References
BALZAROTTI, F., EILERS, Y., GWOSCH, K. C., GYNNA, A. H., WESTPHAL, V., STEFANI, F. D., ELF, J. & HELL, S. W. 2017. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science, 355, 606-612.
BANAZ, N., MAKELA, J. & UPHOFF, S. 2019. Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes. J Phys D Appl Phys, 52, 064002.
BARLAG, B., BEUTEL, O., JANNING, D., CZARNIAK, F., RICHTER, C. P., KOMMNICK, C., GOSER, V., KURRE, R., FABIANI, F., ERHARDT, M., PIEHLER, J. & HENSEL, M. 2016. Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.Sci Rep, 6, 31601.
BEER, T., HANSCH, S., PFEFFER, K., SMITS, S. H. J., WEIDTKAMP-PETERS, S. & SCHMITT, L. 2022. Quantification and Surface Localization of the Hemolysin A Type I Secretion System at the Endogenous Level and under Conditions of Overexpression. Appl Environ Microbiol,88, e0189621.
BERGER, C., RAVELLI, R. B. G., LOPEZ-IGLESIAS, C., KUDRYASHEV, M., DIEPOLD, A. & PETERS, P. J. 2021. Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography. J Struct Biol, 213, 107701.
BETZIG, E., PATTERSON, G. H., SOUGRAT, R., LINDWASSER, O. W., OLENYCH, S., BONIFACINO, J. S., DAVIDSON, M. W., LIPPINCOTT-SCHWARTZ, J. & HESS, H. F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science, 313, 1642-5.
BOLOGNESI, B. & LEHNER, B. 2018. Reaching the limit. Elife, 7.
BUTTNER, M., LAGERHOLM, C. B., WAITHE, D., GALIANI, S., SCHLIEBS, W., ERDMANN, R., EGGELING, C. & REGLINSKI, K. 2021. Challenges of Using Expansion Microscopy for Super-resolved Imaging of Cellular Organelles.Chembiochem, 22, 686-693.
CARSTEN, A., RUDOLPH, M., WEIHS, T., SCHMIDT, R., JANSEN, I., WURM, C. A., DIEPOLD, A., FAILLA, A. V., WOLTERS, M. & AEPFELBACHER, M. 2022. MINFLUX imaging of a bacterial molecular machine at nanometer resolution. Methods Appl Fluoresc, 11.
CASSARO, C. J. & UPHOFF, S. 2022. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells. Methods Mol Biol, 2476, 191-208.
CHEN, F., TILLBERG, P. W. & BOYDEN, E. S. 2015. Optical imaging. Expansion microscopy. Science, 347, 543-8.
DEANE, J. E., ROVERSI, P., CORDES, F. S., JOHNSON, S., KENJALE, R., DANIELL, S., BOOY, F., PICKING, W. D., PICKING, W. L., BLOCKER, A. J. & LEA, S. M. 2006. Molecular model of a type III secretion system needle: Implications for host-cell sensing. Proc Natl Acad Sci U S A,103, 12529-33.
DEGUCHI, T., IWANSKI, M. K., SCHENTARRA, E. M., HEIDEBRECHT, C., SCHMIDT, L., HECK, J., WEIHS, T., SCHNORRENBERG, S., HOESS, P., LIU, S., CHEVYREVA, V., NOH, K. M., KAPITEIN, L. C. & RIES, J. 2023. Direct observation of motor protein stepping in living cells using MINFLUX.Science, 379, 1010-1015.
DIEPOLD, A., KUDRYASHEV, M., DELALEZ, N. J., BERRY, R. M. & ARMITAGE, J. P. 2015. Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.PLoS Biol, 13, e1002039.
DIEPOLD, A., SEZGIN, E., HUSEYIN, M., MORTIMER, T., EGGELING, C. & ARMITAGE, J. P. 2017. A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome. Nat Commun, 8, 15940.
FERNANDEZ, N. L., CHEN, Z., FULLER, D. E. H., VAN GIJTENBEEK, L. A., NYE, T. M., BITEEN, J. S. & SIMMONS, L. A. 2023. DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid. mBio, 14,e0318522.
