Subtilis Colonies Research Articles

Competition for iron shapes metabolic antagonism between Bacillus subtilis and Pseudomonas marginalis.

Siderophores have long been implicated in sociomicrobiology as determinants of bacterial interrelations. For plant-associated genera, like Bacillus and Pseudomonas, siderophores are well known for their biocontrol functions. Here, we explored the functional role of the Bacillus subtilis siderophore bacillibactin (BB) in an antagonistic interaction with Pseudomonas marginalis. The presence of BB strongly influenced the outcome of the interaction in an iron-dependent manner. The BB producer B. subtilis restricts colony spreading of P. marginalis by repressing the transcription of histidine kinase-encoding gene gacS, thereby abolishing production of secondary metabolites such as pyoverdine and viscosin. By contrast, lack of BB restricted B. subtilis colony growth. To explore the specificity of the antagonism, we cocultured B. subtilis with a collection of fluorescent Pseudomonas spp. and found that the Bacillus-Pseudomonas interaction is conserved, expanding our understanding of the interplay between two of the most well-studied genera of soil bacteria.

Pulcherrimin protects Bacillus subtilis against oxidative stress during biofilm development

Pulcherrimin is an iron-binding reddish pigment produced by various bacterial and yeast species. In the soil bacterium Bacillus subtilis, this pigment is synthesized intracellularly as the colorless pulcherriminic acid by using two molecules of tRNA-charged leucine as the substrate; pulcherriminic acid molecules are then secreted and bind to ferric iron extracellularly to form the red-colored pigment pulcherrimin. The biological importance of pulcherrimin is not well understood. A previous study showed that secretion of pulcherrimin caused iron depletion in the surroundings and growth arrest on cells located at the edge of a B. subtilis colony biofilm. In this study, we identified that pulcherrimin is primarily produced under biofilm conditions and provides protection to cells in the biofilm against oxidative stress. We presented molecular evidence on how pulcherrimin lowers the level of reactive oxygen species (ROS) and alleviates oxidative stress and DNA damage caused by ROS accumulation in a mature biofilm. We also performed global transcriptome profiling to identify differentially expressed genes in the pulcherrimin-deficient mutant compared with the wild type, and further characterized the regulation of genes by pulcherrimin that are related to iron homeostasis, DNA damage response (DDR), and oxidative stress response. Based on our findings, we propose pulcherrimin as an important antioxidant that modulates B. subtilis biofilm development.

Scattering angle resolved optical coherence tomography measures morphological changes in Bacillus subtilis colonies.

An unmet need is recognized for early detection and diagnosis of neurological diseases. Many psychological markers emerge years after disease onset. Mitochondrial dysfunction and corresponding neurodegeneration occur before onset of large-scale cell and tissue pathology. Early detection of subcellular morphology changes could serve as a beacon for early detection of neurological diseases. This study is on bacterial colonies, Bacillus subtilis, which are similar in size to mitochondria. This study investigates whether morphological changes can be detected in Bacillus subtilis using scattering angle resolved optical coherence tomography (SAR-OCT). The SAR-OCT was applied to detect scattering angle distribution changes in Bacillus subtilis. The rod-to-coccus shape transition of the bacteria was imaged, and the backscattering angle was analyzed by recording the distribution of the ratio of low- to medium angle scattering (L/M ratio). Bacillus orientation at different locations in colonies was analytically modeled and compared with SAR-OCT results. Significant differences in the distribution of backscattering angle were observed in Bacillus subtilis transitioning from rod-to-coccus shapes. In Bacillus subtilis, the -parameter of the Burr distribution of the SAR-OCT-derived L/M ratio was significantly smaller in coccus compared with rod-shaped bacteria. SAR-OCT-derived L/M ratio varied with bacterial position in the colony and is consistent with predicted orientations from previous studies. Study results support the potential of utilizing SAR-OCT to detect bacterial morphological changes.

SOYBEAN AS AN ALTERNATIVE NUTRIENT MEDIUM FOR Bacillus subtilis GROWTH

Nutrient agar is a commonly used medium for the isolation and growth of a broad range of microorganisms. The feasibility of using soybean as a base medium in the development of alternative growth media was assessed in this study. Nutrient agar was used as a standard guide to evaluating the performance of the formulated soybean agar. Bacillus subtilis was inoculated and allowed to grow on nutrient agar and soybean agar. Their growth was compared within 24 h after inoculation based on the morphology of individual colonies formed on both media and the pattern of bacterial growth. Our results showed that soybean agar had comparable performance to nutrient agar as the morphological characteristics of B. subtilis colonies formed on both media are generally identical in terms of texture, margin, optical properties, colour, elevation, and shape. However, due to the similar appearance of the bacterial colonies and the soybean agar, the colonies formed on the soybean agar were slightly larger than those formed on nutrient agar. In addition, our findings also revealed that agar strips formed the best soybean agar compared to gelatin and agar powder. Ultimately, this study has shown that locally available soybeans and agar strips can be easily formulated as an alternative to commercial nutrient agar and have great potential for bacteriological research.

