Applications of Atomic Force Microscopy and Nanopore Translocation in Nanoscale Microbial Surface and Single Molecules Studies

To understand the mechanism behind real-life phenomena, e.g., bacterial infection, metabolic disorders and cancer, it is becoming more and more necessary to get to the level of individual cells and single molecules. This dissertation focuses on the application of atomic force microscopy and nanopore translocation related techniques to study microbial surface characteristics and single molecule properties at the nanoscale. At the cellular level, surface characteristics of single wild type and phoP mutant Salmonella typhimurium cells were analyzed to get a better understanding about the resistance of Salmonella typhimurium to antibiotics. These bacteria were grown under different 〖Mg〗^(2+) concentrations. 〖Mg〗^(2+) is known to modulate the activities of phoP gene which regulates surface structure modifications of Salmonella typhimurium. Wild type Salmonella typhimurium surfaces were found to have an average roughness of 6.6 ± 0.9 nm for high 〖Mg〗^(2+) and 6.0 ± 1.3 nm for low 〖Mg〗^(2+) concentrations, rougher than the 5.3 ± 1.1 nm (high 〖Mg〗^(2+)) and 5.6 ± 1.5 nm (low 〖Mg〗^(2+)) for phoP mutant. In addition, mutant Salmonella typhimurium have average surface potentials of -40 ± 19 mV (high 〖Mg〗^(2+)) and 20 ± 33 mV (low 〖Mg〗^(2+)), comparing to the -65 ± 23 mV (high 〖Mg〗^(2+)) and -71 ± 27 mV (low 〖Mg〗^(2+)) of wild-type bacteria. These significant surface characteristics differences will provide insights in the important role of the phoP gene in regulating Salmonella typhimurium surface structures. On the single-molecule level, the forming components of chromatin from two esophagus cell lines, one normal (EPC2) and one cancerous (CPD), were studied using atomic force microscopy (AFM) recognition imaging. Both EPC2 and CPD chromatin samples were found to contain histone H3 and SMC2, a subunit of the condensin complex. Western blotting results supported this conclusion. Further, DNA translocation speeds through a nanopore were controlled by utilizing rolling circle replication (RCR) with Φ29 polymerases. This is a major part for future sequencing single glycosaminoglycan (GAG) molecules to resolve their structures. Translocation time on the scale of seconds, which is much longer compared to the translocation of free DNA molecules, had been detected, indicating that the polymerase successfully controlled the translocation process.