FILLOUX, A. 2022. Bacterial protein secretion systems: Game of types.Microbiology (Reading), 168.
FRUH, S. M., MATTI, U., SPYCHER, P. R., RUBINI, M., LICKERT, S., SCHLICHTHAERLE, T., JUNGMANN, R., VOGEL, V., RIES, J. & SCHOEN, I. 2021. Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors. ACS Nano, 15,12161-12170.
GALAN, J. E. 2009. Common themes in the design and function of bacterial effectors. Cell Host Microbe, 5, 571-9.
GOSER, V., KOMMNICK, C., LISS, V. & HENSEL, M. 2019. Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins. mBio, 10.
GOSER, V., SANDER, N., SCHULTE, M., SCHARTE, F., FRANZKOCH, R., LISS, V., PSATHAKI, O. E. & HENSEL, M. 2023. Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nat Commun, 14, 1240.
GOTZ, R., KUNZ, T. C., FINK, J., SOLGER, F., SCHLEGEL, J., SEIBEL, J., KOZJAK-PAVLOVIC, V., RUDEL, T. & SAUER, M. 2020. Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy. Nat Commun, 11, 6173.
GUSTAFSSON, M. G. 2000. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc, 198, 82-7.
GUSTAFSSON, M. G., SHAO, L., CARLTON, P. M., WANG, C. J., GOLUBOVSKAYA, I. N., CANDE, W. Z., AGARD, D. A. & SEDAT, J. W. 2008. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J, 94,4957-70.
HELL, S. W. & WICHMANN, J. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett, 19, 780-2.
HU, J., WORRALL, L. J., HONG, C., VUCKOVIC, M., ATKINSON, C. E., CAVENEY, N., YU, Z. & STRYNADKA, N. C. J. 2018. Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin.Nat Commun, 9, 3840.
JENKINS, J., WORRALL, L. J. & STRYNADKA, N. C. J. 2022. Recent structural advances towards understanding of the bacterial type III secretion injectisome. Trends Biochem Sci, 47, 795-809.
JEONG, D., KIM, M. J., PARK, Y., CHUNG, J., KWEON, H. S., KANG, N. G., HWANG, S. J., YOUN, S. H., HWANG, B. K. & KIM, D. 2022. Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy. BMC Biol, 20, 270.
KHATEB, H., SORENSEN, R. S., CRAMER, K., EKLUND, A. S., KJEMS, J., MEYER, R. L., JUNGMANN, R. & SUTHERLAND, D. S. 2022. The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT. ACS Nano,16, 10392-10403.
KLAR, T. A. & HELL, S. W. 1999. Subdiffraction resolution in far-field fluorescence microscopy. Opt Lett, 24, 954-6.
KUNZ, T. C., RUHLING, M., MOLDOVAN, A., PAPROTKA, K., KOZJAK-PAVLOVIC, V., RUDEL, T. & FRAUNHOLZ, M. 2021. The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy. Front Cell Infect Microbiol, 11, 644750.
LARA-TEJERO, M., KATO, J., WAGNER, S., LIU, X. & GALAN, J. E. 2011. A sorting platform determines the order of protein secretion in bacterial type III systems. Science, 331, 1188-91.
LE, N. H., PINEDO, V., LOPEZ, J., CAVA, F. & FELDMAN, M. F. 2021. Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector. Proc Natl Acad Sci U S A, 118.
LIEW, A. T. F., FOO, Y. H., GAO, Y., ZANGOUI, P., SINGH, M. K., GULVADY, R. & KENNEY, L. J. 2019. Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2.Elife, 8.
LIN, L., CAPOZZOLI, R., FERRAND, A., PLUM, M., VETTIGER, A. & BASLER, M. 2022. Subcellular localization of Type VI secretion system assembly in response to cell-cell contact. EMBO J, 41, e108595.
LISS, V., BARLAG, B., NIETSCHKE, M. & HENSEL, M. 2015. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy. Sci Rep, 5, 17740.
