Evolutionary Divergence of the Wsp Signal Transduction Systems in Beta- and Gammaproteobacteria.

Bacteria rapidly adapt to their environment by integrating external stimuli through diverse signal transduction systems. Pseudomonas aeruginosa, for example, senses surface contact through the Wsp signal transduction system to trigger the production of cyclic di-GMP. Diverse mutations in wsp genes that manifest enhanced biofilm formation are frequently reported in clinical isolates of P. aeruginosa and in biofilm studies of Pseudomonas spp. and Burkholderia cenocepacia. In contrast to the convergent phenotypes associated with comparable wsp mutations, we demonstrate that the Wsp system in B. cenocepacia does not impact intracellular cyclic di-GMP levels, unlike that in Pseudomonas spp. Our current mechanistic understanding of the Wsp system is based entirely on the study of four Pseudomonas spp., and its phylogenetic distribution remains unknown. Here, we present a broad phylogenetic analysis to show that the Wsp system originated in the betaproteobacteria and then horizontally transferred to Pseudomonas spp., the sole member of the gammaproteobacteria. Alignment of 794 independent Wsp systems with reported mutations from the literature identified key amino acid residues that fall within and outside annotated functional domains. Specific residues that are highly conserved but uniquely modified in B. cenocepacia likely define mechanistic differences among Wsp systems. We also find the greatest sequence variation in the extracellular sensory domain of WspA, indicating potential adaptations to diverse external stimuli beyond surface contact sensing. This study emphasizes the need to better understand the breadth of functional diversity of the Wsp system as a major regulator of bacterial adaptation beyond B. cenocepacia and select Pseudomonas spp. IMPORTANCE The Wsp signal transduction system serves as an important model system for studying how bacteria adapt to living in densely structured communities known as biofilms. Biofilms frequently cause chronic infections and environmental fouling, and they are very difficult to eradicate. In Pseudomonas aeruginosa, the Wsp system senses contact with a surface, which in turn activates specific genes that promote biofilm formation. We demonstrate that the Wsp system in Burkholderia cenocepacia regulates biofilm formation uniquely from that in Pseudomonas species. Furthermore, a broad phylogenetic analysis reveals the presence of the Wsp system in diverse bacterial species, and sequence analyses of 794 independent systems suggest that the core signaling components function similarly but with key differences that may alter what or how they sense. This study shows that Wsp systems are highly conserved and more broadly distributed than previously thought, and their unique differences likely reflect adaptations to distinct environments.

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity.

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR RpfR uniquely integrates two major signaling systems-quorum sensing and the motile-sessile switch mediated by cyclic-di-GMP-by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions.

Effect of Novel Quercetin Titanium Dioxide-Decorated Multi-Walled Carbon Nanotubes Nanocomposite on Bacillus subtilis Biofilm Development.

The present work was targeted to design a surface against cell seeding and adhering of bacteria, Bacillus subtilis. A multi-walled carbon nanotube/titanium dioxide nano-power was produced via simple mixing of carbon nanotube and titanium dioxide nanoparticles during the sol-gel process followed by heat treatment. Successfully, quercetin was immobilized on the nanocomposite via physical adsorption to form a quercetin/multi-walled carbon nanotube/titanium dioxide nanocomposite. The adhesion of bacteria on the coated-slides was verified after 24 h using confocal laser-scanning microscopy. Results indicated that the quercetin/multi-walled carbon nanotube/titanium dioxide nanocomposite had more negativity and higher recovery by glass surfaces than its counterpart. Moreover, coating surfaces with the quercetin-modified nanocomposite lowered both hydrophilicity and surface-attached bacteria compared to surfaces coated with the multi-walled carbon nanotubes/titanium dioxide nanocomposite.

Presence of Calcium Lowers the Expansion of Bacillus subtilis Colony Biofilms.

Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca2+ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca2+ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca2+ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin.

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 225 mJ/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.

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins. Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

Specific Bacillus subtilis 168 variants form biofilms on nutrient-rich medium.

Bacillus subtilis is an intensively studied Gram-positive bacterium that has become one of the models for biofilm development. B. subtilis 168 is a well-known domesticated strain that has been suggested to be deficient in robust biofilm formation. Moreover, the diversity of available B. subtilis laboratory strains and their derivatives have made it difficult to compare independent studies related to biofilm formation. Here, we analysed numerous 168 stocks from multiple laboratories for their ability to develop biofilms in different set-ups and media. We report a wide variation among the biofilm-forming capabilities of diverse stocks of B. subtilis 168, both in architecturally complex colonies and liquid-air interface pellicles, as well as during plant root colonization. Some 168 variants are indeed unable to develop robust biofilm structures, while others do so as efficiently as the non-domesticated NCIB 3610 strain. In all cases studied, the addition of glucose to the medium dramatically improved biofilm development of the laboratory strains. Furthermore, the expression of biofilm matrix component operons, epsA-O and tapA-sipW-tasA, was monitored during colony biofilm formation. We found a lack of direct correlation between the expression of these genes and the complexity of wrinkles in colony biofilms. However, the presence of a single mutation in the exopolysaccharide-related gene epsC correlates with the ability of the stocks tested to form architecturally complex colonies and pellicles, and to colonize plant roots.

The impact of manganese on biofilm development of Bacillus subtilis.

Bacterial biofilms are dynamic and structurally complex communities, involving cell-to-cell interactions. In recent years, various environmental signals that induce the complex biofilm development of the Gram-positive bacterium Bacillus subtilis have been identified. These signalling molecules are often media components or molecules produced by the cells themselves, as well as those of other interacting species. The responses can also be due to depletion of certain molecules in the vicinity of the cells. Extracellular manganese (Mn2+) is essential for proper biofilm development of B. subtilis. Mn2+ is also a component of practically all laboratory biofilm-promoting media used for B. subtilis. Comparison of complex colony biofilms in the presence or absence of supplemented Mn2+ using microarray analyses revealed that genes involved in biofilm formation are indeed downregulated in the absence of Mn2+. In addition, Mn2+ also affects the transcription of several other genes involved in distinct differentiation pathways of various cellular processes. The effects of Mn2+ on other biofilm-related traits like motility, antimicrobial production, stress and sporulation were followed using fluorescent reporter strains. The global transcriptome and morphology studies highlight the importance of Mn2+ during biofilm development and provide an overview on the expressional changes in colony biofilms in B. subtilis.

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

UNLABELLED: Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. IMPORTANCE: Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.

From environmental signals to regulators: modulation of biofilm development in Gram-positive bacteria.

Bacterial lifestyle is influenced by environmental signals, and many differentiation processes in bacteria are governed by the threshold concentrations of molecules present in their niche. Biofilm is one such example where bacteria in their sessile state adapt to a lifestyle that causes several adaptive alterations in the population. Here, a brief overview is given on a variety of environmental signals that bias biofilm development in Gram-positive bacteria, including nutrient conditions, self- and heterologously produced substances, like quorum sensing and host produced molecules. The Gram-positive model organism, Bacillus subtilis is a superb example to illustrate how distinct signals activate sensor proteins that integrate the environmental signals towards global regulators related to biofilm formation. The role of reduced oxygen level, polyketides, antimicrobials, plant secreted carbohydrates, plant cell derived polymers, glycerol, and osmotic conditions are discussed during the transcriptional activation of biofilm related genes in B. subtilis.