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.

Biofilm formation is a complex process involving various signaling pathways and changes in gene expression. Many of the sensory mechanisms and regulatory cascades involved have been defined for biofilms formed by diverse organisms attached to solid surfaces. By comparison, our knowledge on the basic mechanisms underlying the formation of biofilms at air-liquid interfaces, that is, pellicles, is much less complete. In particular, the roles of flagella have been studied in multiple solid-surface biofilm models but remain largely undefined for pellicles. In this work, we characterize the contributions of flagellum-based motility, chemotaxis and oxygen sensing to pellicle formation in the Gram-positive Bacillus subtilis. We confirm that flagellum-based motility is involved in, but is not absolutely essential for, B. subtilis pellicle formation. Further, we show that flagellum-based motility, chemotaxis and oxygen sensing are important for successful competition during B. subtilis pellicle formation. We report that flagellum-based motility similarly contributes to pellicle formation and fitness in competition assays in the Gram-negative Pseudomonas aeruginosa. Time-lapse imaging of static liquid cultures demonstrates that, in both B. subtilis and P. aeruginosa, a turbulent flow forms in the tube and a zone of clearing appears below the air-liquid interface just before the formation of the pellicle but only in strains that have flagella.

Motility, Chemotaxis and Aerotaxis Contribute to Competitiveness during Bacterial Pellicle Biofilm Development.

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.

Cupriavidus necator is a non-obligate bacterial predator of Gram-negative and Gram-positive bacteria. In this study, we set out to determine the conditions, which are necessary to observe predatory behavior of C. necator. Using Bacillus subtilis as a prey organism, we confirmed that the predatory performance of C. necator is correlated with the available copper level, and that the killing is mediated, at least in part, by secreted extracellular factors. The predatory activity depends on the nutrition status of C. necator, but does not require a quorum of predator cells. This suggests that C. necator is no group predator. Further analyses revealed that sporulation enables B. subtilis to avoid predation by C. necator. In contrast to the interaction with predatory myxobacteria, however, an intact spore coat is not required for resistance. Instead resistance is possibly mediated by quiescence.

Ramses Gallegos-Monterrosa

Terrestrial Biofilms Group,
Institute of Microbiology,
Terrestrial Biofilms Group, Institute of Microbiology

Friedrich Schiller University, Jena

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Ramses Gallegos-Monterrosa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Terrestrial Biofilms Group,Institute of Microbiology,Terrestrial Biofilms Group, Institute of Microbiology

Friedrich Schiller University, Jena

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Terrestrial Biofilms Group,
Institute of Microbiology,
Terrestrial Biofilms Group, Institute of Microbiology