LIU, S., HOESS, P. & RIES, J. 2022. Super-Resolution Microscopy for Structural Cell Biology. Annu Rev Biophys, 51, 301-326.
LUND, V. A., GANGOTRA, H., ZHAO, Z., SUTTON, J. A. F., WACNIK, K., DEMEESTER, K., LIANG, H., SANTIAGO, C., LEIMKUHLER GRIMES, C., JONES, S. & FOSTER, S. J. 2022. Coupling Novel Probes with Molecular Localization Microscopy Reveals Cell Wall Homeostatic Mechanisms in Staphylococcus aureus. ACS Chem Biol, 17, 3298-3305.
LUNELLI, M., KAMPRAD, A., BURGER, J., MIELKE, T., SPAHN, C. M. T. & KOLBE, M. 2020. Cryo-EM structure of the Shigella type III needle complex. PLoS Pathog, 16, e1008263.
MARLOVITS, T. C., KUBORI, T., SUKHAN, A., THOMAS, D. R., GALAN, J. E. & UNGER, V. M. 2004. Structural insights into the assembly of the type III secretion needle complex. Science, 306, 1040-2.
MARY, C., FOUILLEN, A., BESSETTE, B., NANCI, A. & BARON, C. 2018. Interaction via the N terminus of the type IV secretion system (T4SS) protein VirB6 with VirB10 is required for VirB2 and VirB5 incorporation into T-pili and for T4SS function. J Biol Chem, 293,13415-13426.
MEZA-TORRES, J., LELEK, M., QUEREDA, J. J., SACHSE, M., MANINA, G., ERSHOV, D., TINEVEZ, J. Y., RADOSHEVICH, L., MAUDET, C., CHAZE, T., GIAI GIANETTO, Q., MATONDO, M., LECUIT, M., MARTIN-VERSTRAETE, I., ZIMMER, C., BIERNE, H., DUSSURGET, O., COSSART, P. & PIZARRO-CERDA, J. 2021. Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner. Proc Natl Acad Sci U S A, 118.
MIHAILA, T. S., BATE, C., OSTERSEHLT, L. M., PAPE, J. K., KELLER-FINDEISEN, J., SAHL, S. J. & HELL, S. W. 2022. Enhanced incorporation of subnanometer tags into cellular proteins for fluorescence nanoscopy via optimized genetic code expansion. Proc Natl Acad Sci U S A, 119, e2201861119.
MILETIC, S., FAHRENKAMP, D., GOESSWEINER-MOHR, N., WALD, J., PANTEL, M., VESPER, O., KOTOV, V. & MARLOVITS, T. C. 2021. Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation. Nat Commun, 12, 1546.
MORRIS, M. A., VALLMITJANA, A., GREIN, F., SCHNEIDER, T., ARTS, M., JONES, C. R., NGUYEN, B. T., HASHEMIAN, M. H., MALEK, M., GRATTON, E. & NOWICK, J. S. 2022. Visualizing the mode of action and supramolecular assembly of teixobactin analogues in Bacillus subtilis. Chem Sci,13, 7747-7754.
NAUTH, T., HUSCHKA, F., SCHWEIZER, M., BOSSE, J. B., DIEPOLD, A., FAILLA, A. V., STEFFEN, A., STRADAL, T. E. B., WOLTERS, M. & AEPFELBACHER, M. 2018. Visualization of translocons in Yersinia type III protein secretion machines during host cell infection. PLoS Pathog, 14, e1007527.
OSTERSEHLT, L. M., JANS, D. C., WITTEK, A., KELLER-FINDEISEN, J., INAMDAR, K., SAHL, S. J., HELL, S. W. & JAKOBS, S. 2022. DNA-PAINT MINFLUX nanoscopy. Nat Methods, 19, 1072-1075.
PENDE, N., SOGUES, A., MEGRIAN, D., SARTORI-RUPP, A., ENGLAND, P., PALABIKYAN, H., RITTMANN, S. K. R., GRANA, M., WEHENKEL, A. M., ALZARI, P. M. & GRIBALDO, S. 2021. SepF is the FtsZ anchor in archaea, with features of an ancestral cell division system. Nat Commun,12, 3214.
