scholarly journals RemA (YlzA) and RemB (YaaB) Regulate Extracellular Matrix Operon Expression and Biofilm Formation in Bacillus subtilis

2009 ◽  
Vol 191 (12) ◽  
pp. 3981-3991 ◽  
Author(s):  
Jared T. Winkelman ◽  
Kris M. Blair ◽  
Daniel B. Kearns
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Pellicle Formation ◽  
Multicellular Aggregates ◽  
The Matrix ◽  
Matrix Components ◽  
Operon Expression ◽  
Motility Inhibition

ABSTRACT Biofilms are multicellular aggregates stabilized by an extracellular matrix. In Bacillus subtilis, the biofilm matrix is composed of an extracellular polysaccharide and the secreted protein TasA. Expression of both of the matrix components is repressed by the DNA-binding master regulator, SinR. Here we identify two small protein regulators of the extracellular matrix: RemA (formerly YlzA) and RemB (formerly YaaB). Mutation of RemA or RemB impairs pellicle formation, complex colony architecture, and motility inhibition in a sinR mutant background. Both proteins are required for the activation of the matrix biosynthesis operons and appear to act in parallel to SinR and two other known biofilm regulators, AbrB and DegU.

scholarly journals Amyloid fibers provide structural integrity to Bacillus subtilis biofilms

2010 ◽  
Vol 107 (5) ◽  
pp. 2230-2234 ◽  
Author(s):  
Diego Romero ◽  
Claudio Aguilar ◽  
Richard Losick ◽  
Roberto Kolter
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Biological Activity ◽  
Biofilm Formation ◽  
Structural Integrity ◽  
Wild Type ◽  
Amyloid Fibers ◽  
The Matrix ◽  
Biochemical Analyses ◽  
Main Components

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10–15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

scholarly journals Biofilm hydrophobicity in environmental isolates of Bacillus subtilis

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Margarita Kalamara ◽  
James C. Abbott ◽  
Cait E. MacPhee ◽  
Nicola R. Stanley-Wall
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Biofilm Formation ◽  
Type Species ◽  
Diverse Range ◽  
Content Type ◽  
Link Type ◽  
The Matrix ◽  
Highly Hydrophobic

Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our novel isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason for the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

scholarly journals Biofilm hydrophobicity in environmental isolates of Bacillus subtilis

2021 ◽  
Author(s):  
Margarita Kalamara ◽  
James C. Abbott ◽  
Cait E. MacPhee ◽  
Nicola. R. Stanley-Wall
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Biofilm Formation ◽  
Environmental Isolates ◽  
Diverse Range ◽  
European Nucleotide Archive ◽  
Universal Feature ◽  
The Matrix ◽  
Highly Hydrophobic

AbstractBiofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our new isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason behind the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.RepositoriesRaw sequence reads and annotated assemblies have been submitted to the European Nucleotide Archive under accession PRJEB43128.

scholarly journals Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Yunrong Chai ◽  
Pascale B. Beauregard ◽  
Hera Vlamakis ◽  
Richard Losick ◽  
Roberto Kolter
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Carbon Sources ◽  
Galactose Metabolism ◽  
Content Type ◽  
Leloir Pathway ◽  
The Matrix ◽  
Living Organisms ◽  
Galactose Metabolites ◽  
Biofilm Matrix

ABSTRACTGalactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. InBacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since thegalEmutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to agalEnull mutant in the presence of galactose, we identified mutations insinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role inB. subtilisbiofilm formation and that the expressions of both thegalandepsgenes are interrelated. Finally, we propose thatB. subtilisand other members of theBacillusgenus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.IMPORTANCEBacteria switch from unicellular to multicellular states by producing extracellular matrices that contain exopolysaccharides. In such aggregates, known as biofilms, bacteria are more resistant to antibiotics. This makes biofilms a serious problem in clinical settings. The resilience of biofilms makes them very useful in industrial settings. Thus, understanding the production of biofilm matrices is an important problem in microbiology. In studying the synthesis of the biofilm matrix ofBacillus subtilis, we provide further understanding of a long-standing microbiological observation that certain mutants defective in the utilization of galactose became sensitive to it. In this work, we show that the toxicity observed before was because cells were grown under conditions that were not propitious to produce the exopolysaccharide component of the matrix. When cells are grown under conditions that favor matrix production, the toxicity of galactose is relieved. This allowed us to demonstrate that galactose metabolism is essential for the synthesis of the extracellular matrix.

