This work was supported by start-up funds from the University of Texas Medical branch, Galveston, TX, and a Texas STAR award to L.J.K. We thank Prof. Edward Lemke (European Molecular Biology Laboratory, Heidelberg, Germany) for plasmid pEVOL-PylRS-AF. Images in Figure 1 were created using BioRender.

<ol>
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The authors declare that they have no competing financial interests.

The approach described herein was used to track the precise location of effector proteins injected into the host cell by the bacterial T3SS after infection. T3SSs are employed by intracellular pathogens such as Salmonella, Shigella, and Yersinia to transport virulence components into the host. The development of super-resolution imaging technologies has made it possible to visualize virulence factors at a previously unimaginable resolution12,13,24. However, certain labeling constraints precluded more in-depth investigation, as photoactivatable protein fusions block the T3SS pore and are not secreted. In addition, clustering artifacts and erroneous analysis can result from some fusion protein&#39;s inherent propensity to cluster or aggregate.
In some instances, the fusion protein is also cleaved, as we observed previously with PAmCherry-SsaP12,13. All these constraints are overcome by GCE, which allows for site-specific fluorescent labeling of POIs. It also allows the visualization of proteins that are not plentiful12,13. While there are a number of factors that can affect the efficiency of ncAA incorporation using an orthogonal tRNA/aminoacyl-tRNA synthetase system, it seems that the codon context is the most crucial. The orthogonal tRNA incorporates an ncAA most efficiently when the preferred codon context is AATTAGACT33. In Figure 5, we can compare the crisp labeling resulting from GCE and a site-specific fluorophore (as well as the absence of background) compared to immunostaining of an HA-tag.
As a result, researchers will be able to visualize POIs in pathogens, commensal bacteria, and eukaryotic hosts using the method outlined here. In short, genetically encoded ncAAs provide an alternate approach to effector protein labeling when conventional methods fail. However, one major limitation of this method is the inherent chemical properties of the fluorophore, such as membrane permeability, distribution, and retention, which can impact the efficiency of fluorescent labeling. Fluorescent ncAAs are an alternative option to avoid click reactions12, however these can only be visualized using two-photon microscopy. Readers are referred to a list of cell-membrane-permeable/impermeable dyes cited herein12,38,39,40,41.
In conclusion, we site-specifically labeled and visualized Salmonella secreted effector SseJ by genetically encoding ncAAs in combination with a fluorogenic fluorophore without impairing its biological function. Labeling of SseJ with JF646-Tz was highly specific, and super-resolution imaging can further be employed to visualize secreted effectors inside host cells. Thus, fluorescent labeling of bacterial effectors using dSTORM-compatible dyes and genetic code expansion allowed the subcellular spatial localization of bacterial secreted effectors in host cells, at a resolution below the diffraction limit. As this labeling platform is compatible with live-cell imaging single particle tracking, our method will make it possible to analyze effector proteins of the T3SS system with unprecedented levels of temporal and spatial resolution, which will enhance our molecular understanding of bacterial pathogenesis.
While there are certain limitations, such as poor ncAA incorporation efficiency and the photophysical constraints of the organic fluorophore, we have demonstrated that this approach can help researchers observe and localize a secreted effector inside host cells, even when it is scarce12,13. We anticipate that adaptation of the technology will open up new and exciting opportunities for imaging and research on other effector proteins produced by bacteria during infection. The method is also easily adapted for the labeling of viruses, demonstrating its broad utility.

