofilms were visualized under a Nikon SMZ25 microscope with an ORCA-R2 camera (Zeiss LSM510 CLS microscope, Carl Zeiss, Oberkochen, Germany)
Summary
In a natural setting, routinely encounter a wide variety of stress types such as temperature related (heat shock), oxidative, nitrosative, mechanical, and reductive. To offset sub-optimal growth conditions, bacteria develop a number of different adaptation mechanisms, or “stress responses”. These include, but are not limited to, dormant endospore formation, lowered metabolic rate, altered cell morphology [1], changes in gene expression [2], and protein synthesis. Bacillus subtilis (B. subtilis), a Gram-positive model bacterium in biofilm research, is able to form morphologically complex and exceptionally robust colony-type biofilm [6]. This bacterium is considered to be largely non-pathogenic, it has been linked to food spoilage [7,8]. The specific phenotype of a B. subtilis colony-type biofilm is determined by multiple environmental factors including growth medium, temperature, presence of cofactors, oxygen availability, etc
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