Bacillus species present a major concern in the dairy industry as they can form biofilms in pipelines and on surfaces of equipment and machinery used in the entire line of production. These biofilms represent a continuous hygienic problem and can lead to serious economic losses due to food spoilage and equipment impairment. Biofilm formation by Bacillus subtilis is apparently dependent on LuxS quorum sensing (QS) by Autoinducer-2 (AI-2). However, the link between sensing environmental cues and AI-2 induced biofilm formation remains largely unknown. The aim of this study is to investigate the role of lactose, the primary sugar in milk, on biofilm formation by B. subtilis and its possible link to QS processes. Our phenotypic analysis shows that lactose induces formation of biofilm bundles as well as formation of colony type biofilm. Furthermore, using reporter strain assays, we observed an increase in AI-2 production by B. subtilis in response to lactose in a dose dependent manner. Moreover, we found that expression of eps and tapA operons, responsible for extracellular matrix synthesis in B. subtilis, were notably up-regulated in response to lactose. Importantly, we also observed that LuxS is essential for B. subtilis biofilm formation in the presence of lactose. Overall, our results suggest that lactose may induce biofilm formation by B. subtilis through the LuxS pathway.
Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of B. subtilis colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm – a central “core” region and an “expanding” region surrounding it. Our results demonstrate that complex biofilm of B. subtillis grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.
Many bacteria in biofilm surround themselves by an extracellular matrix composed mainly of extracellular polysaccharide (EP), proteins such as amyloid-like fibers (ALF) and nucleic acids. While the importance of EP in attachment and acceleration of biofilm by a number of different bacterial species is well established, the contribution of ALF to attachment in multispecies biofilm remains unknown. The study presented here aimed to investigate the role of TasA, a precursor for ALF, in cell-cell interactions in dual-species biofilms of Bacillus subtilis and Streptococcus mutans. Expression of major B. subtilis matrix operons was significantly up-regulated in the presence of S. mutans during different stages of biofilm formation, suggesting that the two species interacted and modulated gene expression in each other. Wild-type B. subtilis expressing TasA adhered strongly to S. mutans biofilm, while a TasA-deficient mutant was less adhesive and consequently less abundant in the dual-species biofilm. Dextran, a biofilm polysaccharide, induced aggregation of B. subtilis and stimulated adhesion to S. mutans biofilms. This effect was only observed in the wild-type strain, suggesting that interactions between TasA and dextran-associated EP plays an important role in inter-species interactions during initial stages of multispecies biofilm development.
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