PRINDLE, J. R., WANG, Y., ROCHA, J. M., DIEPOLD, A. & GAHLMANN, A. 2022. Distinct Cytosolic Complexes Containing the Type III Secretion System ATPase Resolved by Three-Dimensional Single-Molecule Tracking in Live Yersinia enterocolitica. Microbiol Spectr ,e0174422.
REMMEL, M., SCHEIDERER, L., BUTKEVICH, A. N., BOSSI, M. L. & HELL, S. W. 2023. Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores. Small, 19, e2206026.
RIDEAU, F., VILLA, A., BELZANNE, P., VERDIER, E., HOSY, E. & ARFI, Y. 2022. Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas. Microbiol Spectr, 10, e0064522.
RIES, J., KAPLAN, C., PLATONOVA, E., EGHLIDI, H. & EWERS, H. 2012. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods, 9, 582-4.
ROCHA, J. M., RICHARDSON, C. J., ZHANG, M., DARCH, C. M., CAI, E., DIEPOLD, A. & GAHLMANN, A. 2018. Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits. Integr Biol (Camb), 10, 502-515.
RUDOLPH, M., CARSTEN, A., KULNIK, S., AEPFELBACHER, M. & WOLTERS, M. 2022. Live imaging of Yersinia translocon formation and immune recognition in host cells. PLoS Pathog, 18, e1010251.
RUST, M. J., BATES, M. & ZHUANG, X. 2006. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods, 3, 793-5.
SAHL, S. J., HELL, S. W. & JAKOBS, S. 2017. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol, 18, 685-701.
SCHMIDT, R., WEIHS, T., WURM, C. A., JANSEN, I., REHMAN, J., SAHL, S. J. & HELL, S. W. 2021. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.Nat Commun, 12, 1478.
SCHNITZBAUER, J., STRAUSS, M. T., SCHLICHTHAERLE, T., SCHUEDER, F. & JUNGMANN, R. 2017. Super-resolution microscopy with DNA-PAINT. Nat Protoc, 12, 1198-1228.
SEINEN, A. B., SPAKMAN, D., VAN OIJEN, A. M. & DRIESSEN, A. J. M. 2021. Cellular dynamics of the SecA ATPase at the single molecule level.Sci Rep, 11, 1433.
SICHEL, S. R., BRATTON, B. P. & SALAMA, N. R. 2022. Distinct regions of H. pylori’s bactofilin CcmA regulate protein-protein interactions to control helical cell shape. Elife, 11.
SINGH, M. K. & KENNEY, L. J. 2021. Super-resolution imaging of bacterial pathogens and visualization of their secreted effectors.FEMS Microbiol Rev, 45.
SODERSTROM, B., RUDA, A., WIDMALM, G. & DALEY, D. O. 2020. An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy. Microbiology (Reading),166, 1129-1135.
STOCKHAMMER, A. 2020. Appreciating the small things in life: STED microscopy in living cells. Journal of Physics D: Applied Physics, 54.
STRACY, M., LESTERLIN, C., GARZA DE LEON, F., UPHOFF, S., ZAWADZKI, P. & KAPANIDIS, A. N. 2015. Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A, 112, E4390-9.
TANK, R. K. G., LUND, V. A., KUMAR, S., TURNER, R. D., LAFAGE, L., PASQUINA LEMONCHE, L., BULLOUGH, P. A., CADBY, A., FOSTER, S. J. & HOBBS, J. K. 2021. Correlative Super-Resolution Optical and Atomic Force Microscopy Reveals Relationships Between Bacterial Cell Wall Architecture and Synthesis in Bacillus subtilis. ACS Nano,15, 16011-16018.
THRALL, E. S., PIATT, S. C., CHANG, S. & LOPARO, J. J. 2022. Replication stalling activates SSB for recruitment of DNA damage tolerance factors. Proc Natl Acad Sci U S A, 119,e2208875119.
