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The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics 1 , 2 . Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor 1 , 3 . In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength 4 – 6 . Here we applied atomic force microscopy 7 – 12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface 13 , 14 , providing information complementary to traditional structural biology approaches. Using high-resolution atomic force microscopy of live cells, the authors present an updated view of the cell walls of both Staphylococcus aureus and Bacillus subtilis .

Investigating the interaction patterns at nano-bio interface is a key challenge for safe use of nanoparticles (NPs) to any biological system. The study intends to explore the role of interaction pattern at the iron oxide nanoparticle (IONP)-bacteria interface affecting antimicrobial propensity of IONP. To this end, IONP with magnetite like atomic arrangement and negative surface potential (n-IONP) was synthesized by co-precipitation method. Positively charged chitosan molecule coating was used to reverse the surface potential of n-IONP, i.e. positive surface potential IONP (p-IONP). The comparative data from fourier transform infrared spectroscope, XRD and zeta potential analyzer indicated the successful coating of IONP surface with chitosan molecule. Additionally, the nanocrystals obtained were found to have spherical size with 10–20 nm diameter. The BacLight fluorescence assay, bacterial growth kinetic and colony forming unit studies indicated that n-IONP (<50 μM) has insignificant antimicrobial activity against Bacillus subtilis and Escherichia coli . However, coating with chitosan molecule resulted significant increase in antimicrobial propensity of IONP. Additionally, the assay to study reactive oxygen species (ROS) indicated relatively higher ROS production upon p-IONP treatment of the bacteria. The data, altogether, indicated that the chitosan coating of IONP result in interface that enhances ROS production, hence the antimicrobial activity.

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.

Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

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.

Chlorogenic acid (CGA), a natural phenolic compound, is an important bioactive compound, and its antibacterial activity has been widely concerned, but its antibacterial mechanism remains largely unknown. Protein leakage and the solution exosmosis conductivity of Bacillus subtilis 24434 (B. subtilis) reportedly display no noticeable differences before and after CGA treatment. The bacterial cells treated with CGA displayed a consistently smooth surface under the electron microscope, indicating that CGA cannot directly disrupt bacterial membranes. However, CGA induced a significant decrease in the intracellular adenosine triphosphate (ATP) concentration, possibly by affecting the material and energy metabolism or cell-signaling transduction. Furthermore, metabolomic results indicated that CGA stress had a bacteriostatic effect by inducing the intracellular metabolic imbalance of the tricarboxylic acid (TCA) cycle and glycolysis, leading to metabolic disorder and death of B. subtilis. These findings improve the understanding of the complex action mechanisms of CGA antimicrobial activity and provide theoretical support for the application of CGA as a natural antibacterial agent.

Growing tissue and bacterial colonies are active matter systems where cell divisions and cellular motion generate active stress. Although they operate in the non-equilibrium regime, these biological systems can form large-scale ordered structures. How mechanical instabilities drive the dynamics of active matter systems and form ordered structures are not well understood. Here, we use chaining Bacillus subtilis , also known as a biofilm, to study the relation between mechanical instabilities and nematic ordering. We find that bacterial biofilms have intrinsic length scales above which a series of mechanical instabilities occur. Localized stress and friction drive buckling and edge instabilities which further create nematically aligned structures and topological defects. We also observe that topological defects control stress distribution and initiate the formation of sporulation sites by creating three-dimensional structures. In this study we propose an alternative active matter platform to study the essential roles of mechanics in growing biological tissue. Active nematics consist of elongated self-driven units and can emerge in bacterial systems. Here the authors investigate such emergence in a growing bacterial biofilm and demonstrate that localized stress and mechanical instabilities drive the formation of large-scale order.

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Bacterial adhesion onto mineral surfaces and subsequent biofilm formation play key roles in aggregate stability, mineral weathering and the fate of contaminants in soils. However, the mechanisms of bacteria-mineral interactions are not fully understood. Atomic force microscopy (AFM) was used to determine the adhesion forces between bacteria and goethite in water and to gain insight into the nanoscale surface morphology of the bacteria-mineral aggregates and biofilms formed on clay-sized minerals. This study yields direct evidence of a range of different association mechanisms between bacteria and minerals. All strains studied adhered predominantly to the edge surfaces of kaolinite rather than to the basal surfaces. Bacteria rarely formed aggregates with montmorillonite, but were more tightly adsorbed onto goethite surfaces. This study reports the first measured interaction force between bacteria and a clay surface and the approach curves exhibited jump-in events with attractive forces of 97 ± 34 pN between E. coli and goethite. Bond strengthening between them occurred within 4 s to the maximum adhesion forces and energies of −3.0 ± 0.4 nN and −330 ± 43 aJ (10 −18  J), respectively. Under the conditions studied, bacteria tended to form more extensive biofilms on minerals under low rather than high nutrient conditions.

In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.