The inhibitory effect of Ukrainian honey on probiotic bacteria

Honey is used in the food industry as a natural sweetener and has therapeutic effects on the human body. Obtaining quality honey involves using organic preventive and treatment agents in beekeeping. The most common of these agents are probiotic supplements. This research aimed to study honey’s interaction with an inhibitory effect on the growth of microorganisms from the probiotic supplement Immunobacterin-D under laboratory and experimental field conditions. At the first stage of the research, we assessed the effects of ten honey varieties (buckwheat, sunflower, meadow and forest plants, linden) on B. subtilis and B. licheniformis from the dry probiotic supplement. The honey-containing nutrient media had an inhibitory effect on the growth of B. subtilis colonies. After 24 hours of cultivation under aerobic conditions, the concentration of B. subtilis decreased, on average, from 5×1012 colony-forming units in 1 g to 3.2×104 and 2.1×105 CFU/g in samples with monofloral and polyfloral honey, respectively. These results emphasize the need for further research on the symbiotic role of microflora in the stability of the microbiota of the hive and bee colony ecosystem. The next stage of the study investigated the probiotic effect on bee colonies in the field. Observations were made on the sanitary conditions of the hives and the behaviour of bees at the Petrodolyna demo apiary. No differences were found at the macro hive-bee colony ecosystem level between control bee colonies (n = 5) and the experimental ones (n = 5) that had received carbohydrate feeding with added probiotics. This confirms the inhibitory effect of honey on the development of bacteria, which eliminates the risk of uncontrolled growth of B. subtilis and B. licheniformis strain colonies inside the hive and the bacteria getting into bee products. The probiotic had positive effects, increasing the live weight of worker bees by 9.15% by the end of the apiary season compared to the control. This can improve the viability of the bees during wintering. At the last stage of the research, the honey obtained from the experimental colonies was checked for the spores of B. subtilis and B. licheniformis using melissopalynology.

The majority of the matrix protein TapA is dispensable for Bacillus subtilis colony biofilm architecture

Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.

The extracellular matrix protects Bacillus subtilis colonies from Pseudomonas invasion and modulates plant co-colonization

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.

Enhanced molecular visualization of root colonization and growth promotion by Bacillus subtilis EA-CB0575 in different growth systems

Bacillus subtilis EA-CB0575 is a plant growth-promoting bacterium (PGPB) associated with banana and tomato crops. Root colonization is an important trait for PGPB microorganisms and potentiates the bacterial effect related to the mechanisms of plant growth promotion. Therefore, detection of bacterial colonization of roots in different culture systems is important in the study of plant–microorganism interactions. In this study, fluorescent in situ hybridization (FISH) and catalyzed reporter deposition–FISH (CARD–FISH) were evaluated to determine the colonization ability of B. subtilis EA-CB0575 on banana and tomato roots planted on solid and liquid Murashige and Skoog medium (MS(S) and MS(L), respectively) and in soil for tomato plants. Results showed B. subtilis colonization 0–30 days post inoculation for banana and tomato plants in different culture systems with differential distribution of bacterial cells along tomato and banana roots. FISH and CARD–FISH methodologies were both successful in detecting B. subtilis colonies, but CARD–FISH proved to be superior due to its enhanced fluorescence signal. The presence of bacteria correlated with the promotion of plant growth in both plant species, providing clues to relate rhizospheric colonization with improvement in plant growth. FISH and CARD–FISH analysis results suggested the presence of native microbiota on the roots of in vitro banana plants, but not on those of tomato plants.

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.

Experimental and theoretical studies on the morphogenesis of bacterial biofilms.

Biofilm morphogenesis not only reflects the physiological state of bacteria but also serves as a strategy to sustain bacterial survival. In this paper, we take the Bacillus subtilis colony as a model system to explore the morphomechanics of growing biofilms confined in a defined geometry. We find that the growth-induced stresses may drive the occurrence of both surface wrinkling and interface delamination in the biofilm, leading to the formation of a labyrinthine network on its surface. The wrinkles are perpendicular to the boundary of the constraint region. The variation in the surface undulations is attributed to the spatial stress field, which is isotropic in the inner regime but anisotropic in the vicinity of the boundary. Our experiments show that the directional surface wrinkles can confer biofilms with anisotropic wetting properties. This study not only highlights the role of mechanics in sculpturing organisms within the morphogenetic context but also suggests a promising route toward desired surfaces at the interface between synthetic biology and materials sciences.

