Subtilis Biofilm Research Articles

Production and analysis of a Bacillus subtilis biofilm comprised of vegetative cells and spores using a modified colony biofilm model

Bacillus subtilis is a spore-forming soil bacterium that is capable of producing robust biofilms. Sporulation can occur in B. subtilis biofilms and it is possible that the spores embedded in the protective matrix could present a significant challenge to disinfecting agents or processes. This article describes a method for the growth and quantification of a reproducible B. subtilis ATCC 35021 biofilm comprised of vegetative cells and spores using a modified colony biofilm model. In this method, membranes were inoculated and incubated for a total of 8 days to promote biofilm formation and subsequent sporulation within the biofilm. Representative samples were taken over the course of the incubation period to evaluate the biofilms using enumerative, microscopic, and spectrometric methods. At various time points, the total numbers of cells and spores were quantified. A Congo red agar (CRA) method was utilized to detect the TasA matrix protein, a primary component of the B. subtilis biofilm matrix. The presence of TasA was also confirmed using mass spectrometry. The biofilm morphologies were correlated to the enumeration data with a variety of correlative imaging techniques: confocal microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). At the end of the incubation period, the biofilm contained >7 logs total colony forming units with spores comprising approximately 10% of the biofilm. The biofilm generated using this method allows researchers to use a new, more robust challenge for efficacy testing of chemical and physical antimicrobial treatments such as antibiotics, disinfectants, or heat.

Role of metabolic spatiotemporal dynamics in regulating biofilm colony expansion

Cell fate determination is typically regulated by biological networks, yet increasing evidences suggest that cell-cell communication and environmental stresses play crucial roles in the behavior of a cell population. A recent microfluidic experiment showed that the metabolic codependence of two cell populations generates a collective oscillatory dynamic during the expansion of a Bacillus subtilis biofilm. We develop a modeling framework for the spatiotemporal dynamics of the associated metabolic circuit for cells in a colony. We elucidate the role of metabolite diffusion and the need of two distinct cell populations to observe oscillations. Uniquely, this description captures the onset and thereafter stable oscillatory dynamics during expansion and predicts the existence of damping oscillations under various environmental conditions. This modeling scheme provides insights to understand how cells integrate the information from external signaling and cell-cell communication to determine the optimal survival strategy and/or maximize cell fitness in a multicellular system.

Effect of metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS) on Bacillus subtilis: biofilm formation, flocculability and surface characteristics.

In order to understand the inhibitory mechanism of metabolic uncoupler in biofilm, this study investigated the effect of TCS on B. subtilis biofilm formation, flocculability, surface characteristics and thermodynamic properties. An optimal concentration of TCS, a metabolic uncoupler, was observed to substantially inhibit biofilm formation and the secretion of extracellular polymeric substances (EPS). The effect of TCS on the zeta potential and flocculability of bacterial suspension implied the addition of 100 μg L−1 TCS increased the net negative charge of cell surface which induced the reduction of B. subtilis flocculability. Meanwhile, the effects of TCS on bacterial surfacial thermodynamic properties were analyzed by the Derjaguin–Landau–Verwey–Overbeek (DLVO) and extend DLVO (XDLVO) theories. As DLVO and XDLVO predicted, the primary energy barrier between bacterial cells incubated with 100 μg L−1 TCS were increased compared to that of control, indicating that B. subtilis incubated with 100 μg L−1 TCS must consume more energy to aggregate or form biofilm.

Bacillus subtilis Biofilm Development - A Computerized Study of Morphology and Kinetics.

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.

Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine.

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death.IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms.

The effect of iron oxide nanoparticles on Bacillus subtilis biofilm, growth and viability

Abstract Bacillus subtilis is one of the key microorganisms for the industrial production of many value added products. The fermentation of this bacterium is associated with biofilm formation that results in major process and operational issues. The study of the effect of magnetic nanoparticles on the bacterial cells can immensely contribute to designing intensified bioprocess methods Present study was aimed at understanding the effect of superparamagnetic iron oxide (naked IONs) and 3-aminopropyltriethoxy silane coated IONs (IONs@APTES) on biofilm formation, growth and viability of Bacillus subtilis. Both IONs were successfully prepared and characterised by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction patterns (XRD) and vibrating sample magnetometry (VSM). IONs@APTES at 100 μg/mL were found to significantly reduce the total biofilm biomass without affecting the cell viability compared with the behavior of naked IONS, which significantly affected the viability of B. subtilis at high concentrations without any significant redution in biofilm biomass. This suggests an active role of IONs@APTES in eliminating the biofilm formation as compared to the naked IONs. This work suggests the possibility of using IONs@APTES coated bacteria for a range of applications for future industrial fermentation with Bacillus subtilis .

Morphologies and phenotypes in Bacillus subtilis biofilms.

