Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes.
ABSTRACT The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids.
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ABSTRACT: The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non-random organization and coherent motion of intracellular components are central for bacterial life-sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy-depleted, stationary-state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation-induced DNA-binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA-Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co-ordinated process, in which the energy-dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.Molecular Microbiology 02/2004; 51(2):395-405. · 4.96 Impact Factor
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ABSTRACT: The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.The EMBO Journal 04/2001; 20(5):1184-91. · 9.82 Impact Factor
Article: Dynamic proteins in bacteria.[show abstract] [hide abstract]
ABSTRACT: Growth of the bacterial cell involves proteins that assemble into dynamic localized structures that are required for cellular morphogenesis and division. During the past year, the continued application of fluorescence microscopy has led to the discovery of novel actin-like filaments involved in cell shape and plasmid DNA segregation, and to new insights into the regulation and dynamics of the Z-ring. Studies on the Min proteins, which rapidly oscillate between the cell poles to spatially regulate Z-ring assembly, has led to a biochemical basis for the oscillation and a suggestion that MinD assembles into dynamic filaments. These studies further demonstrate that the eukaryotic cytoskeleton had its origins in bacteria.Current Opinion in Microbiology 01/2003; 5(6):548-52. · 8.23 Impact Factor