Characterization of Bacillus subtilis Colony Biofilms via Mass Spectrometry and Fluorescence Imaging.

Colony biofilms of Bacillus subtilis are a widely used model for studying cellular differentiation. Here, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to examine cellular and molecular heterogeneity in B. subtilis colony biofilms. From B. subtilis cells cultivated on a biofilm-promoting medium, we detected two cannibalistic factors not found in previous MALDI MSI studies of the same strain under different culturing conditions. Given the importance of cannibalism in matrix formation of B. subtilis biofilms, we employed a transcriptional reporter to monitor matrix-producing cell subpopulations using fluorescence imaging. These two complementary imaging approaches were used to characterize three B. subtilis strains, the wild type isolate NCIB3610, and two mutants, Δspo0A and ΔabrB, with defective and enhanced biofilm phenotypes, respectively. Upon deletion of key transcriptional factors, correlated changes were observed in biofilm morphology, signaling, cannibalistic factor distribution, and matrix-related gene expression, providing new insights on cannibalism in biofilm development. This work underscores the advantages of using multimodal imaging to compare spatial patterns of selected molecules with the associated protein expression patterns, obtaining information on cellular heterogeneity and function not obtainable when using a single method to characterize biofilm formation.

Early Developmental Program Shapes Colony Morphology in Bacteria.

SummaryWhen grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process.

Visualization of Biosurfactant Film Flow in a Bacillus subtilis Swarm Colony on an Agar Plate

Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria.

Plant methyl salicylate induces defense responses in the rhizobacterium Bacillus subtilis.

Bacillus subtilis is a rhizobacterium that promotes plant growth and health. Cultivation of B. subtilis with an uprooted weed on solid medium produced pleat-like architectures on colonies near the plant. To test whether plants emit signals that affect B. subtilis colony morphology, we examined the effect of plant-related compounds on colony morphology. Bacillus subtilis formed mucoid colonies specifically in response to methyl salicylate, which is a plant-defense signal released in response to pathogen infection. Methyl salicylate induced mucoid colony formation by stimulating poly-γ-glutamic acid biosynthesis, which formed enclosing capsules that protected the cells from exposure to antimicrobial compounds. Poly-γ-glutamic acid synthesis depended on the DegS-DegU two-component regulatory system, which activated DegSU-dependent gene transcription in response to methyl salicylate. Bacillus subtilis did not induce plant methyl salicylate production, indicating that the most probable source of methyl salicylate in the rhizosphere is pathogen-infected plants. Methyl salicylate induced B. subtilis biosynthesis of the antibiotics bacilysin and fengycin, the latter of which exhibited inhibitory activity against the plant pathogenic fungus Fusarium oxysporum. We propose that B. subtilis may sense plants under pathogen attack via methyl salicylate, and express defense responses that protect both B. subtilis and host plants in the rhizosphere.

Sensitivity of bacteria to diamond nanoparticles of various size differs in gram-positive and gram-negative cells

In this study, the influence of the size and surface termination of diamond nanoparticles (DNPs) on their antibacterial activity against Escherichia coli and Bacillus subtilis was assessed. The average size and distribution of DNPs were determined by dynamic light scattering and X-ray diffraction techniques. The chemical composition of the DNPs studied by X-ray photoelectron spectroscopy showed that DNPs >5nm and oxidized particles have a higher oxygen content. The antibacterial potential of DNPs was assessed by the viable count method. In general, E.coli exhibited a higher sensitivity to DNPs than B.subtilis. However, in the presence of all the DNPs tested, the B.subtilis colonies exhibited altered size and morphology. Antibacterial activity was influenced not only by DNP concentration but also by DNP size and form. Whereas untreated 5-nm DNPs were the most effective against E.coli, the antibacterial activity of 18-50-nm DNPs was higher against B.subtilis. Transmission electron microscopy showed that DNPs interact with the bacterial surface, probably affecting vital cell functions. We propose that DNPs interfere with the permeability of the bacterial cell wall and/or membrane and hinder B.subtilis colony spreading.

