monitoring-spatial-segregation-surface-colonizing-microbial

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 ...

Friedrich Schiller University, Jena

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.

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

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.

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

The impact of manganese on biofilm development of Bacillus subtilis.

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 has made it difficult to compare independent studies related to biofilm formation. Here, we have analyzed numerous 168 stocks from multiple laboratories for their ability to develop biofilms in different setups 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 interphase 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 undomesticated NCIB 3610 strain. In all studied cases, the addition of glucose to the medium dramatically improves biofilm development of the laboratory strains. Furthermore, the expressions of biofilm matrix component operons, epsA-O and tapA-sipW-tasA were monitored during colony biofilm formation. We find 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 EPS-related gene epsC does correlates with the ability of the tested stocks to form architecturally complex colonies, pellicles, and to colonize plant roots.

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

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/cm(2) 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.

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

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.

Article	Year
From environmental signals to regulators: modulation of biofilm development in Gram-positive bacteria. Journal of basic microbiology\| PubMed ID: 24771632	2014
A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis. mBio\| PubMed ID: 26152584	2015
The impact of manganese on biofilm development of Bacillus subtilis. Microbiology (Reading, England)\| PubMed ID: 27267987	2016
Specific Bacillus subtilis 168 variants do form biofilms on nutrient rich medium. Microbiology (Reading, England)\| PubMed ID: 27655338	2016
Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films. Journal of biophotonics\| PubMed ID: 27714933	2016
Application of quercetin and its bio-inspired nanoparticles as anti-adhesive agents against Bacillus subtilis attachment to surface. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 27770951	2017

Eisha Mhatre

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