We thank Michael VanNieuwenhze (Indiana University) for the gift of the FDAAs used in Figure 2 and Figure 4. We thank members of the Brown lab for feedback during the preparation of this manuscript. Research in the Brown lab on A. tumefaciens cell growth and division is supported by the National Science Foundation (IOS1557806).

1. Growth of A. tumefaciens Strains
<ol>
	<li>Culturing A. tumefaciens strains
	<ol>
		<li>Use a sterile wooden stick or pipet tip to inoculate 1 mL of ATGN growth media (see materials list for the recipe) with a single colony of the desired strain. 
		NOTE: For A. tumefaciens depletion strains, the ATGN should contain 1 mM IPTG as an inducer to maintain biosynthesis of the essential protein.</li>
		<li>Grow the A. tumefaciens strains overnight in ATGN at 28 &#1...

The authors have nothing to disclose.

This protocol contains a series of procedures for the investigation of A. tumefaciens wildtype, mutant, and depletion strains. It is worth noting that all of the procedures listed in the protocol section can be readily adapted for other bacterial strains with additional modifications to account for growth media, temperatures, and growth rates.
The use of target-specific dyes is a valuable tool for providing a detailed characterization of cell-cycle events in bacterial cells. Here, pep...

Progression through the bacterial cell cycle requires the coordination of many processes including membrane and cell wall biosynthesis, DNA replication and segregation, and cell division. To fully understand the complexity of bacterial cell biology, it is necessary to study these essential events; however, this is a non-trivial task since cell viability is compromised when key components of these pathways are mutagenized. Epifluorescence microscopy coupled with target-specific dyes is a powerful approach to probe these essential processes in wildtype and mutant bacterial strains.
Peptidoglycan-specific dyes include fluorescent antibiotics (vancomycin-FL, bocillin-FL) and fluorescent-d-amino acids (for example, 7-hydroxycoumarin-3-carboxylic acid-3-amino-d-alanine, HADA; 4-chloro-7-nitrobenzofurazan-3-amino- d-alanine, NADA; tetramethylrhodamine-3-amino-d-alanine; TADA). In Gram-positive bacteria, the use of sublethal concentrations of fluorescent antibiotic analogs to probe sites of peptidoglycan biosynthesis has been an effective strategy to reveal peptidoglycan insertion patterns1,2,3,4. While fluorescent vancomycin labeling has been used to gain insights into the peptidoglycan insertion patterns in fixed Gram-negative bacteria5, the outer membrane generally provides a permeability barrier that prevents the use of fluorescent antibiotics as a probe for peptidoglycan biosynthesis in live cells. In contrast, short pulses of fluorescent-d-amino acids or d-amino acids with biorthogonal functional groups covalently label regions of recent peptidoglycan insertion in a wide range of living bacterial cells6,7. Patterns of peptidoglycan insertion that have been observed with synthetic d-amino acids include punctate and septal (Escherichia coli and Bacillus subtilis), polar and septal (Agrobacterium tumefaciens and Listeria monocytogenes), septal only (Staphylococcus aureus), and apical (Streptomyces venezuelae)6,7. These observations indicate that bacteria exhibit diverse patterns of cell wall biogenesis and that the use of synthetic d-amino acids as probes for examining growth patterning is a valuable strategy in many bacteria.
Dyes that label bacterial chromosomes include the deoxyribonucleic acid (DNA) specific minor groove binder (4,6-diamidino-2-phenylindole; DAPI) and high affinity cyanine dyes (Green and Orange; see materials list). DAPI staining of fixed cells assists in enumeration of bacteria from environmental samples8, whereas DAPI staining of live cells is used to indicate bacterial viability9. In contrast, cyanine dyes such as Orange and Green are frequently described as membrane impermeant &#34;dead&#34; cell stains to enumerate non-viable cells9. Remarkably, when these reagents are used to probe the morphology of the bacterial nucleoid during cell growth, DAPI, Orange, and Green were all shown to be membrane permeant and capable of labeling live cells10. In live E. coli cells, DAPI staining of DNA appears diffuse due to auto-fluorescence from the cytoplasm and repeated exposures of DAPI-stained cells to ultraviolet (UV) light perturbs the nucleoid structure10. Staining E. coli or B. subtilis with Orange reveals that this dye is membrane permeant and provides long-lasting fluorescence upon binding to DNA in live cells without impacting cell growth, DNA replication, or chromosome segregation10. These observations suggest that cyanine DNA dyes can be used to monitor the morphology of nucleoids during cell growth in many bacteria.