Bacterial infections have long been regarded as a serious hazard to human health. Pathogens use highly evolved, extremely powerful, and intricate defense systems, as well as a variety of bacterial virulence factors (referred to as effector proteins) to evade host immune responses and establish infections1,2. However, the molecular mechanisms underlying these systems and the role of individual effector proteins are still largely unknown due to the dearth of suitable approaches for directly following the crucial protein components and effectors in host cells during pathogenesis.
One typical example is Salmonella enterica serovar Typhimurium, which causes acute gastroenteritis. Salmonella Typhimurium uses type three secretion systems (T3SS) to inject a variety of effector proteins directly into host cells. As soon as Salmonella enters the host cell, it resides in an acidic membrane-bound compartment, termed the Salmonella-containing vacuole (SCV)3,4. The acid pH of the SCV activates the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS and translocates a volley of 20 or more effector proteins across the vacuolar membrane into the host cytosol5,6,7,8. Inside the host, these complex cocktails of effector proteins coordinately manipulate host cell signaling pathways, resulting in the formation of highly dynamic, complex tubular membrane structures extended from the SCV along microtubules, termed Salmonella-induced filaments (SIFs), that enable Salmonella to survive and replicate within the host cells9,10,11.
Methods to visualize, track, and monitor bacterial effector localizations, and examine their trafficking and interactions inside host cells, provides critical insight into the mechanisms underpinning bacterial pathogenesis. Labeling and localization of Salmonella secreted T3SS effector proteins inside host cells has proven to be a technological challenge12,13; Nonetheless, the development of genetically-encoded fluorescent proteins has transformed our ability to study and visualize proteins within living systems. However, the size of fluorescent proteins (~25-30 kDa)15 is often comparable to or even greater than that of the protein of interest (POI; e.g., 13.65 kDa for SsaP, 37.4 kDa for SifA). In fact, fluorescent protein labeling of effectors often blocks the secretion of the labeled effector and jams the T3SS14.
Furthermore, fluorescent proteins are less stable and emit a low number of photons before photobleaching, limiting their use in super-resolution microscopic techniques16,17,18, particularly in photoactivation localization microscopy (PALM), STORM, and stimulated emission depletion (STED) microscopy. While the photophysical properties of organic fluorescent dyes are superior to those of fluorescent proteins, methods/techniques such as CLIP/SNAP19,20, Split-GFP21, ReAsH/FlAsH22,23, and HA-Tags24,25 require an additional protein or peptide appendage that may impair the structure-function of the effector protein of interest by interfering with post-translational modification or trafficking. An alternative method that minimizes necessary protein modification involves the incorporation of ncAAs into a POI during translation through GCE. The ncAAs are either fluorescent or can be made fluorescent via click chemistry12,13,26,27,28.
Using GCE, ncAAs with tiny, functional, bio-orthogonal groups (such as an azide, cyclopropene, or cyclooctyne group) can be introduced at nearly any location in a target protein. In this strategy, a native codon is swapped with a rare codon such as an amber (TAG) stop codon at a specified position in the gene of the POI. The modified protein is subsequently expressed in cells alongside an orthogonal aminoacyl-tRNA synthetase/tRNA pair. The tRNA synthetase active site is designed to receive only one particular ncAA, which is then covalently attached to the 3&#39;-end of the tRNA that recognizes the amber codon. The ncAA is simply introduced into the growth medium, but it must be taken up by the cell and reach the cytosol where the aminoacyl-tRNA synthetase (aaRS) can link it to the orthogonal tRNA; it is then incorporated into the POI at the specified location (see Figure 1)12. Thus, GCE enables site-specific incorporation of a plethora of bio-orthogonal reactive groups such as ketone, azide, alkyne, cyclooctyne, transcyclooctene, tetrazine, norbonene, &#945;, &#946;-unsaturated amide, and bicyclo [6.1.0]-nonyne into a POI, potentially overcoming the limitations of conventional protein labeling methods12,26,27,28.
Recent emerging trends in super-resolution imaging techniques have opened up new avenues to investigate biological structures at the molecular level. In particular, STORM, a single-molecule, localization-based, super-resolution technique, has become an invaluable tool to visualize cellular structures down to ~20-30 nm and is able to investigate biological processes one molecule at a time, thereby discovering the roles of intracellular molecules that are yet unknown in traditional ensemble-averaged studies13. Single-molecule and super-resolution techniques require a small tag with bright, photostable organic fluorophores for the best resolution. We recently demonstrated that GCE can be used for incorporating suitable probes for super-resolution imaging12.
Two of the best choices for protein labeling in cells are bicyclo [6.1.0] nonynelysine (BCN) and trans-cyclooctene-lysine (TCO; shown in Figure 1), which may be genetically encoded using a variant of the tRNA/synthetase pair (here termed tRNAPyl/PylRSAF), where Pyl represents pyrrolysine, and AF represents a rationally designed double mutant (Y306A, Y384F) derived from Methanosarcina mazei that naturally encodes pyrrolysine12,29,30,31. Through the strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reaction, these amino acids react chemoselectively with tetrazine conjugates (Figure 1)12,30,31. Such cycloaddition reactions are exceptionally fast and compatible with living cells; they may also be fluorogenic, if an appropriate fluorophore is functionalized with the tetrazine moiety12,26,32. This paper presents an optimized protocol for monitoring the dynamics of bacterial effectors delivered into host cells using GCE, followed by subcellular localization of secreted proteins in HeLa cells using dSTORM. The results indicate that incorporation of an ncAA via GCE, followed by a click reaction with fluorogenic tetrazine-bearing dyes, represents a versatile method for selective labeling, visualization of secreted proteins, and subsequent sub-cellular localization in the host. All the components and procedures detailed here, however, can be adjusted or substituted so that the GCE system can be adapted to investigate other biological questions.

