The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli.

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

Chemical Analysis of a "Miller-Type" Complex Prebiotic Broth : Part II: Gas, Oil, Water and the Oil/Water-Interface.

We have analyzed the chemical variety obtained by Miller-Urey-type experiments using nuclear magnetic resonance (NMR) spectroscopy and coherent anti-Stokes Raman scattering (CARS) spectroscopy, gas chromatography followed by mass spectrometry (GC/MS) and two-dimensional gas chromatography followed by mass spectrometry (GCxGC/MS). In the course of a running Miller-Urey-type experiment, a hydrophobic organic layer emerged besides the hydrophilic aqueous phase and the gaseous phase that were initially present. The gas phase mainly consisted of aromatic compounds and molecules containing C≡C or C≡N triple bonds. The hydrophilic phase contained at least a few thousands of different molecules, primarily distributed in a range of 50 and 500 Da. The hydrophobic phase is characterized by carbon-rich, oil-like compounds and their amphiphilic derivatives containing oxygen with tensioactive properties. The presence of a wide range of oxidized molecules hints to the availability of oxygen radicals. We suggest that they intervene in the formation of alkylated polyethylene glycol (PEG) in the oil/water interface. CARS spectroscopy revealed distinct vibrational molecular signatures. In particular, characteristic spectral bands for cyanide compounds were observed if the broth was prepared with electric discharges in the gaseous phase. The characteristic spectral bands were absent if discharges were released onto the water surface. NMR spectroscopy on the same set of samples independently confirmed the observation. In addition, NMR spectroscopy revealed overall high chemical variability that suggests strong non-linearities due to interdependent, sequential reaction steps.

Chemical Analysis of a "Miller-Type" Complex Prebiotic Broth: Part I: Chemical Diversity, Oxygen and Nitrogen Based Polymers.

In a famous experiment Stanley Miller showed that a large number of organic substances can emerge from sparking a mixture of methane, ammonia and hydrogen in the presence of water (Miller, Science 117:528-529, 1953). Among these substances Miller identified different amino acids, and he concluded that prebiotic events may well have produced many of Life's molecular building blocks. There have been many variants of the original experiment since, including different gas mixtures (Miller, J Am Chem Soc 77:2351-2361, 1955; Oró Nature 197:862-867, 1963; Schlesinger and Miller, J Mol Evol 19:376-382, 1983; Miyakawa et al., Proc Natl Acad Sci 99:14,628-14,631, 2002). Recently some of Miller's remaining original samples were analyzed with modern equipment (Johnson et al. Science 322:404-404, 2008; Parker et al. Proc Natl Acad Sci 108:5526-5531, 2011) and a total of 23 racemic amino acids were identified. To give an overview of the chemical variety of a possible prebiotic broth, here we analyze a "Miller type" experiment using state of the art mass spectrometry and NMR spectroscopy. We identify substances of a wide range of saturation, which can be hydrophilic, hydrophobic or amphiphilic in nature. Often the molecules contain heteroatoms, with amines and amides being prominent classes of molecule. In some samples we detect ethylene glycol based polymers. Their formation in water requires the presence of a catalyst. Contrary to expectations, we cannot identify any preferred reaction product. The capacity to spontaneously produce this extremely high degree of molecular variety in a very simple experiment is a remarkable feature of organic chemistry and possibly prerequisite for Life to emerge. It remains a future task to uncover how dedicated, organized chemical reaction pathways may have arisen from this degree of complexity.