Nicola R. Stanley-Wall

Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes

Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Critical to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnological uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the molecules and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, experimental techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars.

DegU co-ordinates multicellular behaviour exhibited by Bacillus subtilis

Unicellular organisms use a variety of mechanisms to co‐ordinate activity within a community and accomplish complex multicellular processes. Because some of the processes that are exhibited by one species can be physiologically incompatible, it raises the question of how entry into these different pathways is regulated. In the Gram‐positive bacterium Bacillus subtilis, genetic competence, swarming motility, biofilm formation, complex colony architecture and protease production are all regulated by the response regulator DegU. DegU appears to integrate environmental signals and co‐ordinate multicellular behaviours that are subsequently manifested at different levels of DegU phosphorylation. Data are presented which indicate that: (i) swarming motility is activated by very low levels of DegU∼P that can be generated independently from its cognate sensor kinase DegS; (ii) complex colony architecture is activated by low levels of DegU∼P that are produced in a DegS‐dependent manner to activate transcription of yvcA, a novel gene required for complex colony architecture; and (iii) high levels of DegU∼P inhibit complex colony architecture and swarming motility but are required prior to the activation of exoprotease production. A model is proposed to explain why such a system may have evolved within B. subtilis to control these multicellular processes through a single regulator.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

A pivotal role for the response regulator DegU in controlling multicellular behaviour

Bacteria control multicellular behavioural responses, including biofilm formation and swarming motility, by integrating environmental cues through a complex regulatory network. Heterogeneous gene expression within an otherwise isogenic cell population that allows for differentiation of cell fate is an intriguing phenomenon that adds to the complexity of multicellular behaviour. This review focuses on recent data about how DegU, a pleiotropic response regulator, co-ordinates multicellular behaviour in Bacillus subtilis. We review studies that challenge the conventional understanding of the molecular mechanisms underpinning the DegU regulatory system and others that describe novel targets of DegU during activation of biofilm formation by B. subtilis. We also discuss a novel role for DegU in regulating multicellular processes in the food-borne pathogen Listeria monocytogenes.

YuaB functions synergistically with the exopolysaccharide and TasA amyloid fibers to allow biofilm formation by Bacillus subtilis.

During biofilm formation by Bacillus subtilis, two extracellular matrix components are synthesized, namely, the TasA amyloid fibers and an exopolysaccharide. In addition, a small protein called YuaB has been shown to allow the biofilm to form. The regulatory protein DegU is known to initiate biofilm formation. In this report we show that the main role of DegU during biofilm formation is to indirectly drive the activation of transcription from the yuaB promoter. The N terminus of YuaB constitutes a signal peptide for the Sec transport system. Here we show that the presence of the signal peptide is required for YuaB function. In addition we demonstrate that upon export of YuaB from the cytoplasm, it localizes to the cell wall. We continue with evidence that increased production of TasA and the exopolysaccharide is not sufficient to overcome the effects of a mutation in yuaB, demonstrating the unique involvement of YuaB in forming a biofilm. In line with this, YuaB is not involved in correct synthesis, export, or polymerization of either the TasA amyloid fibers or the exopolysaccharide. Taken together, these findings identify YuaB as a protein that plays a novel role during biofilm formation. We hypothesize that YuaB functions synergistically with the known components of the biofilm matrix to facilitate the assembly of the biofilm matrix.

Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms

Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram‐positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix.

DegU and Spo0A Jointly Control Transcription of Two Loci Required for Complex Colony Development by Bacillus subtilis

Biofilm formation is an example of a multicellular process which depends on cooperative behavior and differentiation within a bacterial population. Our findings indicate that there is a complex feedback loop that maintains the stoichiometry of the extracellular matrix and other proteins required for complex colony development by Bacillus subtilis. Analysis of the transcriptional regulation of two DegU-activated genes that are required for complex colony development by B. subtilis revealed additional involvement of global regulators that are central to controlling biofilm formation. Activation of transcription from both the yvcA and yuaB promoters requires DegU approximately phosphate, but transcription is inhibited by direct AbrB binding to the promoter regions. Inhibition of transcription by AbrB is relieved when Spo0A approximately phosphate is generated due to its known role in inhibiting abrB expression. Deletion of SinR, a key coordinator of motility and biofilm formation, enhanced transcription from both loci; however, no evidence of a direct interaction with SinR for either the yvcA or yuaB promoter regions was observed. The enhanced transcription in the sinR mutant background was subsequently demonstrated to be dependent on biosynthesis of the polysaccharide component that forms the major constituent of the B. subtilis biofilm matrix. Together, these findings indicate that a genetic network dependent on activation of both DegU and Spo0A controls complex colony development by B. subtilis.