<table><tbody><tr><td>10x Tris/Glycine SDS running buffer</td><td>BIO-RAD</td><td>1610732</td><td></td></tr><tr><td>Ammonium chloride</td><td>Fischer Scientific</td><td>A661-500</td><td></td></tr><tr><td>Ammonium sulphate</td><td>Fischer Scientific</td><td>BP212R-1</td><td></td></tr><tr><td>Ampicillin Sodium</td><td>Sigma-Aldrich</td><td>A0166</td><td>5 g</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>ThermiFischer Scientific</td><td>15240096</td><td></td></tr><tr><td>Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td>500 g</td></tr><tr><td>Avanti J-26XP (High-Performance Centrifuge)</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Bacto-Agar</td><td>BD Diagnostics, Franklin Lakes, USA</td><td>214010</td><td></td></tr><tr><td>BDP-FL-tetrazine</td><td>Lumiprobe (USA)</td><td>2.14E+02</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Millipore</td><td>444203</td><td>250 mL</td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>B8026</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503</td><td>500 g</td></tr><tr><td>Casein</td><td>Sigma-Aldrich</td><td>C8654</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C9322</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C1919</td><td></td></tr><tr><td>Click Amino Acid / trans-Cyclooct-2-en &amp;ndash; L - Lysine (TCO*A)</td><td>SiChem GmbH</td><td>SC-8008</td><td>Size: 500 mg</td></tr><tr><td>DAPI (Hoechst33342)</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>DeNovix DS-11+ Spectrophotometer</td><td>DeNovix</td><td></td><td></td></tr><tr><td>DMEM*</td><td>Corning</td><td>10-013-CV</td><td>* Used for maintaining HeLa Cell</td></tr><tr><td>DMEM**</td><td>Gibco</td><td>11965-092</td><td>**Used for bacterial infection in presence of ncAA, see section 5.4.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>250 g</td></tr><tr><td>Donkey anti-rabbit Alexa fluoro555 secondary antibody</td><td>Invitrogen</td><td>A-31572</td><td></td></tr><tr><td>DPBS, 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>E. coli strain BL21 (DE3)</td><td>Novagen (Madison, WI)</td><td></td><td></td></tr><tr><td>EMCCD Camera</td><td>Andor</td><td>iXon Ultra 897-BV</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tube 1.5 mL (PCR clean)</td><td>Eppendorf, Hamburg, Germany</td><td>30123.328</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Fischer</td><td>10082147</td><td></td></tr><tr><td>Fisherbrand Syringe Filters - Sterile (PVDF 0.22 &amp;micro;m)</td><td>Fischer Scientific</td><td>97203</td><td>Pack of 100</td></tr><tr><td>Gene Pulser Xcell Electroporator</td><td>BIO-RAD</td><td>1652660</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1272</td><td>10 mL</td></tr><tr><td>Gibco L-Glutamine (200 mM), 100x</td><td>Fischer Scientific</td><td>25-030-081</td><td></td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G7141-50KU</td><td></td></tr><tr><td>Glycerol</td><td>Fischer Scientific</td><td>BF229-4</td><td></td></tr><tr><td>HeLa cells</td><td>ATTC</td><td>CCL-2</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning</td><td>25-060-C1</td><td>100 mL</td></tr><tr><td>Hydrocloric acid</td><td>Fischer Scientific</td><td>A144-212</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Image processing and analysis:&amp;nbsp; http://rsbweb.nih.gov/ij</td></tr><tr><td>IntantBlue</td><td>Expedeon</td><td>ISB1L</td><td>Coomassie-based