Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases.

Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.

Biocorrosion on Nanofilms Induces Rapid Bacterial Motions via Iron Dissolution.

Stability and reactivity of solid metal or mineral surfaces in contact with bacteria are critical properties for development of biocorrosion protection and for understanding bacteria-solid environmental interactions. Here, we opted to work with nanosheets of iron nanolayers offering arbitrarily large and stable areas of contact that can be simply monitored by optical means. We focused our study on the sediments' bacteria, the strain Shewanella oneidensis WT MR-1, that served as models for previous research on electroactivity and iron-reduction effects. Data show that a sudden uniform corrosion appeared after an early electroactive period without specific affinities and that iron dissolution induced rapid bacterial motions. By extending the approach to mutant strains and three bacterial species, we established a correlation between corrosion onset and oxygen-depletion combined with iron reduction and demonstrated bacteria's extraordinary ability to transform their solid environments.

Degradation Mechanism of Porous Metal-Organic Frameworks by In Situ Atomic Force Microscopy.

In recent years, Metal-Organic Frameworks (MOFs) have attracted a growing interest for biomedical applications. The design of MOFs should take into consideration the subtle balance between stability and biodegradability. However, only few studies have focused on the MOFs' stability in physiological media and their degradation mechanism. Here, we investigate the degradation of mesoporous iron (III) carboxylate MOFs, which are among the most employed MOFs for drug delivery, by a set of complementary methods. In situ AFM allowed monitoring with nanoscale resolution the morphological, dimensional, and mechanical properties of a series of MOFs in phosphate buffer saline and in real time. Depending on the synthetic route, the external surface presented either well-defined crystalline planes or initial defects, which influenced the degradation mechanism of the particles. Moreover, MOF stability was investigated under different pH conditions, from acidic to neutral. Interestingly, despite pronounced erosion, especially at neutral pH, the dimensions of the crystals were unchanged. It was revealed that the external surfaces of MOF crystals rapidly respond to in situ changes of the composition of the media they are in contact with. These observations are of a crucial importance for the design of nanosized MOFs for drug delivery applications.

Direct observation of the cell-wall remodeling in adhering Staphylococcus aureus 27217: An AFM study supported by SEM and TEM.

We took benefit from Atomic Force Microscopy (AFM) in the force spectroscopy mode to describe the time evolution - over 24 h - of the surface nanotopography and mechanical properties of the strain Staphylococcus aureus 27217 from bacterial adhesion to the first stage of biofilm genesis. In addition, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) experiments allowed identifying two types of self-adhering subpopulations (the so-called "bald" and "hairy" cells) and revealed changes in their relative populations with the bacterial culture age and the protocol of preparation. We indeed observed a dramatic evanescing of the "hairy" subpopulation for samples that underwent centrifugation and resuspension processes. When examined by AFM, the "hairy" cell surface resembled to a herringbone structure characterized by upper structural units with lateral dimensions of ∼70 nm and a high Young modulus value (∼2.3 MPa), a mean depth of the trough between them of ∼15 nm and a resulting roughness of ∼5 nm. By contrast, the "bald" cells appeared much softer (∼0.35 MPa) with a roughness one order of magnitude lower. We observed too the gradual detachment of the herringbone patterns from the "hairy" bacterial envelope of cell harvested from a 16 h old culture and their progressive accumulation between the bacteria in the form of globular clusters. The secretion of a soft extracellular polymeric substance was also identified that, in addition to the globular clusters, may contribute to the initiation of the biofilm spatial organization.

Recent advances in studying single bacteria and biofilm mechanics.

Bacterial biofilms correspond to surface-associated bacterial communities embedded in hydrogel-like matrix, in which high cell density, reduced diffusion and physico-chemical heterogeneity play a protective role and induce novel behaviors. In this review, we present recent advances on the understanding of how bacterial mechanical properties, from single cell to high-cell density community, determine biofilm tri-dimensional growth and eventual dispersion and we attempt to draw a parallel between these properties and the mechanical properties of other well-studied hydrogels and living systems.

Hydrolysis of peptidoglycan is modulated by amidation of meso-diaminopimelic acid and Mg2+ in Bacillus subtilis.

The ability of excess Mg2+ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild-type cells remains unaffected with excess Mg2+ , but the proportion of amidated meso-diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg2+ . Growth without excess Mg2+ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild-type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg2+ . Consistently, we find that Mg2+ inhibits autolysis of wild-type cells. We suggest that Mg2+ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.

Modification of the bacterial adhesion of Staphylococcus aureus by antioxidant blooming on polyurethane films.

Medical device-related infections are a major problem in hospital. The risk of developing an infection is linked to the bacterial adhesion ability of pathogen strains on the device and their ability to form a biofilm. Here we focused on polymer surfaces exhibiting a blooming of antioxidant (Irganox 3114® and Irganox 1076®) on their surface. We tried to put into evidence the effect of such a phenomenon on the bacterial adhesion in terms of number of viable cultivable bacteria and bacteria localization on the surface. We showed that the blooming has a tendency to increase the Staphylococcus aureus adhesion phenomenon in part for topographic reasons.

Viruses Occur Incorporated in Biogenic High-Mg Calcite from Hypersaline Microbial Mats.

Using three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake "La Salada de Chiprana", Spain, contain viruses with a diameter of 50-80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>10(10) viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10(9) viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as "nanobacteria" and that viruses may play a role in initiating calcification.

An in vivo study of electrical charge distribution on the bacterial cell wall by atomic force microscopy in vibrating force mode.

We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc.

In-situ determination of the mechanical properties of gliding or non-motile bacteria by atomic force microscopy under physiological conditions without immobilization.

We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.