Faculty Opinions recommendation of Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus.

Bacterial specialized (or secondary) metabolites are structurally diverse molecules that mediate intra- and interspecies interactions by altering growth and cellular physiology and differentiation. Bacillus subtilis , a Gram-positive model bacterium commonly used to study biofilm formation and sporulation, has the capacity to produce over ten specialized metabolites. Some of these B. subtilis specialized metabolites have been investigated for their role in facilitating cellular differentiation, only rarely has the behavior of multiple metabolites been simultaneously investigated. In this study, we explored the interconnectivity of differentiation (biofilm and sporulation) and specialized metabolites in B. subtilis . Specifically, we interrogated how development influences specialized metabolites and vice versa. Using the sporulation-inducing medium DSM, we found that the majority of the specialized metabolites examined are expressed and produced during biofilm formation and sporulation. Additionally, we found that six of these metabolites (surfactin, ComX, bacillibactin, bacilysin, subtilosin A, and plipastatin) are necessary signaling molecules for proper progression of B. subtilis differentiation. This study further supports the growing body of work demonstrating that specialized metabolites have essential physiological functions as cell-cell communication signals in bacteria. Importance/Significance Bacterially produced specialized metabolites are frequently studied for their potential use as antibiotics and antifungals. However, a growing body of work has suggested that the antagonistic potential of specialized metabolites is not their only function. Here, using Bacillus subtilis as our model bacterium, we demonstrated that developmental processes such as biofilm formation and sporulation are tightly linked with specialized metabolite gene expression and production. Additionally, under our differentiation-inducing conditions, six out of the nine specialized metabolites investigated behave as intraspecific signals that impact B. subtilis physiology and influence biofilm formation and sporulation. Our work supports the viewpoint that specialized metabolites have a clear role as cell-cell signaling molecules within differentiated populations of bacteria.

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Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1103630108 ◽

2011 ◽

Vol 108 (48) ◽

pp. E1236-E1243 ◽

Cited By ~ 62

Author(s):

E. A. Shank ◽

V. Klepac-Ceraj ◽

L. Collado-Torres ◽

G. E. Powers ◽

R. Losick ◽

...

Keyword(s):

Bacillus Subtilis ◽

Interspecies Interactions

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A Dual-Species Biofilm with Emergent Mechanical and Protective Properties

Journal of Bacteriology ◽

10.1128/jb.00670-18 ◽

2019 ◽

Vol 201 (18) ◽

Cited By ~ 12

Author(s):

Sarah M. Yannarell ◽

Gabrielle M. Grandchamp ◽

Shih-Yuan Chen ◽

Karen E. Daniels ◽

Elizabeth A. Shank

Keyword(s):

Bacillus Subtilis ◽

Relative Proportion ◽

Viscoelastic Behavior ◽

Natural World ◽

Pantoea Agglomerans ◽

Protective Mechanism ◽

Interspecies Interactions ◽

Protective Properties ◽

Content Type ◽

Microbe Interactions

ABSTRACTMany microbes coexist within biofilms, or multispecies communities of cells encased in an extracellular matrix. However, little is known about the microbe-microbe interactions relevant for creating these structures. In this study, we explored a striking dual-species biofilm betweenBacillus subtilisandPantoea agglomeransthat exhibited characteristics that were not predictable from previous work examining monoculture biofilms. Coculture wrinkle formation required aP. agglomeransexopolysaccharide as well as theB. subtilisamyloid-like protein TasA. Unexpectedly, otherB. subtilismatrix components essential for monoculture biofilm formation were not necessary for coculture wrinkling (e.g., the exopolysaccharide EPS, the hydrophobin BslA, and cell chaining). In addition,B. subtiliscell chaining prevented coculture wrinkling, even though chaining was previously associated with more robust monoculture biofilms. We also observed that increasing the relative proportion ofP. agglomerans(which forms completely featureless monoculture colonies) increased coculture wrinkling. Using microscopy and rheology, we observed that these two bacteria assemble into an organized layered structure that reflects the physical properties of both monocultures. This partitioning into distinct regions negatively affected the survival ofP. agglomeranswhile also serving as a protective mechanism in the presence of antibiotic stress. Taken together, these data indicate that studying cocultures is a productive avenue to identify novel mechanisms that drive the formation of structured microbial communities.IMPORTANCEIn the environment, many microbes form biofilms. However, the interspecies interactions underlying bacterial coexistence within these biofilms remain understudied. Here, we mimic environmentally relevant biofilms by studying a dual-species biofilm formed betweenBacillus subtilisandPantoea agglomeransand subjecting the coculture to chemical and physical stressors that it may experience in the natural world. We determined that both bacteria contribute structural elements to the coculture, which is reflected in its overall viscoelastic behavior. Existence within the coculture can be either beneficial or detrimental depending on the context. Many of the features and determinants of the coculture biofilm appear distinct from those identified in monoculture biofilm studies, highlighting the importance of characterizing multispecies consortia to understand naturally occurring bacterial interactions.

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Predation by Myxococcus xanthus Induces Bacillus subtilis To Form Spore-Filled Megastructures

Applied and Environmental Microbiology ◽

10.1128/aem.02448-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 203-210 ◽

Cited By ~ 25

Author(s):

Susanne Müller ◽

Sarah N. Strack ◽

Sarah E. Ryan ◽

Daniel B. Kearns ◽

John R. Kirby

Keyword(s):

Bacillus Subtilis ◽

Biofilm Formation ◽

Myxococcus Xanthus ◽

Common Mechanism ◽

Alternative Mechanism ◽

Dense Matrix ◽

Interspecies Interactions ◽

Content Type ◽

New Type ◽

Surfactin Production

ABSTRACTBiofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacteriumMyxococcus xanthusandBacillus subtiliswere found to induce the formation of a new type ofB. subtilisbiofilm, termed megastructures. Megastructures are tree-like brachiations that are as large as 500 μm in diameter, are raised above the surface between 150 and 200 μm, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. AsB. subtilisendospores are not susceptible to predation byM. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition,M. xanthusfruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting thatM. xanthusis unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, aB. subtilismutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitatesB. subtilisescape into dormancy via sporulation.

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Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine

Journal of Bacteriology ◽

10.1128/jb.00204-17 ◽

2017 ◽

Vol 199 (22) ◽

Cited By ~ 8

Author(s):

Ramses Gallegos-Monterrosa ◽

Stefanie Kankel ◽

Sebastian Götze ◽

Robert Barnett ◽

Pierre Stallforth ◽

...

Keyword(s):

Bacillus Subtilis ◽

Screening Method ◽

Crop Protection ◽

Soil Bacterium ◽

Signal Recognition ◽

Interspecies Interactions ◽

Content Type ◽

Lysinibacillus Fusiformis ◽

Bacillus Subtilis 168 ◽

Direct Induction

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.

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Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

Journal of Bacteriology ◽

10.1128/jb.02535-14 ◽

2015 ◽

Vol 197 (13) ◽

pp. 2129-2138 ◽

Cited By ~ 41

Author(s):

Matthew J. Powers ◽

Edgardo Sanabria-Valentín ◽

Albert A. Bowers ◽

Elizabeth A. Shank

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Biofilm Formation ◽

Chemical Compounds ◽

Natural World ◽

Specific Gene ◽

Interspecies Interactions ◽

Content Type ◽

Bacterial Development ◽

Subinhibitory Concentrations

ABSTRACTInterspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacteriumBacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identifiedPseudomonas putidaandPseudomonas protegensas bacteria that secrete compounds that inhibit biofilm gene expression inB. subtilis. The active compound produced byP. protegenswas identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies ofB. subtilisgrown adjacent to a DAPG-producingP. protegensstrain had altered colony morphologies relative toB. subtiliscolonies grown next to a DAPG-nullP. protegensstrain (phlDstrain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced inB. subtilisliquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria.IMPORTANCEBiofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that inhibited biofilm gene expression inBacillus subtilis. We identifiedPseudomonas protegensas one such bacterium and found that the biofilm-inhibiting compound it produces was the antibiotic 2,4-diacetylphloroglucinol (DAPG). We showed that even at subinhibitory concentrations, DAPG inhibits biofilm formation and sporulation inB. subtilis. These findings have potential implications for understanding the interactions between these two microbes in the natural world and support the idea that many compounds considered antibiotics can impact bacterial development at subinhibitory concentrations.

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Bacillus subtilis Protects Public Goods by Extending Kin Discrimination to Closely Related Species

mBio ◽

10.1128/mbio.00723-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 8

Author(s):

Nicholas A. Lyons ◽

Roberto Kolter

Keyword(s):

Bacillus Subtilis ◽

Large Strain ◽

Bacterial Biofilms ◽

Kin Discrimination ◽

Interspecies Interactions ◽

Bacterial Populations ◽

Single Strain ◽

Strain Collection ◽

Important Trait ◽

Cooperative Behaviors

ABSTRACT Kin discrimination systems are found in numerous communal contexts like multicellularity and are theorized to prevent exploitation of cooperative behaviors. The kin discrimination system in Bacillus subtilis differs from most other such systems because it excludes nonkin cells rather than including kin cells. Because nonkin are the target of the system, B. subtilis can potentially distinguish degrees of nonkin relatedness, not just kin versus nonkin. We examined this by testing a large strain collection of diverse Bacillus species against B. subtilis in different multicellular contexts. The effects of kin discrimination extend to nearby species, as the other subtilis clade species were treated with the same antagonism as nonkin. Species in the less-related pumilus clade started to display varied phenotypes but were mostly still discriminated against, while cereus clade members and beyond were no longer subject to kin discrimination. Seeking a reason why other species are perceived as antagonistic nonkin, we tested the ability of B. subtilis to steal communally produced surfactant from these species. We found that the species treated as nonkin were the only ones that made a surfactant that B. subtilis could utilize and that nonkin antagonism prevented such stealing when the two strains were mixed. The nonkin exclusion kin discrimination method thus allows effective protection of the cooperative behaviors prevalent in multicellularity while still permitting interactions with more distant species that are not a threat. IMPORTANCE Multicellular systems like bacterial biofilms and swarms rely on cooperative behaviors that could be undermined by exploitative invaders. Discriminating kin from nonkin is one way to help guard against such exploitation but has thus far been examined only intraspecifically, so the phylogenetic range of this important trait is unknown. We tested whether Bacillus subtilis treats other species as nonkin by testing a single strain against a diverse collection of Bacillus isolates. We found that the species in the same clade were treated as nonkin, which then lessened in more distant relatives. Further experiments showed that these nonkin species produced a cooperative good that could be stolen by B. subtilis and that treating each other as nonkin largely prevented this exploitation. These results impact our understanding of interspecies interactions, as bacterial populations can interact only after they have diverged enough to no longer be a threat to their cooperative existences. IMPORTANCE Multicellular systems like bacterial biofilms and swarms rely on cooperative behaviors that could be undermined by exploitative invaders. Discriminating kin from nonkin is one way to help guard against such exploitation but has thus far been examined only intraspecifically, so the phylogenetic range of this important trait is unknown. We tested whether Bacillus subtilis treats other species as nonkin by testing a single strain against a diverse collection of Bacillus isolates. We found that the species in the same clade were treated as nonkin, which then lessened in more distant relatives. Further experiments showed that these nonkin species produced a cooperative good that could be stolen by B. subtilis and that treating each other as nonkin largely prevented this exploitation. These results impact our understanding of interspecies interactions, as bacterial populations can interact only after they have diverged enough to no longer be a threat to their cooperative existences.

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Use of Antibody to aid Visualization of Protein at the Termini of Bacteriophage Ø29 DNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002385 ◽

1980 ◽

Vol 38 ◽

pp. 540-541

Author(s):

Dwight Anderson ◽

Charlene Peterson ◽

Gursaran Notani ◽

Bernard Reilly

Keyword(s):

Electron Microscopy ◽

Bacillus Subtilis ◽

Dna Synthesis ◽

Restriction Endonuclease ◽

Protein Complex ◽

Protein Product ◽

Viral Dna ◽

Small Proteins ◽

Antibody Labeling ◽

Subtilis Bacteriophage

The protein product of cistron 3 of Bacillus subtilis bacteriophage Ø29 is essential for viral DNA synthesis and is covalently bound to the 5’-termini of the Ø29 DNA. When the DNA-protein complex is cleaved with a restriction endonuclease, the protein is bound to the two terminal fragments. The 28,000 dalton protein can be visualized by electron microscopy as a small dot and often is seen only when two ends are in apposition as in multimers or in glutaraldehyde-fixed aggregates. We sought to improve the visibility of these small proteins by use of antibody labeling.

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