Cellular interactions in singles- and mixed-species biofilms of bacillus subtilis

Microbiology has had a profound impact on human societies and interests. It has provided solutions for diseases and changed entire industries. In nature, microorganisms commonly live in sessile communities called biofilms. These communities have high cell densities that promote the development of communications networks based on signaling molecules, and also allow for complex interactions to form among cells forming part of the biofilm. Furthermore, cells living in natural environments are often exposed to members of other species, which may become collaborative partners, or compete for resources. The recent development of better molecular biology tools and more sophisticated microscopy techniques, along with the application of social theory and big-data informatic approaches to the study of large microbial populations has brought forward the novel field of sociomicrobiology, which tries to better understand how microbes interact with one another. This dissertation presents a comprehensive review of the current knowledge of the development of biofilms by the Gram-positive model bacterium Bacillus subtilis, with a focus on the mechanisms and signals that mediate the interactions that this bacterium can establish, both among its own cells and with those of other species. It includes original research on the interactions that B. subtilis can develop with other soil bacteria, both as active members of a predator-prey relationship, and as providers of environmental cues that change the structure of B. subtilis biofilms. Additionally, the present work includes investigations on the genetic differences between B. subtilis strains and strain variants that impact phenotypic social behavior and biofilm formation. This dissertation also includes the first comprehensive inquiry about the total effect that a family of regulatory phosphatases has upon the population heterogeneity of B. subtilis and its adaptability to diverse environments and growth conditions

The ecological impact of a bacterial weapon:microbial interactions and the Type VI secretion system

Bacteria inhabit all known ecological niches and establish interactions with organisms from all kingdoms of life. These interactions are mediated by a wide variety of mechanisms and very often involve the secretion of diverse molecules from the bacterial cells. The Type VI secretion system (T6SS) is a bacterial protein secretion system that uses a bacteriophage-like machinery to secrete a diverse array of effectors, usually translocating them directly into neighbouring cells. These effectors display toxic activity in the recipient cell, making the T6SS an effective weapon during inter-bacterial competition and interactions with eukaryotic cells. Over the last two decades, microbiology research has experienced a shift towards using systems-based approaches to study the interactions between diverse organisms and their communities in an ecological context. Here, we focus on this aspect of the T6SS. We consider how our perspective of the T6SS has developed and examine what is currently known about the impact that bacteria deploying the T6SS can have in diverse environments, including niches associated with plants, insects and mammals. We consider how T6SS-mediated interactions can affect host organisms by shaping their microbiota, as well as the diverse interactions that can be established between different microorganisms through the deployment of this versatile secretion system

Draft Genome Sequence of the Soil Isolate Lysinibacillus fusiformis M5, a Potential Hypoxanthine Producer

Lysinibacillus fusiformis strain M5 is a potential hypoxanthine producer that was isolated from clay soil. Here, we present the draft genome sequence that was annotated in order to facilitate future studies of L. fusiformis M5

Lysinibacillus fusiformis M5 induces increased complexity in Bacillus subtilis 168 colony biofilms via hypoxanthine:Running Title: L. fusiformis M5 interaction with B. subtilis 168

ABSTRACT 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. </jats:p

The Ectomycorrhizospheric Habitat of Norway Spruce and Tricholoma vaccinum: Promotion of Plant Growth and Fitness by a Rich Microorganismic Community

The contribution of the mycorrhizospheric microbes in a stand of ectomycorrhizal Norway spruce (Picea abies) featuring mycorrhiza with the basidiomycete Tricholoma vaccinum was addressed by microbiome analysis and in vitro reconstruction of microbial as well as plant-microbe interactions. The protective role of the mycorrhizal fungus with respect to pathogen attack could be validated against Botrytis cinerea and Heterobasidion annosum in co-cultures revealing reduced pathogen growth, higher survival rate of the spruce trees and reduced symptoms on needles upon symbiosis with T. vaccinum. The community structure was shown to yield a high diversity in ECM forming basidiomycetes of Thelephorales and Agaricales associated with a rich bacterial diversity dominated by Rhizobiales with the most abundant Nitrobacter winogradski (3.9%). Isolated bacteria were then used to address plant growth promoting abilities, which included production of the phytohormone indole-3-acetic acid (performed by 74% of the bacterial isolates), siderophores (22%), and phosphate mobilization (23%). Among the isolates, mycorrhiza helper bacteria (MHB) were identified, with Bacillus cereus MRZ-1 inducing hyperbranching in T. vaccinum, supporting tree germination, shoot elongation, and root formation as well as higher mycorrhization rates. Thus, a huge pool of potential MHB and fungal community with widely distributed auxin-production potential extended the ability of T. vaccinum to form ectomycorrhiza. The forest community profited from the mycorrhizal fungus T. vaccinum, with spruce survival enhanced by 33% in microcosms using soil from the native habitat. A higher fungal abundance and diversity in cases where the tree had died during the experiment, showing that decomposition of plant litter from a dead tree supported a different community. T. vaccinum thus actively structured the community of microorganisms in its habitat

Rhs NADase effectors and their immunity proteins are exchangeable mediators of interbacterial competition in Serratia

Abstract Many bacterial species use Type VI secretion systems (T6SSs) to deliver anti-bacterial effector proteins into neighbouring bacterial cells, representing an important mechanism of inter-bacterial competition. Specific immunity proteins protect bacteria from the toxic action of their own effectors, whilst orphan immunity proteins without a cognate effector may provide protection against incoming effectors from non-self competitors. T6SS-dependent Rhs effectors contain a variable C-terminal toxin domain (CT), with the cognate immunity protein encoded immediately downstream of the effector. Here, we demonstrate that Rhs1 effectors from two strains of Serratia marcescens, the model strain Db10 and clinical isolate SJC1036, possess distinct CTs which both display NAD(P)+ glycohydrolase activity but belong to different subgroups of NADase from each other and other T6SS-associated NADases. Comparative structural analysis identifies conserved functions required for NADase activity and reveals that unrelated NADase immunity proteins utilise a common mechanism of effector inhibition. By replicating a natural recombination event, we show successful functional exchange of CTs and demonstrate that Db10 encodes an orphan immunity protein which provides protection against T6SS-delivered SJC1036 NADase. Our findings highlight the flexible use of Rhs effectors and orphan immunity proteins during inter-strain competition and the repeated adoption of NADase toxins as weapons against bacterial cells

A duo of Potassium-responsive Histidine Kinases govern the multicellular destiny of Bacillus subtilis

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.Fil: Grau, Roberto Ricardo. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Oña, Paula. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kunert, Maritta. Instituto Max Planck Institut Fur Chemische Okologie; AlemaniaFil: Leñini, Cecilia Andrea. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gallegos Monterrosa, Ramses. Universitat Jena; AlemaniaFil: Mhatre, Eisha. Universitat Jena; AlemaniaFil: Vileta, Darío. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Donato, Veronica. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hölscher, Theresa. Universitat Jena; AlemaniaFil: Boland, Wilhem. Instituto Max Planck Institut Fur Chemische Okologie; AlemaniaFil: Kuipers, Oscar P.. University of Groningen; Países BajosFil: Kovács, Ákos T.. Universitat Jena; Alemani