Long-Term Visualization of Micro Vortices in Bacillus Subtilis Bacterial Biofilm on Agar Plate using Particle Image Velocimetry

The collective dynamics of bacteria has a crucial role in the development of biofilm. Although the behavior of the fluidic biofilm was studied by many research groups, detailed mechanics of its motion still remains elusive. Previously, we developed a method for visualization of vortical flow in the Bacillus Subtilis (B. subtilis) colony using 200-nm fluorescent microbeads, which were initially embedded in the agar plate and distributed spontaneously at the upper surface of the growing colony. Here, we conducted a long-term live imaging of B. subtilis colony with the fluorescent beads and obtained high-resolution velocity maps of microscale vortices in the biofilm using particle image velocimetry (PIV). At the tip of the colony, a distinct periodic fluctuation of average speed and vorticity revealed by Micro-PIV analysis was correlated with switching between bacterial swarming and growth phases. Our advanced imaging tool helps to explain the effect of micro vorticies in the biofilm on the collective dynamics of bacteria.

형광 미세입자를 이용한 박테리아 군집의 3차원 형상 분석 및 유동성 생물막의 가시화

세균의 집단 행동은 생물막의 형성에 중요한 역할을 하고 있다. 본 연구에서는 대장균(E. coli) 및 고초균(B. subtilis) 군집에서 형성된 유동성 생물막의 유체역학적 특성을 비교하고자 초기에 아가 플레이트 내에서 층을 이루다가 성장하는 군집 표면 위로 자발적으로 분포되는 200 nm의 형광입자를 가시화하였다. 대장균 군집에서는 유동하지 않는 200 nm 크기의 형광입자를 이용하여 성장하는 세균 군집의 3차원 형상 프로파일을 측정하였다. 고초균 군집의 경계에서 나타나는 와류 패턴은 고초균이 분비하는 계면활성제 내에서 유동하는 형광입자를 추적하여 가시화하였다. 본 연구는 세균의 생리 기능을 조절하는 새로운 물리적인 요소를 밝혀내고 세균의 증식 및 군집 이동에 영향을 미치는 유동성 생물막의 효과를 파악하는 첫걸음이 될 것이다.

Imaging secondary metabolism of Streptomyces sp. Mg1 during cellular lysis and colony degradation of competing Bacillus subtilis

Soil streptomycetes are saprotrophic bacteria that secrete numerous secondary metabolites and enzymes for extracellular functions. Many streptomycetes produce antibiotics thought to protect vegetative mycelia from competing organisms. Here we report that an organism isolated from soil, Streptomyces sp. Mg1, actively degrades colonies and causes cellular lysis of Bacillus subtilis when the organisms are cultured together. We predicted that the inhibition and degradation of B. subtilis colonies in this competition depends upon a combination of secreted factors, including small molecule metabolites and enzymes. To begin to unravel this complex competitive phenomenon, we use a MALDI imaging mass spectrometry strategy to map the positions of metabolites secreted by both organisms. In this report, we show that Streptomyces sp. Mg1 produces the macrolide antibiotic chalcomycin A, which contributes to inhibition of B. subtilis growth in combination with other, as yet unidentified factors. We suggest that efforts to understand competitive and cooperative interactions between bacterial species benefit from assays that pair living organisms and probe the complexity of metabolic exchanges between them.

Visualization of Vortical Flow Patterns in a Thin Fluidic Biofilm Surrounding a Bacillus Subtilis Bacterial Colony

The collective behavior of bacteria plays an important role in the biofilm development. It is essential to investigate the dynamics of bacterial colonization and the interactions between the bacteria and the physical parameters of the substrate and fluidic biosurfactants as well. Recently, studies on prokaryotic cell biology have been facilitated with the advancement of quantitative microscopic imaging techniques. In this study, the fluidic properties of the biofilms formed in Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis) colonies were compared by visualizing 200-nm fluorescent beads which were initially embedded in the agar plate and distributed spontaneously on the upper surface of the growing colonies. We focus on finding the effect of the substrate stiffness on their swarming motility and on the hydrodynamics of the fluidic biofilm by long-term live cell imaging of B.subtilis cells grown on an agar gel substrate. The viable cell number and motility of the bacteria in the colony were measured by phase contrast imaging and multiple particle tracking techniques. It was found that the bacteria in the biofilm have a property to switch between the growth and swarming phase. Vortical flow patterns near the edge of the colony were observed clearly by tracking of the beads moving in the biofilm of the B.subtilis colony, whereas there was no movement of the beads in the E.coli colony. Further analysis with a cross-correlation method revealed the detailed velocity vector field of the thin biofilm surrounding the B.subtilis cells. The present study should be a first step to find the effect of the fluidic biofilm on the growth and the swarming dynamics of bacteria, and to uncover a novel physical factor that regulates bacterial cell physiology.