TROUVE, J., GLUSHONKOV, O. & MORLOT, C. 2021. Metabolic biorthogonal labeling and dSTORM imaging of peptidoglycan synthesis in Streptococcus pneumoniae. STAR Protoc, 2, 101006.
TRUCKENBRODT, S., MAIDORN, M., CRZAN, D., WILDHAGEN, H., KABATAS, S. & RIZZOLI, S. O. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep, 19.
UPHOFF, S., REYES-LAMOTHE, R., GARZA DE LEON, F., SHERRATT, D. J. & KAPANIDIS, A. N. 2013. Single-molecule DNA repair in live bacteria.Proc Natl Acad Sci U S A, 110, 8063-8.
VEENENDAAL, A. K., HODGKINSON, J. L., SCHWARZER, L., STABAT, D., ZENK, S. F. & BLOCKER, A. J. 2007. The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol, 63, 1719-30.
VINCENT, M. S., COMAS HERVADA, C., SEBBAN-KREUZER, C., LE GUENNO, H., CHABALIER, M., KOSTA, A., GUERLESQUIN, F., MIGNOT, T., MCBRIDE, M. J., CASCALES, E. & DOAN, T. 2022. Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium. PLoS Biol, 20, e3001443.
WAGNER, S., GRIN, I., MALMSHEIMER, S., SINGH, N., TORRES-VARGAS, C. E. & WESTERHAUSEN, S. 2018. Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells. FEMS Microbiol Lett, 365.
WANG, Y., FU, M., WU, B., HUANG, M., MA, T., ZANG, H., JIANG, H., ZHANG, Y. & LI, C. 2022. Insight into biofilm-forming patterns: biofilm-forming conditions and dynamic changes in extracellular polymer substances. Environ Sci Pollut Res Int, 29, 89542-89556.
WIMMI, S., BALINOVIC, A., JECKEL, H., SELINGER, L., LAMPAKI, D., EISEMANN, E., MEUSKENS, I., LINKE, D., DRESCHER, K., ENDESFELDER, U. & DIEPOLD, A. 2021. Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH. Nat Commun, 12, 1625.
WOLFF, J. O., SCHEIDERER, L., ENGELHARDT, T., ENGELHARDT, J., MATTHIAS, J. & HELL, S. W. 2023. MINFLUX dissects the unimpeded walking of kinesin-1. Science, 379, 1004-1010.
WORRALL, L. J., HONG, C., VUCKOVIC, M., DENG, W., BERGERON, J. R. C., MAJEWSKI, D. D., HUANG, R. K., SPRETER, T., FINLAY, B. B., YU, Z. & STRYNADKA, N. C. J. 2016. Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body. Nature, 540,597-601.
ZHANG, Y., LARA-TEJERO, M., BEWERSDORF, J. & GALAN, J. E. 2017. Visualization and characterization of individual type III protein secretion machines in live bacteria. Proc Natl Acad Sci U S A,114, 6098-6103.
ZILKENAT, S., FRANZ-WACHTEL, M., STIERHOF, Y. D., GALAN, J. E., MACEK, B. & WAGNER, S. 2016. Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach. Mol Cell Proteomics,15, 1598-609.
Acknowledgements:
We thank Jost Enninga for helpful discussions and Antonio Virgilio Failla for critical reading of the manuscript. The selected papers on SRM/SMT and their applications in bacteria that we mention and cite in this article do not claim to be complete. We apologize to all colleagues whose papers we have not cited here due to space limitations or because we simply did not discover them during our extensive literature searches.
Funding:
This study was supported by a grant from the Joachim Herz Foundation to Alexander Carsten and by the DFG-funded RTG 2771 Humans and Microbes. Acquisition of the MINFLUX microscope was made possible through funding from the European Regional Development Fund (ERDF) under the Operational Programme Hamburg ERDF 2014-2020, REACT-EU, awarded by the Hamburgische Investitions- und Förderbank (grant number 51164232).
Conflict of interest:
The authors declare no conflict of interest.