A mechanical signal transmitted by the flagellum controls signalling in Bacillus subtilis

In the natural environment bacteria predominantly live adhered to a surface as part of a biofilm. While many of the components needed for biofilm assembly are known, the mechanism by which microbes sense and respond to contact with a surface is poorly understood. Bacillus subtilis is a Gram‐positive model for biofilm formation. The DegS–DegU two‐component system controls several multicellular behaviours in B. subtilis, including biofilm formation. Here we identify the B. subtilis flagellum as a mechanosensor that activates the DegS–DegU regulatory pathway. Inhibition of flagellar rotation by deletion or mutation of the flagellar stator gene, motB, results in an increase in both degU transcription and DegU∼P driven processes, namely exoprotease production and poly‐γ‐dl‐glutamic acid biosynthesis. Similarly, inhibition of flagellar rotation by engaging the flagellar clutch or by tethering the flagella with antibodies also promotes an increase in degU transcription that is reflective of increased DegU∼P levels in the cell. Collectively, these findings strongly indicate that inhibition of flagellar rotation acts as a mechanical trigger to activate the DegS–DegU two‐component signal transduction system. We postulate that inhibition of flagellar rotation could function as a mechanical trigger to activate bacterial signal transduction cascades in many motile bacteria upon contact with a surface.

Evolution and Multiplicity of Arginine Decarboxylases in Polyamine Biosynthesis and Essential Role in Bacillus subtilis Biofilm Formation

Arginine decarboxylases (ADCs; EC 4.1.1.19) from four different protein fold families are important for polyamine biosynthesis in bacteria, archaea, and plants. Biosynthetic alanine racemase fold (AR-fold) ADC is widespread in bacteria and plants. We report the discovery and characterization of an ancestral form of the AR-fold ADC in the bacterial Chloroflexi and Bacteroidetes phyla. The ancestral AR-fold ADC lacks a large insertion found in Escherichia coli and plant AR-fold ADC and is more similar to the lysine biosynthetic enzyme meso-diaminopimelate decarboxylase, from which it has evolved. An E. coli acid-inducible ADC belonging to the aspartate aminotransferase fold (AAT-fold) is involved in acid resistance but not polyamine biosynthesis. We report here that the acid-inducible AAT-fold ADC has evolved from a shorter, ancestral biosynthetic AAT-fold ADC by fusion of a response regulator receiver domain protein to the N terminus. Ancestral biosynthetic AAT-fold ADC appears to be limited to firmicute bacteria. The phylogenetic distribution of different forms of ADC distinguishes bacteria from archaea, euryarchaeota from crenarchaeota, double-membraned from single-membraned bacteria, and firmicutes from actinobacteria. Our findings extend to eight the different enzyme forms carrying out the activity described by EC 4.1.1.19. ADC gene clustering reveals that polyamine biosynthesis employs diverse and exchangeable synthetic modules. We show that in Bacillus subtilis, ADC and polyamines are essential for biofilm formation, and this appears to be an ancient, evolutionarily conserved function of polyamines in bacteria. Also of relevance to human health, we found that arginine decarboxylation is the dominant pathway for polyamine biosynthesis in human gut microbiota.

A degradation product of the salicylic acid pathway triggers oxidative stress resulting in down-regulation of Bacillus subtilis biofilm formation on Arabidopsis thaliana roots

Bacillus subtilis, a plant growth promoting rhizobacteria (PGPR), induces growth response and protection against pathogenic organisms through colonization and biofilm formation on the Arabidopsis thaliana root surface. In the current investigation, we utilized various Arabidopsis defense pathway mutants in a series of studies and showed that the plants recognize B. subtilis by a chemical-dependent cascade, which is independent of the salicylic acid (SA), jasmonic acid (JA), or ethylene pathways. These experiments revealed the importance of root surface chemistry in colonization and biofilm formation by B. subtilis. It was found that B. subtilis FB17 could not form biofilms on the roots of NahG, a transgenic Arabidopsis line for salicylate hydroxylase that produces catechol as the degradation product of SA. These findings suggest that catechol may play a direct role in inhibiting B. subtilis FB17 biofilm formation on the NahG root surface, possibly through induction of reactive oxygen species (ROS) in the roots. Using both in vitro microtitre plate and in planta assays we confirmed that catechol inhibited biofilm formation, but not the planktonic growth, of B. subtilis. Inhibition of biofilm formation was shown to be the result of a physiological response by B. subtilis to the presence of catechol, which resulted in the down-regulation of transcription of the yqxM-sipW-tasA and epsA-O operons, both of which are required for biofilm formation by B. subtilis. These data indicate that the suppression of biofilm formation on NahG plants was strongly influenced by the root-derived catechol production through ROS-mediated down-regulation of B. subtilis biofilm genes.