In this study, we explored Bacillus subtilis biofilm growth under various conditions such as the use of substrates with different stiffnesses and nutrient levels using a well-developed optical imaging technique to spatially and temporally track biofilm growth. We also developed a quantitative method to characterize B. subtilis biofilm morphologies under various growth conditions. To determine biofilm rim irregularities, we used the dimensionless P2A ratio, defined as P2/4πA, where P is the perimeter and A is the area of the biofilm. To estimate biofilm thickness from transmission images, we developed a calibration procedure based on Beer- Lambert's law and cross sectioning. Furthermore, to determine the distributions of different B. subtilis cell phenotypes during biofilm growth, we used a triple-fluorescence-labeled B. subtilis strain that expressed motility, matrix production, and sporulation. Based on this work, we are able to tune biofilm growth by changing its growing environment.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR. Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR. Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Habits of Highly Effective Biofilms: Ion Signaling

How do neighboring bacterial biofilms sense and communicate with each other? In a recent paper, Liu etal. (2017) demonstrate how electrical signaling allows communication of metabolic states between adjacent B.subtilis biofilms, providing a possible generalizable mechanism for communication in multispecies biofilms with interdependent metabolism.

De novo evolved interference competition promotes the spread of biofilm defectors

Biofilms are social entities where bacteria live in tightly packed agglomerations, surrounded by self-secreted exopolymers. Since production of exopolymers is costly and potentially exploitable by non-producers, mechanisms that prevent invasion of non-producing mutants are hypothesized. Here we study long-term dynamics and evolution in Bacillus subtilis biofilm populations consisting of wild-type (WT) matrix producers and mutant non-producers. We show that non-producers initially fail to incorporate into biofilms formed by the WT cells, resulting in 100-fold lower final frequency compared to the WT. However, this is modulated in a long-term scenario, as non-producers evolve the ability to better incorporate into biofilms, thereby slightly decreasing the productivity of the whole population. Detailed molecular analysis reveals that the unexpected shift in the initially stable biofilm is coupled with newly evolved phage-mediated interference competition. Our work therefore demonstrates how collective behaviour can be disrupted as a result of rapid adaptation through mobile genetic elements.

Bacillus subtilis utilizes the DNA damage response to manage multicellular development

Bacteria switch between free-living and a multicellular state, known as biofilms, in response to cellular and environmental cues. It is important to understand how these cues influence biofilm development as biofilms are not only ubiquitous in nature but are also causative agents of infectious diseases. It is often believed that any stress triggers biofilm formation as a means of bacterial protection. In this study, we propose a new mechanism for how cellular and environmental DNA damage may influence biofilm formation. We demonstrate that Bacillus subtilis prevents biofilm formation and cell differentiation when stressed by oxidative DNA damage. We show that during B. subtilis biofilm development, a subpopulation of cells accumulates reactive oxygen species, which triggers the DNA damage response. Surprisingly, DNA damage response induction shuts off matrix genes whose products permit individual cells to stick together within a biofilm. We further revealed that DDRON cells and matrix producers are mutually exclusive and spatially separated within the biofilm, and that a developmental checkpoint protein, Sda, mediates the exclusiveness. We believe this represents an alternative survival strategy, ultimately allowing cells to escape the multicellular community when in danger.

Involvement of etfA gene during CaCO3 precipitation in Bacillus subtilis biofilm

ABSTRACTThe eftA gene in Bacillus subtilis has been suggested to be involved in the oxidation/reduction reactions during fatty acid metabolism. Interestingly etfA deletion in B. subtilis results in impairment in CaCO3 precipitation on the biofilm. Comparisons between the wild type B. subtilis 168 and its etfA mutant during in vitro CaCO3 crystal precipitation (calcite) revealed changes in phospholipids membrane composition with accumulation of up to 10% of anteiso-C17:0 and 11% iso-C17:0 long fatty acids. Ca2+ nucleation sites such as dipicolinic acid and teichoic acids seem to contribute to the CaCO3 precipitation. etfA mutant strain showed up to 40% less dipicolinic acid accumulation compared with B. subtilis 168, while a B. subtilis mutant impaired in teichoic acids synthesis was unable to precipitate CaCO3. In addition, B. subtilis etfA mutant exhibited acidity production leading to atypical flagella formation and inducing extensive lateral growth on the biofilm when grown on 1.4% agar. From the ecological point of view, this study shows a number of physiological aspects that are involved in CaCO3 organomineralization on biofilms.

Bacillus subtilis biofilm development in the presence of soil clay minerals and iron oxides

Clay minerals and metal oxides, as important parts of the soil matrix, play crucial roles in the development of microbial communities. However, the mechanism underlying such a process, particularly on the formation of soil biofilm, remains poorly understood. Here, we investigated the effects of montmorillonite, kaolinite, and goethite on the biofilm formation of the representative soil bacteria Bacillus subtilis. The bacterial biofilm formation in goethite was found to be impaired in the initial 24 h but burst at 48 h in the liquid–air interface. Confocal laser scanning microscopy showed that the biofilm biomass in goethite was 3–16 times that of the control, montmorillonite, and kaolinite at 48 h. Live/Dead staining showed that cells had the highest death rate of 60% after 4 h of contact with goethite, followed by kaolinite and montmorillonite. Atomic force microscopy showed that the interaction between goethite and bacteria may injure bacterial cells by puncturing cell wall, leading to the swarming of bacteria toward the liquid–air interface. Additionally, the expressions of abrB and sinR, key players in regulating the biofilm formation, were upregulated at 24 h and downregulated at 48 h in goethite, indicating the initial adaptation of the cells to minerals. A model was proposed to describe the effects of goethite on the biofilm formation. Our findings may facilitate a better understanding of the roles of soil clays in biofilm development and the manipulation of bacterial compositions through controlling the biofilm in soils.

