SMP, SR and AKS acknowledge the fellowships received from the National Institute of Science Education and Research, Department of Atomic Energy. RS acknowledges intramural funding support from the Department of Atomic Energy, and this work is supported through a research grant to RS (BT/PR42977/MED/29/1603/2022) from the Department of Biotechnology (DBT). The authors also acknowledge V Badireenath Konkimalla for his comments, suggestions, and discussions throughout the development of the protocol.

1. Expression of GFP-tagged bacterial cytoskeleton proteins in S. pombe
NOTE: Please see Table 1 for information on all plasmids and strains used here. Please see Table 2 for all media compositions.
<ol>
	<li>Perform cloning of E. coli MreB with an N-terminal GFP fusion (GFP-MreB) and S. aureus FtsZ carrying a C-terminal GFP (SaFtsZ-GFP) into the S. pombe expression vector, pREP42 with a medium-strength thiamine-repressible promoter, nmt4118,19 as previously described14,15. Maintain, amplify, and isolate the plasmids from E. coli strains (DH10&#946;) as described in20,21. 
	NOTE: Other E. coli strains such as DH5&#945;, XL1Blue, TOP10, etc. or other commercially available competent cells routinely used for molecular cloning may also be used.</li>
	<li>Transformation of plasmids pREP42-GFP-EcMreB and pREP42-SaFtsZ-GFP into S. pombe
	<ol>
		<li>Transform the plasmids pCCD3 (pREP42-GFP-EcMreB) and pCCD713 (pREP42-SaFtsZ-GFP) into S. pombe strain (h-leu1-32 ura4-D18) using the lithium acetate method22, as mentioned in the below steps.</li>
		<li>Day 1 - Primary culture: Inoculate a loopful of freshly streaked out S. pombe culture in 3 mL of autoclaved yeast extract and supplements (YES) broth. Incubate it in an orbital shaker at 30 &#176;C overnight (O/N).</li>
		<li>Day 2:
		<ol>
			<li>Secondary culture: Add about 500 &#181;L of primary culture to 30 mL of autoclaved YES broth. Incubate at 30 &#176;C for 3 - 4 h with shaking till the OD600 reaches 0.4 - 0.6. 
			NOTE: For each transformation, 30 mL is used.</li>
			<li>Pellet the 30 mL culture at 2,500 x g for 6-8 min at room temperature. Discard the supernatant and wash the cells with 50 mL of sterile distilled water (D/W). Pellet down again and discard supernatant. Resuspend the cells in 1 mL of sterile D/W and transfer it to a 2 mL centrifuge tube.&#160;Centrifuge as above and discard supernatant.</li>
			<li>Add 1 mL of 0.1 M lithium acetate, Tris-EDTA (LiAc-TE) solution and centrifuge as above. Discard the supernatant and resuspend in 1 mL of 0.1 M LiAc-TE. Centrifuge and discard supernatant, leaving 100 &#181;L of solution behind.</li>
			<li>Add 10 - 20 &#181;g of carrier DNA (Salmon sperm DNA; denatured and flash cooled on ice) and 2 - 3 &#181;g of the plasmid DNA, which needs to be transformed. Mix gently. Incubate at room temperature for 10 min.</li>
			<li>Add 260 &#181;L of 40 % PEG/LiAc-TE; mix gently. Incubate for 60 min in the shaker or a thermomixer at 30 &#176;C with gentle mixing. Add 43 &#181;L of DMSO; mix gently.</li>
			<li>Heat shock at 42 &#176;C for 10 min in the thermomixer. Pellet at 2,500 x g for 6-8 min and discard supernatant. Wash the pellet 1x with 1 mL of sterile D/W.</li>
			<li>Pellet, discard supernatant and resuspend the pellet in 200 &#181;L of sterile D/W. Plate 100 &#181;L on Edinburgh minimal medium (EMM) plates containing 5 &#181;g/ mL thiamine (to represses nmt41 promoter) and amino-acid supplements adenine (0.225 mg/ mL), histidine (0.225 mg/ mL) and leucine (0.225 mg/ mL) but lacking uracil (selection marker for pREP42 plasmid). 
