Abstract
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.
Highlights
Atomic force microscopy (AFM) is a very powerful tool to get precious information about the nanoscale surface architecture of cells, the localization and interactions of their individual constituents with various internal and external agents
In order to increase the adhesion of microorganisms, such as bacteria, to surfaces for AFM imaging in liquid, many protocols have been proposed
We successively studied two categories of bacteria: first Rhodococcus wratislaviensis [37], a bacterium currently used for degradation of mixtures of hydrocarbons, known to be non-motile, and, second, Nostoc bacteria able to glide on solid samples without the help of flagella or pili
Summary
Atomic force microscopy (AFM) is a very powerful tool to get precious information about the nanoscale surface architecture of cells, the localization and interactions of their individual constituents with various internal and external agents. Many methods based on the use of various chemical ligands between the micro-organisms and the sample have been proposed such as polyphenolic proteins extracted from the marine mussel [10] Another very common technique consists of pretreating the support with polycations, such as poly-L-lysine [11] or derivatives. Mechanical entrapment in aluminum oxide filters [14] or in porous membranes such as isopore polycarbonate membranes [15] were developed to avoid slow poisoning by chemical immobilizing and proved to be effective Such entrapment methods are mainly suitable for imaging and force measurements on spherical cells. This technique may impede the monitoring of active processes such as cell division and bacteria may be in a state of mechanical stress far away from their standard living conditions. It consists in assembling the living cells on specific areas within the patterns of micro-structured, functionalized poly-dimethylsiloxane stamps using convective/capillary deposition [17]
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