Abstract The categorization of microbial strains is conventionally based on the molecular method, and seldom are the morphological characteristics in the bacterial strains studied. In this research, we revealed the macromolecular structures of the bacterial surface via AFM mechanical mapping, whose resolution was not only determined by the nanoscale tip size but also the mechanical properties of the specimen. This technique enabled the nanoscale study of membranous structures of microbial strains with simple specimen preparation and flexible working environments, which overcame the multiple restrictions in electron microscopy and label-enable biochemical analytical methods. The characteristic macromolecules located among cellular surface were considered as surface layer proteins and were found to be specific to the Escherichia coli genotypes, from which the averaged molecular sizes were characterized with diameters ranging from 38 to 66 nm, and the molecular shapes were kidney-like or round. In conclusion, the surface macromolecular structures have unique characteristics that link to the E. coli genotype, which suggests that the genomic effects on cellular morphologies can be rapidly identified using AFM mechanical mapping. Graphical AbstractQuantification of surface macromolecules of E. coli cells using AFM mechanical mapping. Surface macromolecules of cellular surface of three E. coli genotypes, MG1655, CFT073, and RS218, were characterized with the sizes ranging from 38 to 66 nm and with round or kidney-like shapes. The topography images were colored with adhesion mapping with the scale bars = 200 nm.
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