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Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.

A six-laboratory session biofilm-focused biochemistry laboratory module is described. Through just six four-hour laboratory sessions over three weeks, students first master background knowledge and fundamental experimental techniques to explore questions involving bacterial biofilms at the interface of microbiology and biochemistry and then execute self-designed experiments to make novel discoveries. Foundational biofilm experiments in the course-based undergraduate research experiences (CURE) module yield visually striking data with distinct phenotypes that are easy to differentiate and present numerous avenues for experimental exploration and discovery. The module was designed to incorporate the five components of a CURE course: broadly relevant or important work, use of scientific processes, collaboration, discovery, and iteration. The six-session CURE module is attractive to incorporate into a Chemistry or Biochemistry curriculum that may not have flexibility to incorporate a full quarter or semester long CURE.

Taxol (paclitaxel, PTX) is an important anticancer drug that owes it unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. Experimental results from [sup.2.H]{[sup.19.F}] rotational-echo double resonance nuclear magnetic resonance (REDOR NMR) are presented that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.

Solid-state NMR is used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to five different vancomycin, eremomycin, and chloroeremomycin derivatives. The (super 13)C{(super 9)F} rotational-echo double-resonance (REDOR) results indicate a single fluorine dephasing center in each peptidoglycan complex.

Research has been conducted on Staphylococcus aureus intact cells harvested during stationary or exponential growth conditions. The study of these cells carried out via the use of cross-polarization magic-angle spinning and rotational-echo double resonance NMR experiments in the presence of vancomycin allows to determine peptidoglycan cross-link concentrations and suggests that vancomycin interrupts the synthesis of peptidoglycans via interference with transglycosylation.

Results demonstrate that the vancomycin analogue LY329332 binds to peptidoglycan of Staphylococcus aureus in a monomeric form as determined by the rotational-echo double resonance magic angle spinning nuclear magnetic resonance spectoscopy. Characterization of the in situ structure of the antibiotic binding site in the cell wall peptidoglycan is presented.

Fluorescence and NMR spectroscopy have been used to examine microtubule-bound paclitaxel conformation. Results indicate that this amino group has not affected the ligand-receptor association.