			NOTE: Alternately, plate 70 &#181;L and 130 &#181;L in two different plates. Two different volumes (70 &#181;L and 130 &#181;L) are plated to obtain isolated colonies on at least one of the plates depending on the transformation efficiencies, which can vary from experiment to experiment.</li>
			<li>Incubate the plates at 30 &#176;C for 2-3 days till colonies appear. Mix a single colony with 100 &#181;L of sterile D/W and spread out on a fresh EMM plate containing thiamine but lacking uracil as described above.</li>
			<li>Incubate at 30 &#176;C for 2-3 days until a complete lawn appears. Scrap the grown lawn of cells using an inoculating loop and resuspend it to 1 mL of YES media containing 30% glycerol in a cryo-vial.</li>
			<li>Flash freeze the cryo-vial in liquid nitrogen and store at -80 &#176;C to preserve the frozen yeast stocks.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Expression of bacterial cytoskeleton proteins in fission yeast
	<ol>
		<li>Aseptically streak a patch from the glycerol stock onto a fresh yeast-specific plate to obtain enough culture for further experiments. 
		NOTE: In the case of pREP42, we used an EMM agar plate (minimal media) containing adenine, histidine, and leucine (as using S. pombe strain (h- leu1-32 ura4-D18)) without uracil (uracil present in pREP42 as a selection marker). We add 15 - 20 &#181;M thiamine to the agar plates (as pREP42 has thiamine repressible nmt41 promoter) to repress the expression of the gene of interest when growing on the plate.</li>
		<li>Inoculate a small loopful of the inoculum from the streaked patch into 5 mL of yeast-specific EMM media lacking thiamine and incubate it for 10 - 12 h at 30 &#176;C. 
		NOTE: The expression of FtsZ and MreB from different species under the nmt41 promoter in the S. pombe can vary, typically from 16 to 30 h. The optimal time for protein expression of GFP-EcMreB and SaFtsZ-GFP in S. pombe is 20 - 24 h and 16 - 20 h at 30 &#176;C, respectively, without thiamine.</li>
	</ol>
	</li>
</ol>
2. Treatment of S. pombe cultures expressing GFP-tagged bacterial cytoskeletal proteins
NOTE: A number of different drug molecules are tested on the overnight-grown yeast culture in a 96-well plate.
<ol>
	<li>Inoculate a fresh culture by transferring 50 &#181;L of the overnight culture into each well of a 96-well plate containing 150 &#181;L of fresh yeast EMM media using the semi-automated, 96-well multichannel pipetting instrument.</li>
	<li>While pipetting in and out for proper mixing using a semi-automated, 96-well multichannel pipette, slowly introduce different increasing concentrations of drugs into the growing culture, each done in triplicate as represented in the schematic in Figure 1.</li>
	<li>As a positive control, use the already known drugs, PC190723 for (S. aureus FtsZ) and A22 (for E. coli MreB) in duplicate. 
	NOTE: As previously reported, PC190723 and A22 were used at a concentration of 56.2 &#181;M17 and 72.6 &#181;M14,17, respectively. Meanwhile, A22 treatment of SaFtsZ and PC190723 treatment of EcMreB served as negative controls, respectively.</li>
	<li>Use DMSO as a solvent control at three different concentrations according to the lowest and highest concentrations of drugs. 
	NOTE: The solvent in which drugs are dissolved is used as a solvent control.</li>
	<li>Incubate the control and treated cultures at 30 &#176;C for 6-10 h (till the cells show the expression of bacterial fluorescent proteins) and then image using epifluorescence microscopy.</li>
</ol>
3. Visualization of the polymers
<ol>
	<li>Apply a coating of 20 &#181;L of 1 mg/mL concanavalin A to each well of the optically transparent bottom 96-well plate (black-walled for fluorescence imaging) and incubate for 20 min at room temperature. Aspirate liquid and let it air dry for 10 min.</li>
	<li>Transfer 20 &#181;L of cells from the culture plate into each respective well and allow to sit for 10 min. Wash the cell 3x-4x with the sterile EMM media.</li>
	<li>With the objective lens to be used in place (see step 3.6), use the navigator mode in the acquisition panel of the image acquisition&#160;software for 96-well plate alignment and well coverage. Align well edges on the navigator.</li>
	<li>Next, select the well coverage parameters, which include a multi-position selection of the region of interest in the well, and use adaptive autofocus control with on-demand mode to keep the sample in focus during imaging.</li>
	<li>Acquire images using differential interference contrast (DIC) and fluorescence. Set excitation and emission filters of 475/28 nm and 525/48 nm, respectively to image the GFP-tagged bacterial proteins expressed in yeast. Obtain Z-stacks at a step size of 0.2 &#181;m through the thickness of the yeast cells (5 &#181;m).</li>
	<li>Capture images using an inverted epifluorescence microscope equipped with a 100x, 1.4-NA oil-immersion objective and a 2,000 x 2,000 sCMOS camera (6.5 &#181;m x 6.5 &#181;m pixel size). Use a LED illumination system for excitation of the fluorophore. 