scholarly journals The Exo-Polysaccharide Component of Extracellular Matrix is Essential for the Viscoelastic Properties of Bacillus subtilis Biofilms

2020 ◽  
Vol 21 (18) ◽  
pp. 6755 ◽  
Author(s):  
Santosh Pandit ◽  
Mina Fazilati ◽  
Karolina Gaska ◽  
Abderahmane Derouiche ◽  
Tiina Nypelö ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Mechanical Stress ◽  
Viscoelastic Properties ◽  
Mechanical Stability ◽  
Growth Dynamics ◽  
Interfacial Rheology ◽  
The Matrix ◽  
Infection Processes ◽  
Biofilm Matrix

Bacteria are known to form biofilms on various surfaces. Biofilms are multicellular aggregates, held together by an extracellular matrix, which is composed of biological polymers. Three principal components of the biofilm matrix are exopolysaccharides (EPS), proteins, and nucleic acids. The biofilm matrix is essential for biofilms to remain organized under mechanical stress. Thanks to their polymeric nature, biofilms exhibit both elastic and viscous mechanical characteristics; therefore, an accurate mechanical description needs to take into account their viscoelastic nature. Their viscoelastic properties, including during their growth dynamics, are crucial for biofilm survival in many environments, particularly during infection processes. How changes in the composition of the biofilm matrix affect viscoelasticity has not been thoroughly investigated. In this study, we used interfacial rheology to study the contribution of the EPS component of the matrix to viscoelasticity of Bacillus subtilis biofilms. Two strategies were used to specifically deplete the EPS component of the biofilm matrix, namely (i) treatment with sub-lethal doses of vitamin C and (ii) seamless inactivation of the eps operon responsible for biosynthesis of the EPS. In both cases, the obtained results suggest that the EPS component of the matrix is essential for maintaining the viscoelastic properties of bacterial biofilms during their growth. If the EPS component of the matrix is depleted, the mechanical stability of biofilms is compromised and the biofilms become more susceptible to eradication by mechanical stress.

scholarly journals Salmonella Extracellular Matrix Components Influence Biofilm Formation and Gallbladder Colonization

2016 ◽  
Vol 84 (11) ◽  
pp. 3243-3251 ◽  
Author(s):  
Haley E. Adcox ◽  
Erin M. Vasicek ◽  
Varun Dwivedi ◽  
Ky V. Hoang ◽  
Joanne Turner ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Typhoid Fever ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Bacterial Survival ◽  
Content Type ◽  
Extracellular Matrix Components ◽  
Matrix Components

Salmonella enterica serovar Typhi, the causative agent of typhoid fever in humans, forms biofilms encapsulated by an extracellular matrix (ECM). Biofilms facilitate colonization and persistent infection in gallbladders of humans and mouse models of chronic carriage. Individual roles of matrix components have not been completely elucidated in vitro or in vivo . To examine individual functions, strains of Salmonella enterica serovar Typhimurium, the murine model of S . Typhi, in which various ECM genes were deleted or added, were created to examine biofilm formation, colonization, and persistence in the gallbladder. Studies show that curli contributes most significantly to biofilm formation. Expression of Vi antigen decreased biofilm formation in vitro and virulence and bacterial survival in vivo without altering the examined gallbladder pro- or anti-inflammatory cytokines. Oppositely, loss of all ECM components (Δ wcaM Δ csgA Δ yihO Δ bcsE ) increased virulence and bacterial survival in vivo and reduced gallbladder interleukin-10 (IL-10) levels. Colanic acid and curli mutants had the largest defects in biofilm-forming ability and contributed most significantly to the virulence increase of the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant strain. While the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant was not altered in resistance to complement or growth in macrophages, it attached and invaded macrophages better than the wild-type (WT) strain. These data suggest that ECM components have various levels of importance in biofilm formation and gallbladder colonization and that the ECM diminishes disseminated disease in our model, perhaps by reducing cell attachment/invasion and dampening inflammation by maintaining/inducing IL-10 production. Understanding how ECM components aid acute disease and persistence could lead to improvements in therapeutic treatment of typhoid fever patients.

