Super-resolution fluorescence microscopy in bacterial cell biology
Deciphering the precise timing and localization of peptidoglycan synthesis in Gram-negative and Gram-positive bacteria is a long-standing goal in molecular bacteriology and may be of medical importance, for example, in the development of new antibiotics. In one study, fluorescent D-amino acids were incorporated into bacterial peptidoglycans and imaged by STED nanoscopy at below 100 nm resolution (Soderstrom et al., 2020). In another study, bioorthogonal metabolic labeling of peptidoglycan in Streptococcus pneumoniae was successfully combined with dSTORM (Trouve et al., 2021). Cephalosporin-, metabolic- and hydroxylamine-based fluorescent probes in combination with d-STORM revealed the molecular details of how peptidoglycan dynamics in Staphylococcus aureus are controlled during growth and division (Lund et al., 2022). 3D-SIM demonstrated that SepF transiently co-localizes with FtsZ at the septum of the archaeonMethanobrevibacter smithii . It was found that SepF is the relevant FtsZ anchor and possibly primes the future division plane (Pende et al., 2021). Combinations of STORM or SIM super-resolution fluorescence microscopy with atomic force microscopy (AFM), named STORMForce or SIMForce respectively, revealed the intricate spatiotemporal 3D dynamics of peptidoglycan synthesis during growth and division in Bacillus subtilis (Tank et al., 2021). 3D-SIM helped identify the molecular function and interaction partners of aHelicobacter pylori bactofilin (CcmA), that plays a crucial role in generating the distinctive helical shape of the bacteria. CcmA aids coordinate cell shape-determining proteins and peptidoglycan synthesis machinery to organize cell wall synthesis and curvature (Sichel et al., 2022). SIM of fluorescently labeled teixobactin, a recently introduced antibiotic, allowed visualization of teixobactin interactions and structural organization in the Gram-positive cell wall. This provided insights into the mechanism of action of teixobactin and could contribute to the development of antibiotics with similar properties (Morris et al., 2022). PALM and dSTORM were employed to study the subcellular localization of proteins at the surface and in the cytoplasm of Mycoplasma spp., which are the smallest known bacteria with sizes of 300 to 800 nm. Because of their tiny genomes and sizes, these human and animal pathogens represent important model organisms for synthetic biology, and super-resolution microscopy techniques will greatly improve the understanding of their biology (Rideau et al., 2022). S. aureus fibronectin binding receptor organization and adhesion to patches of fibronectin of systematically varied size (100-1000 nm) was investigated using DNA-PAINT. The results suggest that for strong adhesion of S. aureus fibronectin patches of 300 nm or larger and the involvement of two or more receptors are required (Khateb et al., 2022). Different SRM and single-molecule tracking (SMT) techniques have been employed to visualize activities of bacterial RNA-polymerases, DNA-binding proteins, the exact spatial organization of DNA replication and transcription, and DNA repair processes in real time (Cassaro and Uphoff, 2022, Uphoff et al., 2013, Stracy et al., 2015). DNA methyltransferases (MTases) have central functions in restriction modification systems, cell cycle regulation, and the control of gene expression. SMLM and SMT of the MTase DnmA revealed its preferential localization to the nucleoid and the replisome region as well as its intracellular dynamics (Fernandez et al., 2023). Single-molecule localization microscopy in E. coli cells was employed to investigate enrichment of the translesion synthesis polymerase Pol IV at stalled replication forks in the presence of DNA damage. It turned out that alterations in the dynamics of single-stranded DNA-binding proteins at the replication fork likely contribute to the Pol IV enrichment (Thrall et al., 2022). Microbial biofilms play an important role in both, human infections and biotechnological processes. SR-SIM revealed the distribution of extracellular polysaccharides and DNA in resident biofilms (Wang et al., 2022). The production of extracellular vesicles (EVs) by Gram-positive bacteria was investigated by STORM. The results showed that EV can be formed by membrane blebbing and explosive cell lysis, suggesting that cell wall degradation plays a significant role in their biogenesis (Jeong et al., 2022). Listeriolysin S (LLS) a thiazole/oxazole-modified microcin from Listeria monocytogenes was localized to the bacterial cell membrane and cytoplasm using PAINT/dSTORM. This contributed to the understanding of LLS as a contact-dependent bacteriocin (Meza-Torres et al., 2021).