	NOTE: Any epifluorescence microscope system that has a motorized stage for a 96-well plate with precise multi-point positioning should be suitable.Acquire all the cell images of control and drug-treated with a similar exposure time (usually in the range of 0.3 - 0.5 s) at a binning of 1 x 1 and illumination at 15% - 20% to minimize the experiment variables and maintain consistency.</li>
</ol>
4. Quantification of the images using ImageJ
<ol>
	<li>Process cell images and measure the number of spots per cell and density for FtsZ17. In the case of MreB, measure density and anisotropy23 and compare between control and treated cells using Fiji (v2.0.0-rc-69/1.52p)24.</li>
	<li>Using the images from the DIC channel, first outline the individual yeast cells using the freehand drawing tool and save as a region of interest (ROI) in the ROI manager. This step is not yet automated, but recently developed tools using machine learning25,26 could be attempted and incorporated in the near future.</li>
	<li>Mask the outside area of the cell and fill black as described in27.</li>
	<li>Use the OPS threshold IJ1 analyse macro, a built-in feature in Fiji, to count the number of spots24.</li>
	<li>Use the otsu method for auto-thresholding and mask the segmented particles. Run the plug-in analyse particles using a macro.</li>
	<li>For measuring polymer density (amount of cytoskeleton per unit area in a cell), process images as described, including masking and skeletonization steps27,28. Use Lpx 2Dfilter in the lpx plug-in to skeletonize the images.</li>
	<li>For details, check this recent publication17. Perform EcMreB polymer quantification as previously mentioned in23, use anisotropy to quantify spatial organization using FibrilTool29 as described previously23. 
	NOTE: These methods of analysis can be used to quantify any other bacterial cytoskeleton proteins which are treated or untreated by the drugs.</li>
</ol>

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ancient+yeast+for+young+geneticists:+A+primer+on+the+Schizosaccharomyces+pombe+model+system.">An ancient yeast for young geneticists: A primer on the Schizosaccharomyces pombe model system.</a> Genetics. 201 (2), 403-423 (2015).</li><li>Aoi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+first+and+the+second+meiotic+divisions+using+a+marker-less+drug-hypersensitive+fission+yeast.">Dissecting the first and the second meiotic divisions using a marker-less drug-hypersensitive fission yeast.</a> Cell Cycle. 13 (8), 1327-1334 (2014).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+sensitivity+of+HIV-1+protease+inhibitors+by+restriction+of+cellular+efflux+system+in+a+fission+yeast+model.">Improving drug sensitivity of HIV-1 protease inhibitors by restriction of cellular efflux system in a fission yeast model.</a> Pathogens. 11 (7), 804 (2022).</li></ol>

All the authors declare no conflicts of interest.

Antimicrobial resistance (AMR) is a serious global health threat, and there is an urgent need for new antibiotics with novel targets. The bacterial cytoskeleton has emerged as an attractive target for developing new antibiotics, with small molecule inhibitors of the cell division protein FtsZ, such as TXA709, already in Phase-I clinical trials30. Several methods have been developed to identify inhibitors of FtsZ polymerization7,31. We have recently used the eukaryotic fission yeast in a cell-based assay to show that the FtsZ stabilizing drug, PC190723, can target HpFtsZ17. Moreover, we also showed that although drugs such as berberine and sanguinarine affected the polymerization of FtsZ, they also affected the morphology of yeast cells17. Prompted by these studies and observations, we set out to develop a single-step cell-based assay to screen for small molecule modulators of the bacterial cytoskeleton using the fission yeast expression platform. Since the screen relies upon inhibition of the assembly of GFP-tagged cytoskeletal proteins, small molecules sharing similar fluorophore properties may give rise to false positives. Avoiding long-pass filters and choosing narrow band-pass filters may help reduce these false positives arising from fluorescent small molecules. Here, we show the steps involved in setting up a medium throughput screen using an epifluorescence microscope equipped with a 96-well plate stage holder that can be used to screen for drugs targeting SaFtsZ or EcMreB. The workflow involves semi-automated image acquisition and analysis, which can easily be scaled to high-throughput screens in the future using robotic liquid handling systems and image processing pipelines.
