Final Report Summary - MICROBIALSPM (Microbial recognition and adhesion on the nano scale using BIO-SPM)
Our main goal in this proposal was the elucidation of fundamental interaction process between microbial systems and a living organism. This research directly led us to a so far missing understanding of the detailed mechanism of bacterial pathogen infection. The newly gained knowledge is highly important for the development of antibacterial drugs against pathogen-related diseases and for the ultra-sensitive detection of pathogens using bio-sensors.
Bacteria have existed for several billion years by adapting to changes in their environment. Understanding how bacteria acquire new functions in response to environmental changes will advance our fundamental knowledge thereby enhancing our ability to design and tailor changes in biological structure. We investigated local physical and biochemical variations in the bacterial outer membrane of live bacteria, at nano-meter resolutions, as bacteria interact with both abiotic and biotic surfaces. For these studies, we used Escherichia coli (E. coli), primarily because it has been well studied and the genome sequence has been determined.
Biological scanning probe microscopy (SPM) is the tool of choice for these studies because it is the only instrument that allows studying living microbial organisms in their natural environment at the nano-meter scale resolution. A broad range of scanning microscopic techniques including force spectroscopy, topography and recognition imaging, Kelvin probe force microscopy, and scanning microwave microscopy was utilized in these studies for looking into the dynamics of individual protein domains, local binding sites, and locations of charge centers of complex proteins at sub-nanometer, pico-Newton, and nano-Ampere resolution.
The main task of this proposal was to understand changes induced in microbial surface structures when microbes contact and colonize on abiotic and biotic surfaces. Under different defined environmental conditions and using various SPM techniques, we conducted our studies using E. coli for gaining a comprehensive understanding of the changes that occur in the outer membrane of E. coli from the initial contact with surfaces, and through colonization and initial biofilm formation.
We investigated the nano-characterization of bacterial structures in variations of growing culture conditions with respect to the biotic and the abiotic environment and followed morphological and functional changes of bacterial surface structures using high resolution AFM imaging and force spectroscopy. We furthermore elucidated the physical mechanisms in which growing conditions can control the expression of protein domains on bacterial outer membrane. Moreover, KPFM and SMM was used as a tool for measuring variations of local electrostatic interaction with respect to heterogeneous charge adsorptions onto bacteria cell envelopes and for studying structural and functional characteristics of the bacterial membrane:
a. Bio-SPM was used to measure topographical changes of living bacteria and single bio-molecular interactions in native and liquid conditions with nano-meter accuracy.
b. BIO-SPM was further used to map chemical and mechanical surface properties so as to elucidate dynamic bio-chemical processes on surface.
c. This study allowed for measuring the specific binding forces of individual adhesions and for mapping their force distributions on the surface of living bacteria using SPM tips bearing biologically active molecules, thereby yielding recognition images with nano-meter and pico-Newton resolution.
d. KPFM and SMM investigated the surface charge distribution of bacterial membranes, from which electrostatic interactions between specific expressed protein domains on bacterial surfaces and interfaces were identified.
The novel technology developed in this project will be of eminent importance for both molecular microbiologists as well as biophysicists for the investigation of biological processes from a completely different and new perspective, reaching unprecedented levels of sensitivity, detail and complexity.
In particular, in the context of nano-scale electrical detection methods for biological systems combined with the electrical analysis using semi-conductor technology, force spectroscopy and KPFM will serve as basis for the design of next generation semi-conductor nano-device bio-sensors and analysis methods in Europe.
In the biological point of view, we are able to follow dynamic processes in living bacteria with nanometric spatial resolution and single molecule detection sensitivity. This technological breakthrough will have a profound impact on our understanding of membrane organization in relation to function and disease, most probably leading to new discoveries in microbiology.
Moreover, potential users of this technology will also belong to material science, surface chemistry, and biotechnology and pharmaceutical industries. In particular, these industries will be strongly interested since new avenues for studying effects of drugs at the single cell level with nano-meter resolution will be opened.
Bacteria have existed for several billion years by adapting to changes in their environment. Understanding how bacteria acquire new functions in response to environmental changes will advance our fundamental knowledge thereby enhancing our ability to design and tailor changes in biological structure. We investigated local physical and biochemical variations in the bacterial outer membrane of live bacteria, at nano-meter resolutions, as bacteria interact with both abiotic and biotic surfaces. For these studies, we used Escherichia coli (E. coli), primarily because it has been well studied and the genome sequence has been determined.
Biological scanning probe microscopy (SPM) is the tool of choice for these studies because it is the only instrument that allows studying living microbial organisms in their natural environment at the nano-meter scale resolution. A broad range of scanning microscopic techniques including force spectroscopy, topography and recognition imaging, Kelvin probe force microscopy, and scanning microwave microscopy was utilized in these studies for looking into the dynamics of individual protein domains, local binding sites, and locations of charge centers of complex proteins at sub-nanometer, pico-Newton, and nano-Ampere resolution.
The main task of this proposal was to understand changes induced in microbial surface structures when microbes contact and colonize on abiotic and biotic surfaces. Under different defined environmental conditions and using various SPM techniques, we conducted our studies using E. coli for gaining a comprehensive understanding of the changes that occur in the outer membrane of E. coli from the initial contact with surfaces, and through colonization and initial biofilm formation.
We investigated the nano-characterization of bacterial structures in variations of growing culture conditions with respect to the biotic and the abiotic environment and followed morphological and functional changes of bacterial surface structures using high resolution AFM imaging and force spectroscopy. We furthermore elucidated the physical mechanisms in which growing conditions can control the expression of protein domains on bacterial outer membrane. Moreover, KPFM and SMM was used as a tool for measuring variations of local electrostatic interaction with respect to heterogeneous charge adsorptions onto bacteria cell envelopes and for studying structural and functional characteristics of the bacterial membrane:
a. Bio-SPM was used to measure topographical changes of living bacteria and single bio-molecular interactions in native and liquid conditions with nano-meter accuracy.
b. BIO-SPM was further used to map chemical and mechanical surface properties so as to elucidate dynamic bio-chemical processes on surface.
c. This study allowed for measuring the specific binding forces of individual adhesions and for mapping their force distributions on the surface of living bacteria using SPM tips bearing biologically active molecules, thereby yielding recognition images with nano-meter and pico-Newton resolution.
d. KPFM and SMM investigated the surface charge distribution of bacterial membranes, from which electrostatic interactions between specific expressed protein domains on bacterial surfaces and interfaces were identified.
The novel technology developed in this project will be of eminent importance for both molecular microbiologists as well as biophysicists for the investigation of biological processes from a completely different and new perspective, reaching unprecedented levels of sensitivity, detail and complexity.
In particular, in the context of nano-scale electrical detection methods for biological systems combined with the electrical analysis using semi-conductor technology, force spectroscopy and KPFM will serve as basis for the design of next generation semi-conductor nano-device bio-sensors and analysis methods in Europe.
In the biological point of view, we are able to follow dynamic processes in living bacteria with nanometric spatial resolution and single molecule detection sensitivity. This technological breakthrough will have a profound impact on our understanding of membrane organization in relation to function and disease, most probably leading to new discoveries in microbiology.
Moreover, potential users of this technology will also belong to material science, surface chemistry, and biotechnology and pharmaceutical industries. In particular, these industries will be strongly interested since new avenues for studying effects of drugs at the single cell level with nano-meter resolution will be opened.