stain</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>Janelia Fluoro 646-tetrazine</td><td>Tocris Bioscience</td><td>7279</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>60615</td><td>5 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500 g</td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>&amp;mu;Manager (v. 1.4.2)</td><td></td><td></td><td>https://micro-manager.org/Download_Micro-Manager_Latest_Release</td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td>250 g</td></tr><tr><td>Micro pulser cuvette</td><td>BIO-RAD</td><td>165-2086</td><td>0.2 cm electrode gap, pkg. of 50</td></tr><tr><td>Nikon N-STORM</td><td>Nikon Instruments Inc.</td><td></td><td>https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution</td></tr><tr><td>Nunc EasYFlask Cell Culture Flasks</td><td>ThermiFischer Scientific</td><td>156499</td><td></td></tr><tr><td>Omnipur Casamino Acid</td><td>Calbiochem</td><td>2240</td><td>500 g</td></tr><tr><td>Paraformaldehhyde (PFA)</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>PBS (10x)</td><td>Roche</td><td>11666789001</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution (100x)</td><td>GenDEPOT&amp;nbsp;</td><td>CA005-010</td><td>100 mL</td></tr><tr><td>pEVOL-PylRS-AF</td><td></td><td></td><td>For plasmid construction and map see following references&lt;br/&gt; 1. Angew Chem Int Ed Engl. 2011, 50(17), 3878-81.&lt;br/&gt; 2. Angew Chem Int Ed Engl., 2012, 51, 4166-70.</td></tr><tr><td>Plasmid Mini-prep Kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>plasmid pWSK29-sseJ10TAG-HA</td><td></td><td></td><td>Ref.: elife. 2021, 10, e67789.</td></tr><tr><td>plasmid pWSK29-sseJ-HA</td><td></td><td></td><td>Ref.: elife. 2021, 10, e67789.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Vector map of PWSK29: https://www.addgene.org/172972/</td></tr><tr><td>Pluronic F-127</td><td>Millipore</td><td>540025</td><td>Protein grade, 10% Solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Fischer Scientific</td><td>P285-500</td><td></td></tr><tr><td>Potassium sulphate</td><td>Acros Organic</td><td>424220250</td><td></td></tr><tr><td>Protease Inhibitor Cocktail Set I - Calbiochem</td><td>Sigma-Aldrich</td><td>539131</td><td>100x Solution</td></tr><tr><td>Rabbit anti-HA primary antibody</td><td>Sigma-Aldrich</td><td>H6908</td><td></td></tr><tr><td>S. enterica. serovar Typhimurium 14028s</td><td></td><td></td><td>Ref.: PLoS Biol. 2015, 13, e1002116.</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036-50G-F</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fischer Scientific</td><td>SS255-1</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM), 100x</td><td>Corning</td><td>25-060-C1</td><td>100 mL</td></tr><tr><td>Sonic Dismembrator Model 100</td><td>Fischer Scientific</td><td>24932</td><td></td></tr><tr><td>STED microsocpe (Leica TCS SP8 STED 3X system)</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/</td></tr><tr><td>ThunderSTORM</td><td></td><td></td><td>https://zitmen.github.io/thunderstorm/</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Trypsin-EDTA (1x), 0.25%&amp;nbsp;&amp;nbsp;</td><td>GenDEPOT&amp;nbsp;</td><td>CA014-010</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416</td><td>100 mL</td></tr><tr><td>X-well tissue culture chamber slides</td><td>SARSTEDT</td><td>94.6190.802</td><td></td></tr></tbody></table>

super-resolution imaging of bacterial secreted proteins using genetic code expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This protocol paper describes a GCE-based method for site-specific fluorescent labeling and visualization of Salmonella secreted effectors, as depicted in Figure 1. Chemical structure of the ncAA bearing trans-cyclooctene bioorthogonal group (TCO) and the fluorescent dye are shown in Figure 1A. SseJ labeling was achieved by genetic incorporation of bioorthogonal ncAAs at an amber stop codon (see Figure 1B) using an orthogonal aminoacyl-tRNA synthetase/tRNA pairs via GCE technology. Treatment of TCO-labeled effectors with the tetrazine containing a fluorophore (JF646-Tz or BDP-Tz) provided an alternate protein labeling strategy that enabled the observation of translocated Salmonella secreted effectors many hours after infection of the host (Figure 1B,C). We began by identifying possible amino acid residues and selected position 10 of SseJ for ncAA inclusion based on surface accessibility and existing knowledge of SseJ functional residues. Prior structural data and surface exposure projections can aid in the selection of specific locations.
The placement of the amber codon within the gene encoding the POI affects the efficacy of TCO incorporation. As a result, a series of mutants with amber codons in various positions of the gene of the POI should be created and analyzed for optimum integration efficiency. Selection of the amber codon position is empirical. In general, the altered residues should be non-essential for POI function, devoid of post-translational modifications, and accessible for fluorescent probes. The expression plasmid should be a low copy number so that it mimics the native copy number of the POI. The codon context affects how effectively an ncAA is incorporated by an orthogonal tRNA/aminoacyl-tRNA synthetase system. While AAT-TAG-ACT is the preferred context of the orthogonal tRNA, the position of Phenylalanine-10 of sseJ was changed to TAG, ensuring the AAT-TAG-ACT context for enabling the most effective incorporation of the ncAA. First, we tested whether the mutants were functional in E. coli using a high copy number plasmid to express sseJ. As evidenced by SDS-PAGE, SseJ-F10TCO-HA mutant expression was dependent on the presence of TCO*A and the corresponding optimal tRNA/aaRS (Figure 2A). SseJ-F10FCO-HA was labeled with JF646-Tz using SPIEDAC click chemistry in E. coli. The selective fluorescent labeling of SseJ-F10TCO-HA in E. coli in vitro was confirmed by fluorescence microscopy analysis (Figure 2B). Using GCE, TCO*A was thus site-specifically incorporated at an amber codon in sseJ. Attempts to directly identify genomic off-target incorporation of ncAAs using tRNAPyl/PylRSAF&#160;system produced no evidence of their existence. However, we recommend using in-gel fluorescence, Western blot, fluorescent fusion protein, in vitro fluorescence labeling, and other methods to assess the efficiency, specificity, degree of off-target incorporation, and toxicity of the pEVOL plasmid, as documented in previously reported papers cited herein12,29.
Once we established that the suppressed TAG in sseJ was functional, we cloned sseJ-F10TCO-HA along with its native promoter into the low copy number plasmid pWSK29, ensuring an AAT-TAG-ACT motif for fluorescent labeling and visualization. In vitro selective labeling of SseJ-F10TCO-HA in Salmonella cells was confirmed using fluorescence microscopy (Figure 3). We then infected HeLa cells with the wild-type Salmonella strain complemented with psseJ-HA or psseJ10TAG-HA, in the absence or presence of TCO*A to determine whether the TCO-incorporated SseJ could be translocated into host cells. Infected HeLa cells were fixed with PFA and immunostained with anti-HA antibody 16 h post infection to highlight the SIFs decorated by SseJ. As shown in Figure 4C, SseJ-F10TCO-HA was obviously secreted, SseJ-dependent SIFs were formed, and they looked similar to those observed in wild-type infected cells (Figure 4A). However, SseJ-dependent SIFs were not observed in the absence of TCO*A (Figure 4B). These observations demonstrated that the expression of sseJ-F10TCO-HA using GCE rescued SseJ-dependent SIFs. Additionally, the results also indicated that SseJ-F10TCO-HA was a fully functional virulence factor for Salmonella.
After confirming that the GCE-labeled SseJ was functional and secreted, we used the SPIEDAC reaction to label SseJ-F10TCO-HA with JF646-Tz in HeLa cells. To do so, we infected HeLa cells with wild-type Salmonella complemented with psseJ-F10TCO-HA, in the presence of TCO*A. After labeling with JF646-Tz using two alternative protocols described in protocol section 5, cells were washed extensively to remove excess dye, fixed with PFA, and immunostained with an anti-HA antibody to visualize the secreted SseJ-F10TCO-HA. The SseJ-F10TCO-HA secreted into the cytoplasm of HeLa cells was labeled with JF646-Tz (Figure 5A,B, red) and clearly co-localized with the HA-tag (green). The observation that the two labels overlapped with one another (one a fluorescent dye, the other labeled by immunostaining), further emphasizes the specificity of GCE labeling.
To ensure that the fluorescence signal observed in those HeLa cells was specific only to the secreted effector SseJ, we infected cells with wild-type Salmonella (carrying psseJ-HA) in the presence of TCO*A and pEVOL-PylRS-AF. A click dye was used to observe whether there was any background labeling in the HeLa cells. SseJ was released into the cytoplasm of the host cell, with no intracellular or non-specific fluorescent signals (Figure 5C), demonstrating that the fluorescence signal was specific to SseJ. After determining that ncAAs were incorporated specifically at an amber codon, and that secreted SseJ could be visualized in the host cell via the click reaction, the next goal was to create a super-resolution image of secreted SseJ in HeLa cells (Figure 6). In Figure 6, we demonstrate the power of GCE and the use of the JF646 dye to generate site-specific super-resolution images of the Salmonella secreted effector SseJ in HeLa cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64382/64382fig01.jpg" /> 
Figure 1:&#160;Scheme for site-specific fluorescent labeling of SPI-2 effectors. (A) TCO*A and JF-646-tetrazine. (B) The schematic depicts the incorporation of an ncAA in an SPI-2 effector. In the bacterial cell, a plasmid containing the gene for an effector POI (purple) and an orthogonal suppressor tRNA (red)/aminoacyl synthetase (green) pair are introduced. A native codon is swapped with an amber (TAG, red) stop codon in the effector gene sequence at a permissive location. The ncAA (dark red circle) is simply introduced into the growth medium. It is then picked up by the cell and reaches the cytosol, where it is linked to the orthogonal tRNA by the aminoacyl-tRNAsynthetase(aaRS). The ncAA-acylated tRNA with a CUA anticodon enters the ribosomal machinery as a result of the complementary amber codon on the effector mRNA (purple), incorporating the attached ncAA into the effector. The ncAA is carried by the full-length polypeptide chain of the effector site, which then folds and assembles into a functional effector protein. The newly produced effector is translocated into the host cell through the T3SS. An externally supplied fluorophore can be used to label a secreted SPI-2 effector integrated with ncAA. (C) Copper-free click reaction scheme with a fluorogenic tetrazine dye. Through the SPIEDAC click reaction, an ncAA with a strained alkene group incorporated into a POI (SseJ) reacts with a tetrazine-coupled dye (JF646-Tz). The fluorogenic, tetrazine-coupled dye JF646-Tz becomes fluorescent (red) only after successful labeling. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; ncAA = non-canonical amino acid; T3SS = type three secretion system; SPI-2 = Salmonella pathogenicity island 2; POI = protein of interest; SPIEDAC = strain-promoted inverse electron-demand Diels-Alder cycloaddition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64382/64382fig02.jpg" /> 
Figure 2:TCO*A is site-specifically incorporated into SseJ-F10TCO-HA expressed in E. coli. (A) Coomassie-stained SDS-PAGE confirms the selective incorporation of TCO*A into SseJ-F10TCO-HA (indicated by red arrow) in E. coli. (B) Expression of SseJ-F10TCO-HA in E. coli analyzed by fluorescence microscopy in the absence (top) or presence (bottom) of 1 mM TCO*A. E. coli cells expressing SseJ-F10TCO-HA in the absence or presence of TCO*A are incubated with BDP-Fl-tetrazine and imaged for BDP fluorescence (green). SseJ fluorescence (green) is only observed in the presence of the incorporated TCO*A label; note the absence of background fluorescence in the top panel. Scale bar = 2 &#181;m. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; BF = brightfield; HA = hyaluronic acid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64382/64382fig03.jpg" /> 
Figure 3: TCO*A is site-specifically incorporated into SseJ-F10TCO-HA expressed in Salmonella. Fluorescence microscopy is used to examine the expression of SseJ-F10TCO-HA in Salmonella in the absence (top) or presence (bottom) of 1 mM TCO*A. Salmonella expressing SseJ F10TCO-HA are treated with JF646-Tz and imaged for JF646 fluorescence (magenta), in the presence or absence of TCO*A. Fluorescence of SseJ (magenta) is detected only when TCO*A is present. Scale bar = 2 &#181;m. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; BF = brightfield; HA = hyaluronic acid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64382/64382fig04.jpg" /> 
Figure 4: Secreted SseJ-J10TCO-HA is functional. (A) HeLa cells are infected with Salmonella harboring psseJ-HA for 16 h, fixed, and immunostained with anti-HA antibody (red), LPS (green), and DAPI (blue). As expected, the formation of SseJ-dependent SIFs is observed in infected cells. (B) In the absence of TCO*A, the amber codon is not suppressed, and SseJ-dependent SIFs are absent in infected HeLa cells. HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the absence of TCO*A for 16 h, fixed, and immunostained with anti-HA antibody (red), LPS (green), and DAPI (blue). (C) SseJ-dependent SIFs are observed in the presence of TCO*A. Infected HeLa cells with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 &#181;M TCO*A are fixed and immunostained with anti-HA antibody SseJ (red), LPS (green), and DAPI (blue). SseJ-dependent SIFs are observed in the left panel, clearly indicating that SseJ was rescued by the expression of sseJ-F10TCO-HA using GCE. Scale bar = 10 &#181;m. Abbreviations: HA = hyaluronic acid; LPS = lipopolysaccharide; DAPI = 4&#39;,6-diamidino-2-phenylindole; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64382/64382fig05.jpg" /> 
Figure 5:&#160;Labeling specificity of SseJ with JF646-Tz dye. (A) HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 &#181;M TCO*A. TCO-tagged secreted SseJ-F10TCO-HA is labeled with 2 &#181;M JF646-Tz in 1x PBS containing 1% casein sodium salt from bovine milk, extensively washed, fixed, and immunostained with anti-HA antibody to visualize the secreted SseJ (green). SseJ-F10TCO-HA is secreted and labeled with JF646-Tz dye (red, left panel). SseJ labeled via GCE (red) and HA (green) are clearly co-localized (right panel). (B) Using a slightly different labeling protocol, TCO-tagged secreted SseJ-F10TCO-HA is also labeled with 2 &#181;M JF646-Tz in complete medium DMEM (without FBS), extensively washed, and fixed, and subjected to anti-HA immunofluorescence staining. Secreted SseJ-F10TCO-HA is labeled with JF646-Tz dye (red, left panel) and co-localized with HA (green). (C) To further establish that the fluorescence signal is unique to SseJ and not a product of other SPI-2 released proteins, HeLa cells are infected with wild-type Salmonella carrying psseJ-HA and pEVOL-PylRS-AF, in the presence of ncAA (TCO*A). At 16 h post infection, infected HeLa cells are treated with 2 &#181;M JF646-Tz in complete medium (without FBS and TCO*A), and the excess dye is thoroughly removed using new growth medium. HeLa cells are PFA fixed and thoroughly washed with PBS before being immunostained for SseJ (green). The lack of visible background fluorescence signal in the left panel (JF646-Tz) indicates that almost no off-target labeling occurred within the host cells. Scale bar = 10 &#181;m. Abbreviations: HA = hyaluronic acid; PBS = phosphate-buffered saline; FBS = fetal bovine serum; DAPI = 4&#39;,6-diamidino-2-phenylindole; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion; SPI-2 = Salmonella pathogenicity island 2; BF = brightfield. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64382/64382fig06.jpg" /> 
Figure 6:&#160;Super-resolution imaging of GCE-labeled SseJ in HeLa cells. HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 &#181;M TCO*A. TCO-tagged secreted SseJ-F10TCO-HA is labeled with 2 &#181;M JF646-Tz under physiological conditions as described in the protocol, then extensively washed. Images are acquired using Nikon N-STORM. (A) Brightfield image of a HeLa cell under observation. Scale bar = 10 &#181;m. (B) Diffraction-limited, widefield image of secreted SseJ labeled with TCO*A and JF-646. Scale bar = 10 &#181;m. (C) Corresponding dSTORM image of the SseJ-decorated SIFs. Scale bar = 10 &#181;m. C(i) Inset shows a magnified view of super-resolved SseJ in the boxed region. Scale bar = 1 &#181;m. Abbreviations: HA = hyaluronic acid; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion; WF = widefield; BF = brightfield. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1.&#160;Solutions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64382/64382_Table 1.xlsx" target="_blank">Please click here to view a larger version of this table.</a>

Watch this Scientific Journal Video about Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion at JoVE.com

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

super-resolution-imaging-bacterial-secreted-proteins-using-genetic

Nanyang Technological University

Research

JoVE Journal

Biology

1.4K Views.  University of Texas Medical Branch. Genetische code-uitbreiding, of GCE, overwint beperkingen van andere etiketteringsstrategieën. Het stelt ons in staat om specifiek gelabelde eiwitten te plaatsen, en we hebben heldere organische fluoroforen die hun visualisatie mogelijk maken, zodat we strategieën kunnen vermijden die bijvoorbeeld fluorescerende eiwitfusies maken, die de eiwitfunctie kunnen verstoren. Deze methode kan op elk ander systeem worden toegepast.We hebben het bijvoorbeeld gebruikt om Zika CO-eiwit te labele...

Video: Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioengineering

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter&#39;s role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

Biochemistry

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Immunology and Infection

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

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