Further, the screen should be useful for other bacterial cytoskeletal proteins, such as the actin-fold containing plasmid maintenance protein ParM32 and against any protein that assembles into polymeric structures in fission yeast. However, optimal expression levels for each protein of interest will need to be standardized prior to use in any drug screens. While for FtsZs, we have found nmt41 to be an optimal promoter, for other proteins of interest, a stronger or weaker version of the nmt1 promoter (Promoter strength (and leakiness): nmt1 &gt; nmt41 &gt; nmt81) or other constitutive promoters such as the yeast adh1 or act118,33 may be tried to obtain optimum levels of expression and assembly of polymeric structures. Moreover, while using episomal plasmids, copy number variations from cell to cell also pose problems for uniform gene expression. To circumvent these issues, several vectors that can be integrated into the fission yeast genome (SIVs for stable integration vectors) carrying promoters of various strengths have been recently developed34. The use of these vectors may offer more uniform and quantitative results as compared to the episomal vectors used in this study here. The use of SIVs could avoid the large heterogeneity in expression levels, seen as a variation in the fluorescence intensities across the cells and should be better suited for high-throughput screens.
Furthermore, drugs like PC190723 act to stabilize the FtsZ polymers, resulting in the early assembly of FtsZ filaments compared to untreated cells. While the screen is suitable for screening drugs that inhibit polymerization of bacterial cytoskeletal proteins, because of visual assessment of diffuse fluorescence at the endpoint, screening of drugs stabilizing FtsZ polymers requires further optimization of protein expression levels and the time needed to achieve maximal number of cells having polymers. It is thus critical to have a reasonable estimate of the time needed for protein expression to achieve the critical concentration required for assembly and decide the endpoint for the assay accordingly. In case of drugs that might act to decrease the critical concentration required for polymerization of the protein, the choice of end-point would typically be a time, wherein a considerable population of untreated cells exhibit diffuse fluorescence, but cultures treated with drugs would display polymer assemblies.
One of the limitations which is also a time-intensive step of the workflow is the requirement to identify and segment individual cells for quantitative image analysis. Currently, in the proposed scheme, this is a manual step to identify individual yeast cells and assign the region of interest (ROIs) for the quantification of the effect of the drugs. However, we envisage that the recently developed machine learning algorithms using training sets such as those implementable in ilastik35 should minimize the need for manual intervention.
While our approach here using the yeast platform is useful for identifying hits and potential small molecules targeting the bacterial cytoskeleton, one will eventually need to test their antibacterial activity using the standard minimal inhibitory concentration tests and efficacy in animal models. Nonetheless, the biggest challenge to the global increase in antimicrobial resistance (AMR) still lies in the outer membrane permeability of the Gram-negative bacteria and the numerous efflux pumps in many pathogenic bacterial species. Moreover, efflux pumps in fission yeast may also pose similar problems and higher drug concentrations might be required for the screening process. Alternately, fission yeast strains that are hyper-sensitive to drugs (S. pombe MDR-supML), as a result of the deletion of the seven multi-drug resistance genes, including four transporter genes and a transcription factor, may be used as hosts for screening32,33. Future developments in technologies that combine the targeted antibacterial discovery and address resistance due to general phenomena such as membrane permeability will be necessary to tackle the growing antimicrobial resistance.

The widespread resistance to nearly all antibiotics presently employed to combat bacterial infections has created an immediate necessity for novel categories of antibiotics. A 2019 report indicated that antibiotic-resistant infections resulted in the loss of 1.27 million lives, contributing to an overall tally of 4.95 million deaths when considering complications from resistant bacterial infections1. While still effective in clinical practice, the current arsenal of antibiotics predominantly targets a narrow spectrum of cellular processes, primarily focusing on cell wall, DNA, and protein synthesis. Over the past half-century, fewer than 30 proteins have been commercially exploited as targets for the development of new anti-bacterials2,3. This limited range of viable targets significantly creates constraints to discovering new antibiotics or their derivatives for combating antibiotic-resistant bacteria. Thus, to overcome the emerging antibiotic resistance problem, there is a need for the development of new antibiotics with novel targets and mechanisms of action.
An antibacterial target should ideally be an essential component of bacterial cell growth, be conserved throughout the phylogenetically diverse species, show least eukaryotic homology, and be accessible to antibiotics4. Since the discovery of bacterial cytoskeletal proteins involved in cell division and cell shape maintenance, they have emerged as a promising focal point for developing antibacterial compounds5. These proteins are essential for bacterial viability and play a pivotal role in maintaining cell shape (MreB, CreS), division (FtsZ, FtsA), and DNA segregation (ParM, TubZ, PhuZ, AlfA), akin to the cytoskeleton in eukaryotic cells. Notably, FtsZ exhibits a remarkably high level of conservation across a wide range of prokaryotic organisms, while MreB is found in nearly all rod-shaped bacteria. Such wide distribution and relevance in cell viability make these proteins a fascinating target in antibiotic research5,6,7.
