Progress and prospects for small-molecule probes of bacterial imaging

Abstract

Fluorescence microscopy is an essential tool for the exploration of cell growth, division, transcription and translation in eukaryotes and prokaryotes alike. Despite the rapid development of techniques to study bacteria, the size of these organisms (1–10 μm) and their robust and largely impenetrable cell envelope present major challenges in imaging experiments. Fusion-based strategies, such as attachment of the protein of interest to a fluorescent protein or epitope tag, are by far the most common means for examining protein localization and expression in prokaryotes. While valuable, the use of genetically encoded tags can result in mislocalization or altered activity of the desired protein, does not provide a readout of the catalytic state of enzymes and cannot enable visualization of many other important cellular components, such as peptidoglycan, lipids, nucleic acids or glycans. Here, we highlight the use of biomolecule-specific small-molecule probes for imaging in bacteria.

Towards the generalized iterative synthesis of small molecules

“…In fact, there is probably not a household on the planet that is not positively impacted by this class of chemical matter on a daily basis. Small molecules also possess substantial untapped potential to perform many frontier functions, 1 including modulating protein-protein interactions, 2 allosterically modifying protein function, 3 acting as prostheses on the molecular scale, 4,5 serving as next-generation biological probes, 6–9 enabling miniaturized diagnostics, 10 transducing energy, 11–16 emitting light, 17 initiating self-healing, 18 acting as molecular magnets, 19 enabling next generation computing, 20 and superconducting. 21–22 However, largely due to limitations in synthesis, much of this functional potential remains largely untapped.…”

Imaging Bacterial Cell Wall Biosynthesis

“…These experiments revealed that nascent PG insertion is heterogeneous but correlated with localization of the MreB cytoskeletal protein, suggesting that MreB may have a prominent role in coordinating new PG synthesis. For additional information on different types of PG probes, the reader is referred to a review by Kocaoglu & Carlson (53). …”

Complementary Approaches to Imaging Subcellular Lipid Architectures in Live Bacteria Using Phosphorescent Iridium Complexes and Raman Spectroscopy

“…A typical bacterial cell has a size of less than 10 μm, that is close to, or smaller than, the spatial resolution of many conventional microscopes. Moreover, the small size of these organisms requires more excitation energy that often causes increased photobleaching . The main obstacle to imaging subcellular components in bacteria is represented by their robust cell envelope .…”

“…Moreover,t he smalls ize of these organismsr equires more excitation energy that often causes increasedp hotobleaching. [8][9][10][11] The main obstacle to imaging subcellular components in bacteria is represented by their robust cell envelope. [10] Most of the current protocols to imageb acteria rely on the use of ar elatively limited number of commercial organic dyes [12][13][14][15] or on the genetic manipulation of target proteins.…”