Phospholipid-specific stryl dyes such as N-(3-triethylammoniumpropyl) -4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide (4-64; see materials list) are cationic compounds and associate preferentially with negatively charged phospholipids such as cardiolipin and phosphatidylglycerol11. Distinct patterns are observed when 4-64 is used to label the membrane of different bacteria. In Escherichia coli, 4-64 is enriched in the poles, in bands along the lateral wall, and in the division sites of late pre-divisional cells12. In Bacillus subtilis, 4-64 labeling enables the visualization of lipid spirals13. In Agrobacterium tumefaciens, 4-64 labels the outer membrane and is observed in a characteristic &#34;horseshoe&#34; pattern in which the growth pole is devoid of labeling14,15. These observations indicate that these bacteria exhibit heterogeneous lipid distributions due to the presence of lipid domains which contribute to cellular asymmetry. Changes in 4-64 labeling patterns such as the presence of diffuse labeling, blebs or vesicles, invaginations, or membrane shrinkage can be informative in characterizing mutants that impact the distribution or biosynthesis of lipids.
Beyond staining cells, determining the function of proteins participating in essential processes is necessary. The characterization of essential proteins is technically challenging because it is not possible to delete essential genes and study the phenotypic consequences. Thus, alternative approaches that deplete the protein have emerged. For example, an essential gene can be put under the control of an inducible promoter rather than its native promoter. Inducible promoters are responsive to small molecules such as; zinc16, isopropyl &#946;-d-1-thiogalactopyranoside (IPTG)17,18,19,20,21, arabinose22, vanillate17,23, and xylose23, thus the transcription of the target gene ceases and the protein of interest is depleted when the inducer is removed. Alternative approaches for depleting essential proteins of interest include synthetic riboswitches24 which use small molecule-RNA interactions to hinder transcription of target genes, CRISPR interference25,26 to block transcription of target genes, and inducible protein degradation27,28 which uses peptide tags to target proteins for degradation by the ClpXP protease. Depletion strains provide only a short time for characterization before the cells lose viability, therefore, microscopic imaging of cells over time during protein depletion is a powerful approach for characterization. Indeed, microscopy of living bacterial cells has enabled researchers to gain insights into fundamental biological processes, including the mechanisms of cell shape maintenance, secretion, and compartmentalization29.
A. tumefaciens is a bacterial plant pathogen30 and natural genetic engineer31,32. Thus, mechanisms related to pathogenicity, including host-pathogen interactions33,34,35, secretion36, and host transformation30,31,37 have been extensively investigated. To design strategies that prevent A. tumefaciens mediated disease or enhance plant transformation, the processes essential for A. tumefaciens survival need to be better understood. The use of target-specific dyes and the recent development of a protein depletion strategy for A. tumefaciens18 provides a means to investigate essential processes.
Here, detailed protocols for microscopic analysis of wildtype, mutant, and protein depletion strains of A. tumefaciens are provided. The first two protocols describe how to prepare cells and label them with target-specific dyes. The third protocol provides step-by-step directions for preparing agarose pads (Figure 1) and imaging the bacterial cells (Figure 2, Figure 3, Figure 4). These protocols may also be suitable for other bacteria with additional adaptations to account for different media conditions, growth rates, oxygen requirements, and cell structures.

<table>
	<tbody>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens C58</td>
			<td>ATCC</td>
			<td>33970</td>
			<td>Watson B, Currier TC, Gordon MP, Chilton MD, Nester EW. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol 123:255-264.</td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens C58&#916;tetRA::mini-Tn7T-GM-Ptac-ctrA &#916;ctrA</td>
			<td></td>
			<td></td>
			<td>Figueroa-Cuilan W, Daniel JJ, Howell M, Sulaiman A, Brown PJB. 2016. Mini-Tn7 insertion in an artificial attTn7 site enables depeltion of the essentail master regulator CtrA in the phytopathogen Agrobacterium tumefaciens. Appl Environ Microbiol. 82:5015-5025.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATGN Minimal Medium</td>
			<td></td>
			<td></td>
			<td>To 1 L of sterilized water add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose. For plates, add 15 g Bacto Agar to 1 L of water and autoclave. Cool to 55 &#176;C and add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose.</td>
		</tr>
		<tr>
			<td>20X AT Buffer</td>
			<td></td>
			<td></td>
			<td>Add 214 g/L KH2PO4 to water and adjust pH to 7.0 with sodium hydroxide. Autoclave.</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher BioReagents</td>
			<td>BP359</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Chemical</td>
			<td>P288</td>
			<td></td>
		</tr>
		<tr>
			<td>20X AT Salts</td>
			<td></td>
			<td></td>
			<td>Add 40 g/L (NH4)2SO4, 3.2 g/L MgSO4&#8226;7H2O, 0.2 g/L CaCl2&#8226;2H2O, and 0.024 g/L MnSO4&#8226;H2O to water. Autoclave.</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Fisher Chemical</td>
			<td>A701</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fisher BioReagents</td>
			<td>BP213</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>Fisher BioReagents</td>
			<td>BP510</td>
			<td></td>
		</tr>
		<tr>
			<td>MnSO4&#8226;H2O</td>
			<td>Fisher Chemical</td>
			<td>M114</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16</td>
			<td>Prepare 40% stock in water. Filter sterilize.</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP1423</td>
			<td>Add 15 g to 1 L of water when preparing plates.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optional Media Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>GoldBio</td>
			<td>K-120</td>
			<td>Prepare as a 100 mg/ml stock solution in water and filter sterilize. Use at final concentration of 200 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>GoldBio</td>
			<td>I2481C5</td>
			<td>Prepare as a 1 M stock solution in water and filter sterilize. Use at final concentration of 1 mM as needed for induction.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550D</td>
			<td>25 X 75 X 1.0 mm. Clean with Sparkle glass cleaner.</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisherbrand</td>
			<td>12-541-B</td>
			<td>22 X 22 mm. No. 1.5. Clean with Sparkle glass cleaner.</td>
		</tr>
		<tr>
			<td>Sparkle Glass Cleaner</td>
			<td>Home Depot</td>
			<td>203261385</td>
			<td>Ammonia and alcohol free.</td>
		</tr>
		<tr>
			<td>Ultra Pure Agarose</td>
			<td>Invitrogen</td>
			<td>16500-100</td>
			<td>Add to water, PBS, or media to a final concentration of 1 - 1.5%. Melt in microwave and place on 70 C</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher BioReagents</td>
			<td>BP399500</td>
			<td>10X solution to be diluted to 1X with sterile water.</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-999</td>
			<td>Laboratory film used as gasket in agarose pad preparation.</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td></td>
			<td></td>
			<td>Add equal weights of lanolin, parafin wax, and petroleum jelly to a conical tube. Heat tube in 70 &#176;C dry, bead or water bath to melt and mix. Apply VALAP while still molten.</td>
		</tr>
		<tr>
			<td>Lanolin Butter</td>
			<td>SAAQIN</td>
			<td>SQ-LAB-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum Jelly</td>
			<td>Target Corp.</td>
			<td>06-17644</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>Crafty Candles</td>
			<td>263012</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Target-specific dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher BioReagents</td>
			<td>BP231-1</td>
			<td>Use to dilute stock solutions of dyes as needed.</td>
		</tr>
		<tr>
			<td>FDAAs (NADA, HADA,TADA)</td>
			<td></td>
			<td></td>
			<td>FDAAs can be synthesized or acquired through agreement with Mike VanNieuwenhze (Indiana University). Prepare 100 mM stock solution in DMSO. Use at a final concentration of 5 mM.</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>62247</td>
			<td>Prepare 1 mg/ml stock solution in DMSO. Use at final concentration of 1 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Invitrogen</td>
			<td>S11368</td>
			<td>Stock concentration is 5 mM in DMSO. Use at final concentration of 5 &#181;M.</td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T3166</td>
			<td>Prepare 8 mg/ml stock solution in DMSO. Use at final concentration of 8 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bath</td>
			<td>Sheldon Manufacturing, Inc.</td>
			<td>52120-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallic thermal beads</td>
			<td>Lab Armor</td>
			<td>42370-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope equipped with an EMCDD camera</td>
			<td></td>
			<td></td>
			<td>Nikon Eclipse TiE equipped with a QImaging Rolera em-c2 1K electron-multiplying charge-coupled-device (EMCCD) camera is used in this work.</td>
		</tr>
	</tbody>
</table>

live cell fluorescence microscopy to observe essential processes during microbial cell growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Target-specific labeling of wildtype A. tumefaciens cells 
In order to illustrate that cell morphology is not impacted by the wash steps or treatment with 1% DMSO (which is used to dilute the fluorescent dyes), cells were imaged directly from culture (Figure 2A, far left panel), after washing the cells by centrifugation as described in 1.2 (Figure 2A, left panel), o...

Watch this Scientific Journal Video about Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth at JoVE.com

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

The overall goal of this microscopy-based approach is to observe a central processes during growth of bacterial cells. This method enables us to address important questions in bacteriology, such as how essential processes are coordinated during bacterial cell growth and division. The main advantage of this technique is we're able to use fluorescent reagents to visualize bacterial cell surfaces and structures in living cells.Here, we use this method to provide insight into essential processes in Agrobacterium tumefaciens. However, this process could also be adopted to other bacteria to study their growth as well. To begin, use a sterile wooden stick or pipette tip to inoculate three milliliters of ATGN Growth Medium, with a single colony of a wild-type or mutant strain of A.Tumifaciens.Grow the culture at 28 degrees celsius and 225 RPM overnight. The following day, use a spectrophotometer to measure the OD600. Then, dilute the cell culture to an OD600 of approximately 0.2 and continue to grow the cells to an OD600 of 0.6.Dispense one milliliter of exponential culture into three microfuge tubes. And pellet the cells in a desktop centrifuge at 7, 000 x g for five minutes. Re-suspend each cell pellet in one milliliter of PBS, and then add either one microliter of DMSO Orange stock solution or DAPI stock solution.Mix gently by pipetting, and incubate the re-suspended cells in the dark for five minutes. Pellet the cells by centrifugation at 7, 000 x g for five minutes, and re-suspend the pellet in one milliliter of PBS to remove excess reagent. Then, wash the cells two more times, and re-suspend the pellet in 50 microliters of PBS or medium.For simultaneous time-lapse imaging, combine ten microliters of cells from each treatment in a new microfuge tube. To prepare agarose pads for cell imaging, use a cover slip as a guide, and cut a 22 by 22 millimeter square of laboratory film by running a scalpel around the edges. Cut a square out of the center of the laboratory film, leaving an approximately two to five millimeter border to serve as a gasket for the agarose pad.Then, discard the center cut-out. Place the film gasket onto a glass slide, cleaned with an ammonia and alcohol-free cleaner. And use a heat block set to 70 degrees celsius or a flame to heat the slide until the film is slightly melted onto the glass.Next, prepare agarose solution by mixing approximately 0.075 grams of agarose and five milliliters of medium in a small flask. Heat the solution in a microwave with periodic swirling to mix, until the agarose is dissolved and the solution is clear. Keep the agarose solution at 55-70 degrees celsius and use it for construction of multiple agarose pads within 48 hours.Pipette the warm agarose medium into the center of the gasket. Then, place a cover slip over the gasket to evenly distribute the agarose. Place the slide on a cool level surface to solidify for approximately two minutes.Now, carefully slide the cover slip off the agarose pad, then allow the agarose pad to air-dry for one to two minutes at room temperature, until the surface of the pad appears dry. Proper construction of the agarose pad is crucial for acquiring high-quality images. Don't try to image on a bad pad.If the agarose pad dries out, ripples or rips, it's best not to be lazy and instead, make a new pad. Then, using a scalpel, remove a small strip of agarose to create an air-pocket approximately two by seven to ten millimeters. A.Tumefacien cells tend to grow best near the air-pocket.Spot 0.8 to 1 microliter of cells on the agarose pad. Then gently place a cover slip over the top of the agarose pad to distribute the cells across the surface of the pad. For long-term time-lapse imaging, use melted Valap to seal the edges of the cover slip.Be sure to seal along all the edges and corners of the cover slip with Valap. Failure to do so can result in drifting during the agarose pad time-lapse imaging. To carry out epifluorescence microscopy, place immersion oil on the desired objective and place the inverted slide into the slide-holder on the stage.Use the focusing knobs to bring the cells into focus. Acquire images in Phase, or DIC and fluorescence, using the desired fluorescence filter for fluorescence images. Optionally, acquire multiple x, y positions by randomly selecting ten fields of cells in close proximity to the air pocket of the agarose pads.Set up the time sequence acquisition to image in-Phase, or DIC, at the desired time interval. As shown here, cells were imaged directly from culture after washing by centrifugation and after incubating the cells with 0.1 percent DMSO, the results showed DMSO alone does not impact cell morphology. Both Orange and DAPI labeled DNA within live A.Tumefaciens cells.In late pre-divisional cells, two distinct nucleoloids are observed with Orange staining, whereas DNA labeling appears more diffuse with DAPI staining. In this time-lapse experiment with an equal proportion of unlabeled, DAPI labeled, and Orange labeled wild-type A.Tumefaciens, all cells grew when imaged with Phase-contrast microscopy, indicating that neither stain impaired cell growth. The cells also grew under Phase contrast in epifluoresence microscopy using the TRITC filter.When imaged with the DAPI filter, however, cells stopped growing within an hour. Following similar methods, other fluorescent reagents can also be used to visualize the bacterial cell wall or membranes. Furthermore, many of these fluorescent dyes can be combined to provide a comprehensive image of the bacterial cell surface.Visualization of essential processes in live bacteria is exciting, because it opens up the possibility of studying these processes in conditional mutants or non-model bacteria. This allows us to enhance our studies of diverse bacterial species.

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15.5K Görüntüleme.  University of Missouri. The overall goal of this microscopy-based approach is to observe a central processes during growth of bacterial cells. This method enables us to address important questions in bacteriology, such as how essential processes are coordinated during bacterial cell growth and division. The main advantage of this technique is we're able to use fluorescent reagents to visualize bacterial cell surfaces and structures in living cells.Here, we use this method to provide insight into essential pro...