It is crucial to adopt a multi-pronged approach that combines in vivo observations, in vitro interactions, and enzymatic experiments to thoroughly validate bacterial cytoskeleton proteins as the primary target of a potential inhibitor7. Laborious procedures or substantial cost implications encumber many available assays for this purpose. These are notable obstacles to their widespread utilization in screening lead compounds that could impact the bacterial cytoskeleton. Among these, microscopy stands out as an exceptionally efficient and rapid method for assessing the effectiveness of compounds by directly examining changes in cell morphology. Yet, the hetero-association of target protein with other cytoskeleton complexes, indirect effects due to off-target binding and changes in membrane potential, difficulty in penetrating the cell efficiently, and presence of efflux pumps, especially in Gram-negative bacteria, make it collectively complex to pinpoint the precise cause of bacterial cell deformation8,9,10.
Schizosaccharomyces pombe, or fission yeast as it&#39;s commonly known, is a rod-shaped unicellular eukaryotic organism. Fission yeast is widely used as a model organism in cellular and molecular biology due to the extraordinary conservation in cellular processes such as the cell cycle and division, cellular organization, and chromosome replication with higher eukaryotes, including humans11,12. Furthermore, Errington and colleagues expressed a pole-localized bacterial cytoskeletal protein DivIVA in fission yeast to demonstrate that DivIVA accumulated on negatively curved surfaces13. Again, Balasubramanian and group had first established fission yeast as a cellular model system to bring new insights into the mechanism, assembly, and dynamicity of the E. coli actin cytoskeleton protein like MreB14 and tubulin homolog FtsZ15. They also demonstrate the ability of A22 to efficiently impede the polymerization of MreB through epifluorescence microscopy when expressed in yeast14. Following this, other groups have also successfully employed fission yeast to study the assembly properties of chloroplast FtsZ1 and FtsZ2 proteins16. More recently, we have established a proof-of-concept of the viability of the use of fission yeast as a cellular platform to specifically screen for bacterial cytoskeleton inhibitors by conducting a comprehensive assessment of the impact of three known FtsZ inhibitors-sanguinarine, berberine, and PC190723-on FtsZ proteins derived from two pathogenic bacteria, namely Staphylococcus aureus and Helicobacter pylori17. Additionally, this single-step cell-based assay proves instrumental in minimizing the risk of identifying compounds that may be potentially toxic to eukaryotic cells.
In this report, utilizing the fission yeast system, we propose a systematic workflow using the standard 96-well plate for semi-automated screening and quantification of the effect of small molecule inhibitors targeting FtsZ from Staphylococcus aureus and MreB from Escherichia coli. Here, we set up and optimize the semi-automated workflow using the established inhibitors PC190723 and A22 that specifically target FtsZ and MreB, respectively. This workflow uses an epifluorescence microscope equipped with a motorized high-precision stage and automated image acquisition in a standard 96-well plate to improve upon current standardization. Hence, it can be applied to medium- as well as high-throughput screens of synthetic chemical libraries and circumvents some of the challenges listed above.

<table><tbody><tr><td>96 Well CC2 Optical CVG Sterile, w/Lid. Black</td><td>Thermo Scientific&amp;trade;</td><td>160376</td><td></td></tr><tr><td>96-well plate</td><td>Corning&amp;nbsp;</td><td>&amp;nbsp;CLS3370</td><td></td></tr><tr><td>A22 Hydrochloride</td><td>Sigma&amp;nbsp;</td><td>SML0471</td><td>Dissolved in DMSO</td></tr><tr><td>Adenine</td><td>FormediumTM</td><td>DOC0229</td><td>225 mg/L of media&amp;nbsp;</td></tr><tr><td>Concanavalin A&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C5275-5MG</td><td></td></tr><tr><td>DMSO</td><td>Sigma&amp;nbsp;</td><td>317275</td><td></td></tr><tr><td>Edinburg minimal medium (EMM Agar or EMM Broth)</td><td>FormediumTM</td><td>PMD0210</td><td>See below for composition</td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>EDS-500G</td><td></td></tr><tr><td>epMotion&amp;reg; 96 with 2-position slider</td><td>Eppendorf</td><td>5069000101</td><td></td></tr><tr><td>Histidine</td><td>FormediumTM</td><td>DOC0144</td><td>225 mg/L of media&amp;nbsp;</td></tr><tr><td>Leica DMi8 inverted fluorescence microscope</td><td>Leica Microsystems</td><td></td><td>German company</td></tr><tr><td>Leucine</td><td>FormediumTM</td><td>DOC0157</td><td>225 mg/L of media&amp;nbsp;</td></tr><tr><td>Lithium acetate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>517992-100G</td><td></td></tr><tr><td>PC190723</td><td>Merck&amp;nbsp;</td><td>344580</td><td>Dissolved in DMSO</td></tr><tr><td>Polyethylene glycol (PEG)</td><td>Sigma&amp;nbsp;</td><td>202398</td><td></td></tr><tr><td>Thiamine</td><td>Sigma</td><td>T4625</td><td>Filter sterilised</td></tr><tr><td>Tris-Hydrochloride</td><td>MP</td><td>194855</td><td></td></tr><tr><td>Uracil</td><td>FormediumTM</td><td>DOC0214</td><td>225 mg/L of media, Store solution at 36&amp;deg;C</td></tr><tr><td>YES (Yeast extract + supplements) Agar</td><td>FormediumTM</td><td>PCM0410</td><td>See below for composition</td></tr><tr><td>YES (Yeast extract + supplements) Broth</td><td>FormediumTM</td><td>PCM0310</td><td>See below for composition</td></tr><tr><td>&lt;strong&gt;Yeast (&lt;em&gt;S. pombe&lt;/em&gt;) media&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Yeast extract + supplements (YES)&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td><td>&lt;strong&gt;g/L&lt;/strong&gt;</td><td></td></tr><tr><td></td><td>Yeast extract</td><td>5</td><td></td></tr><tr><td></td><td>Dextrose</td><td>30</td><td></td></tr><tr><td></td><td>Agar</td><td>17</td><td></td></tr><tr><td></td><td>Adenine</td><td>0.05</td><td></td></tr><tr><td></td><td>L-Histidine</td><td>0.05</td><td></td></tr><tr><td></td><td>L-Leucine</td><td>0.05</td><td></td></tr><tr><td></td><td>L-Lysine HCl</td><td>0.05</td><td></td></tr><tr><td></td><td>Uracil</td><td>0.05</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Edinburg minimal medium (EMM)&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td><td>&lt;strong&gt;g/L&lt;/strong&gt;</td><td>&lt;strong&gt;concentration&lt;/strong&gt;</td></tr><tr><td></td><td>potassium hydrogen phthallate&amp;nbsp;</td><td>3</td><td>14.7mM</td></tr><tr><td></td><td>Na2HPO4&amp;nbsp;</td><td>2.2</td><td>15.5 mM</td></tr><tr><td></td><td>NH4Cl&amp;nbsp;</td><td>5</td><td>93.5 mM</td></tr><tr><td></td><td>glucose</td><td>2% (w/v) or 20 g/L</td><td>&amp;nbsp;111 mM</td></tr><tr><td></td><td>Salts (stock x 50)</td><td>20 mL/L (v/v)</td><td></td></tr><tr><td></td><td>Vitamins (stock x 1000)</td><td>1 mL/L (v/v)</td><td></td></tr><tr><td></td><td>Minerals (Stock x 10,000)</td><td>0.1 mL/L (v/v)</td><td></td></tr><tr><td>&lt;strong&gt;Salts x 50&amp;nbsp;&lt;/strong&gt;</td><td>52.5 g/l MgCl2.6H20 (0.26 M)&amp;nbsp;</td><td>52.5</td><td>0.26 M</td></tr><tr><td></td><td>0.735 mg/l CaCl2.2H20 (4.99 mM)&amp;nbsp;</td><td>0.000735</td><td>4.99 mM</td></tr><tr><td></td><td>50 g/l KCl (0.67 M)&amp;nbsp;</td><td>50</td><td>0.67 M</td></tr><tr><td></td><td>2 g/l Na2SO4 (14.l mM)</td><td>2</td><td>14.1 mM</td></tr><tr><td>&lt;strong&gt;Vitamins x 1000&amp;nbsp;&lt;/strong&gt;</td><td>1 g/l pantothenic acid&amp;nbsp;</td><td>1</td><td>4.20 mM</td></tr><tr><td></td><td>10 g/l nicotinic acid&amp;nbsp;</td><td>10</td><td>81.2 mM</td></tr><tr><td></td><td>10 g/l inositol&amp;nbsp;</td><td>10</td><td>55.5 mM</td></tr><tr><td></td><td>10 mg/l biotin&amp;nbsp;</td><td>0.01</td><td>40.8 &amp;micro;M</td></tr><tr><td>&lt;strong&gt;Minerals x 10,000&amp;nbsp;&lt;/strong&gt;</td><td>boric acid</td><td>5</td><td>80.9 mM</td></tr><tr><td></td><td>MnSO4&amp;nbsp;&amp;nbsp;</td><td>4</td><td>23.7 mM</td></tr><tr><td></td><td>ZnSO4.7H2O</td><td>4</td><td>13.9 mM</td></tr><tr><td></td><td>FeCl2.6H2O&amp;nbsp;&amp;nbsp;</td><td>2</td><td>7.40 mM</td></tr><tr><td></td><td>molybdic acid&amp;nbsp;</td><td>0.4</td><td>2.47 mM</td></tr><tr><td></td><td>KI&amp;nbsp;</td><td>1</td><td>6.02 mM</td></tr><tr><td></td><td>CuSO4.5H2O&amp;nbsp;</td><td>0.4</td><td>1.60 mM</td></tr><tr><td></td><td>citric acid&amp;nbsp;</td><td>10</td><td>47.6 mM</td></tr><tr><td>&lt;strong&gt;Strains/ Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td>&lt;strong&gt;Description&lt;/strong&gt;</td><td>&lt;strong&gt;Reference&lt;/strong&gt;</td><td></td></tr><tr><td>CCD190</td><td>&lt;em&gt;Escherichia coli&lt;/em&gt; DH10&amp;beta;&amp;nbsp;</td><td>Invitrogen</td><td></td></tr><tr><td>CCDY4&amp;nbsp;</td><td>MBY3532; CCDY346/pREP42- GFP-EcMreB</td><td>Srinivasan et al., 2007</td><td></td></tr><tr><td>CCDY340</td><td>CCDY346/pREP42- SaFtsZ-GFP</td><td>Sharma et al., 2023</td><td></td></tr><tr><td>CCDY346</td><td>MBY192; &lt;em&gt;Schizosaccharomyces pombe&lt;/em&gt; [ura4-D18, leu1-32, h-]</td><td>Dr. Mithilesh Mishra (DBS, TIFR)</td><td></td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pCCD3</td><td>pREP42-GFP-EcMreB</td><td>Srinivasan et al., 2007</td><td></td></tr><tr><td>pCCD713</td><td>pREP42-SaFtsZ-GFP</td><td>Sharma et al., 2023</td><td></td></tr></tbody></table>

fission yeast as a platform for antibacterial drug screens targeting bacterial cytoskeleton proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Setting up the 96-well plate for the screening of drugs 
Use of S. pombe to express a C- terminally GFP tagged S. aureus FtsZ from a vector (pREP42) containing the medium-strength thiamine repressible promoter nmt41has been previously established17 and similarly, the E. coli MreB tagged with N- terminal GFP was also expressed in S. pombe14. We have also shown that PC190723, a specific inhibitor of SaFtsZ and A22, a MreB inhibitor, can exert their effects on the respective bacterial cytoskeletal proteins in a specific manner when expressed in yeast14,17.
Here, we propose to use the yeast expression system to develop a medium or high-throughput screening of antibacterial drugs targeting the bacterial cytoskeleton proteins. An epifluorescence microscope with a motorized stage can be automated to image a 96-well plate, and commercial drug libraries are also readily available in 96-well plate formats. We, therefore, prefer to use a 96-well plate for the proposed screening of drugs. The plates are set as shown in Figure 1. The first row (Figure 1A) consists of SaFtsZ-GFP expressing yeast culture. The second row (Figure 1B) consists of yeast cultures expressing GFP-EcMreB. The first two columns of the first two rows (A1:B2) are set as media blank. The following six columns of the first two rows (A3:B12) are set as DMSO control with three different concentrations according to the drug dilutions in duplicate. Next, PC190723 (56.2 &#181;M) and A22 (72.6 &#181;M) are added to yeast cells expressing SaFtsZ-GFP or GFP-EcMreB. PC190723 and A22 serve as positive controls for FtsZ and MreB, respectively. All other wells (C1:F12) are utilized for adding various drugs (at three different concentrations and in duplicate) used in the screen to identify effectors of the bacterial cytoskeleton proteins, SaFtsZ or EcMreB. Cultures of yeast expressing the GFP-tagged SaFtsZ or MreB are dispensed into these 96-well plates and grown until the effects of the drugs on the assembly of polymers are visualized.