Species-Independent Attraction to Biofilms through Electrical Signaling

Bacteria residing within biofilm communities can coordinate their behavior through cell-to-cell signaling. However, it remains unclear if these signals can also influence the behavior of distant cells that are notpart of the community. Using a microfluidic approach, we find that potassium ion channel-mediated electrical signaling generated by a Bacillus subtilis biofilm can attract distant cells. Integration of experiments and mathematical modeling indicates that extracellular potassium emitted from the biofilm alters the membrane potential of distant cells, thereby directing their motility. This electrically mediated attraction appears to be a generic mechanism that enables cross-species interactions, as Pseudomonas aeruginosa cells also become attracted to the electrical signal released by the B.subtilis biofilm. Cells within a biofilm community can thus not only coordinate their own behavior but also influence the behavior of diverse bacteria at a distance through long-range electrical signaling. PAPERCLIP.

Modeling of Biofilm Growth on Ager Substrate Using the Extended Finite Element Method

Various types of bacterial form adhesive aggregates on surfaces known as biofilms, which are of interest to researchers in a great variety of fields. In this paper, a continuum model based on diffusion-reaction is proposed and extended finite element method (XFEM) coupled to level set is employed to simulate the biofilm growth on ager substrate. Numerical results show the quantitative relationship between colony morphology and nutrient deletion. Comparing with experiment observation, we approve that diffusion limited regime is more appropriate than growth limited regime to describe Bacillus subtilis biofilm growth on MSgg ager substrate. Our model can be extended to investigate detailed biological process in biofilm growth such as cell subdivision and osmotic pressure induced transportation.

Inhibited transport of graphene oxide nanoparticles in granular quartz sand coated with Bacillus subtilis and Pseudomonas putida biofilms

Increasing production and use of graphene oxide nanoparticles (GONPs) boost their wide dissemination in the subsurface environments where biofilms occur ubiquitously, representative of the physical and chemical heterogeneities. This study aimed at investigating the influence of Gram-positive Bacillus subtilis (BS) and Gram-negative Pseudomonas putida (PP) biofilms on the transport of GONPs under different ionic strengths (0.1, 0.5, and 1.0 mM CaCl2) at neutral pH 7.2 in water-saturated porous media. Particularly, the X-ray micro-computed tomography was used to quantitatively characterize the pore structures of sand columns in the presence and absence of biofilms. Our results indicated that the presence of biofilms reduced the porosity and narrowed down the pore sizes of packed columns. Transport experiments in biofilm-coated sand showed that biofilms, irrespective of bacterial species, significantly inhibited the mobility of GONPs compared to that in cleaned sand. This could be due to the Ca2+ complexation, increased surface roughness and charge heterogeneities of collectors, and particularly enhanced physical straining caused by biofilms. The two-site kinetic retention model-fitted value of maximum solid-phase concentration (Smax2) for GONPs was higher for biofilm-coated sand than for cleaned sand, demonstrating that biofilms act as favorable sites for GONPs retention. Our findings presented herein are important to deepen our current understanding on the nature of particle-collector interactions.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films.

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day-old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225mJ/cm2 induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser-based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

Application of quercetin and its bio-inspired nanoparticles as anti-adhesive agents against Bacillus subtilis attachment to surface

The aim of this study was directed to reveal the repulsive effect of coated glass slides by quercetin and its bio-inspired titanium oxide and tungsten oxide nanoparticles on physical surface attachment of Bacillus subtilis as an ab-initio step of biofilm formation. Nanoparticles were successfully synthesized using sol–gel and acid precipitation methods for titanium oxide and tungsten oxide, respectively (in the absence or presence of quercetin). The anti-adhesive impact of the coated-slides was tested through the physical attachment of B. subtilis after 24h using Confocal Laser Scanning Microscopy (CLSM). Here, quercetin was presented as a bio-route for the synthesis of tungsten mixed oxides nano-plates at room temperature. In addition, quercetin had an impact on zeta potential and adsorption capacity of both bio-inspired amorphous titanium oxide and tungsten oxide nano-plates. Interestingly, our experiments indicated a contrary effect of quercetin as an anti-adhesive agent than previously reported. However, its bio-inspired metal oxide proved their repulsive efficiency. In addition, quercetin-mediated nano-tungsten and quercetin-mediated amorphous titanium showed anti-adhesive activity against B. subtilis biofilm.

Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.

Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms.