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

2017 ◽  
Vol 199 (22) ◽  
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.

scholarly journals Lysinibacillus fusiformisM5 induces increased complexity inBacillus subtilis168 colony biofilms via hypoxanthine

2017 ◽  
Author(s):  
Ramses Gallegos-Monterrosa ◽  
Stefanie Kankel ◽  
Sebastian Götze ◽  
Robert Barnett ◽  
Pierre Stallforth ◽  
...  
Keyword(s):  
Soil Bacteria ◽  
Screening Method ◽  
Crop Protection ◽  
Soil Bacterium ◽  
Signal Recognition ◽  
Interspecies Interactions ◽  
Related Gene ◽  
Histidine Kinases ◽  
Multispecies Biofilms ◽  
Direct Induction

ABSTRACTIn recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, or systems biology amongst 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 withB. subtilis. We identified the soil bacteriumLysinibacillus fusiformisM5 as inducer of wrinkle-formation inB. subtiliscolonies 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 operonsepsA-OandtasA-sipW-tapA. Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation ofB. subtilisbiofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression, but rather caused by the excess of hypoxanthine withinB. subtiliscells, which may lead to cell stress and death.IMPORTANCEBiofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be favorable, 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 showcaseL. fusiformisas a soil bacterium that is able to live alongsideB. subtilisand modify the architecture of its biofilms.

scholarly journals A Dual-Species Biofilm with Emergent Mechanical and Protective Properties

2019 ◽  
Vol 201 (18) ◽  
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.

scholarly journals Burkholderia thailandensis Methylated Hydroxyalkylquinolines: Biosynthesis and Antimicrobial Activity in Cocultures

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Jennifer R. Klaus ◽  
Charlotte Majerczyk ◽  
Stephanie Moon ◽  
Natalie A. Eppler ◽  
Sierra Smith ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Soil Bacterium ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
New Information ◽  
Therapeutic Development ◽  
Carbon Side Chain

ABSTRACT The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In coculture experiments, B. thailandensis strain E264 secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis strain 168. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC, and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576 (“hmqL”). The results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NOs). Synthetic HMAQ-NO is active against B. subtilis 168, showing ∼50-fold more activity than HMAQ. Both the methyl group and the length of the carbon side chain account for the high activity of HMAQ-NO. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. IMPORTANCE The soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones that are mostly methylated 4-hydroxyalkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxyalkenylquinolines have antimicrobial activity against other species. We show that Bacillus subtilis strain 168 is particularly susceptible to N-oxidated methylalkenylquinolines (HMAQ-NOs). We confirmed that HMAQ-NO biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxyalkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

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

2014 ◽  
Vol 81 (1) ◽  
pp. 203-210 ◽  
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.

scholarly journals Negative Interplay between Biofilm Formation and Competence in the Environmental Strains of Bacillus subtilis

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Qianxuan She ◽  
Evan Hunter ◽  
Yuxuan Qin ◽  
Samantha Nicolau ◽  
Eliza A. Zalis ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Differentiation ◽  
Biofilm Formation ◽  
Competence Development ◽  
Bacterial Biofilm ◽  
Soil Bacterium ◽  
Natural Competence ◽  
Content Type ◽  
Pathway Regulation

The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm.

scholarly journals Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

2015 ◽  
Vol 197 (13) ◽  
pp. 2129-2138 ◽  
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.

scholarly journals Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms

2020 ◽  
Vol 202 (18) ◽  
Author(s):  
Giulia Orazi ◽  
Fabrice Jean-Pierre ◽  
George A. O’Toole
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Respiratory Infections ◽  
Multiple Infection ◽  
Bacterial Biofilms ◽  
Interspecies Interactions ◽  
Chronic Infections ◽  
Content Type

ABSTRACT The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

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