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).