scholarly journals KinD Is a Checkpoint Protein Linking Spore Formation to Extracellular-Matrix Production in Bacillus subtilis Biofilms

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Claudio Aguilar ◽  
Hera Vlamakis ◽  
Alejandra Guzman ◽  
Richard Losick ◽  
Roberto Kolter
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Cell Types ◽  
Deficient Mutant ◽  
Content Type ◽  
Checkpoint Protein ◽  
Extracellular Matrix Production ◽  
The Matrix ◽  
Matrix Production ◽  
Low Levels

ABSTRACTBacillus subtiliscells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion ofkinDsuppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.IMPORTANCEA question in the field of biofilm development has remained virtually unaddressed: how do the biofilm cells sense the completion of the synthesis of extracellular matrix? The presence of an extracellular matrix that holds the cells together is a defining feature of biofilms. How cells sense that matrix has been assembled and how this signal is transduced have not been investigated.Bacillus subtilisprovides an excellent system in which to address this question, as the molecular pathways involved in regulation of differentiation are well characterized. We provide the first evidence for a protein that senses the presence of matrix. We identify a membrane sensor histidine kinase, KinD, that alters its activity, depending on the presence or absence of the extracellular matrix. This activity feeds back to the master regulator Spo0A to regulate expression of genes involved in producing matrix and genes necessary for the progression into sporulation.

scholarly journals The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 682-691 ◽  
Author(s):  
Jan Gerwig ◽  
Taryn B. Kiley ◽  
Katrin Gunka ◽  
Nicola Stanley-Wall ◽  
Jörg Stülke
Keyword(s):  
Bacillus Subtilis ◽  
Tyrosine Kinases ◽  
Biofilm Formation ◽  
Protein Tyrosine Kinase ◽  
Specific Interaction ◽  
Significant Proportion ◽  
Extracellular Polysaccharide ◽  
Extracellular Proteins ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

scholarly journals Quorum sensing in Bacillus subtilis slows down biofilm formation by enabling sporulation bet hedging

2019 ◽  
Author(s):  
Mihael Spacapan ◽  
Tjaša Danevčič ◽  
Polonca Štefanic ◽  
Ines Mandic-Mulec
Keyword(s):  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bet Hedging ◽  
Wild Type ◽  
Master Regulator ◽  
Matrix Components ◽  
Sporulation Initiation ◽  
Biofilm Development ◽  
Biofilm Matrix

1.2ABSTRACTThe ComQXPA quorum sensing (QS) system of Bacillus subtilis, a Gram-positive, industrially relevant, endospore forming bacterium, promotes surfactin production. This lipopeptide increases transcription of several genes involved in biofilm matrix synthesis via the Spo0A-P master regulator. We hypothesized that the inactivation of the QS system will therefore result in decreased rates of floating biofilm formation. We find that this is not the case and that the QS deficient mutant forms pellicles with a faster rate and produces more biofilm matrix components than the wild type. As Spo0A-P is the master regulator of sporulation initiation we hypothesized that the ComQXPA dependent signaling promotes sporulation and consequently slows the growth rate of the wild type strain. Indeed, our results confirm that cells with the inactive QS initiate endospore formation in biofilms later and more synchronously than the wild type, as evidenced by spore frequencies and the PspoIIQ promoter activity. We argue, that the QS system acts as a switch that promotes stochastic sporulation initiation and consequently bet hedging behavior. By committing a subpopulation of cells to sporulation early during growth, wild type population grows slower and produces thinner biofilms but also assures better survival under stressful conditions.1.1IMPORTANCEBacillus subtilis is widely employed model organism to study biofilm formation and sporulation in Gram-positive bacteria. The ComQXPA quorum sensing (QS) system indirectly increases the transcription of genes involved in biofilm matrix formation, which predicts a positive role of this QS in biofilm development Here we show that QS mutants actually form more matrix components per pellicle than the wild type and that their pellicles are thicker and form with a faster rate. We explain this, by showing that cells with an inactive QS exhibit a delay in sporulation entry, which is also more synchronous relative to the wild type. We argue, that the ComQXPA QS system acts as a switch that contributes to the stochastic sporulation initiation and though this path promotes bet hedging behavior. This finding is important in terms of “quorum quenching” strategies aiming to down modulate biofilm development through inhibition of QS signaling and underscores the richness of QS regulated phenotypic outcomes among bacterial species.

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