Assessment of growth effects on yeast cells 
After incubating the 96-well containing sub-cultured cells at 30 &#176;C for 6 to 10 h, the optical density (OD) of the yeast culture is measured using a microplate reader. The measurement of OD helps assess any growth inhibitory effect of the drugs against the eukaryotic yeast cells and possibly deleterious effects on human cells. Thus, this assay can be used to screen out multiple drugs based on their toxicity in the yeast system. However, neither PC190723 nor A22 exhibits any growth effects or toxicity to yeast cells, and OD600 of the cultures expressing SaFtsZ-GFP treated with DMSO, PC190723, or A22 were 0.38 &#177; 0.03, 0.44 &#177; 0.02 and 0.43 &#177; 0.06 (N &#8805; 4), respectively. Likewise, the OD600 of the cultures expressing GFP-EcMreB and treated with DMSO, PC190723, or A22 were 0.38 &#177; 0.04, 0.44 &#177; 0.07 and 0.41 &#177; 0.08 (N &#8805; 4), respectively.
Visualization of the effect of the drugs on the polymeric structures assembled by SaFtsZ and EcMreB expressed in S. pombe 
In order to assess the effect of the drugs on polymerization of SaFtsZ or EcMreB, an aliquot of the yeast cells from the above-mentioned 96-well plate containing drugs is transferred to another 96-well plate with optically clear glass bottom 96-well suitable for fluorescence imaging. This 96-well plate is also coated with concanavalin A to allow adhesion of the yeast cells expressing the GFP-tagged SaFtsZ or EcMreB. The 96-well plate is imaged using an epifluorescence microscope covering all the 96-wells of the plate at the predetermined positions as controlled by the image acquisition software (Figure 2). In the control wells (DMSO treated), yeast cells expressing SaFtsZ-GFP showed polymeric structures in the form of spots or patches distributed throughout the cells, after 16 - 18 h of growth in the absence of thiamine. However, at 10 - 12 h after growth in the lack of thiamine, cells only exhibited diffuse fluorescence or few patches, suggesting that FtsZ-GFP expression had not reached the critical concentration required for polymerization (Figure 3A). On the contrary, the FtsZ stabilizing drug, PC190723, showed a considerable increase in the polymeric structures (spots or patches) of SaFtsZ-GFP in comparison to DMSO control after 12 h of growth (Figure 3B), serving as the positive control. In contrast, cells treated with A22 showed no difference in the SaFtsZ-GFP structures assembled (Figure 3C). However, unlike in cultures treated with PC190723, SaFtsZ-GFP assembled into patches in untreated cultures only when induced for longer periods, showing that PC190723 acted to reduce the critical concentration of polymerization of SaFtsZ (Figure 3D). Likewise, GFP-EcMreB expressed in S. pombe formed linear arrays of long filaments along the longitudinal axis of fission yeast (Figure 4A). While PC190723 was found to have no effect on EcMreB polymerization(Figure 4B), treatment of cells with A22 resulted in diffused fluorescence throughout the cytoplasm of the yeast cells (Figure 4C). The images from the rest of the 96-well plates are visually inspected for any stabilization or inhibitory effects of the drugs on the SaFtsZ or EcMreB, as the case may be.
The impact of the drugs on the assembly of bacterial cytoskeletal proteins can then be quantified using image analysis tools and plug-ins in ImageJ or Fiji, as previously reported for SaFtsZ17 and EcMreB21. The automated image acquisition in 96-well plate format and implementation of custom macros and scripts for image processing can accelerate the imaging time and quantification of the effects of the drugs. Thus, using the single-celled eukaryotic yeast, S. pombe, as the host system, we propose that a medium or high-throughput screen for small molecules targeting the bacterial cytoskeletal proteins can be successfully carried out, eventually leading to the discovery of new antibiotics.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66657/66657fig01.jpg" /> 
Figure 1: Schematic showing the use of fission yeast cells to screen for drugs targeting the assembly of bacterial cytoskeletal proteins SaFtsZ or EcMreB. FtsZ from S. aureus and MreB from E. coli were cloned into the yeast expression vector, pREP42 having GFP tag at C- and N- terminus, respectively and transformed into S. pombe for the phenotypic and drug study. The cultures were grown for 8 - 12 h. Subsequently, the cultures were sub-cultured in 96-well plates with appropriate controls and the different drugs being screened. Further, the plate was incubated at 30 &#176;C for 6 - 8 h till the OD600 reaches 0.5-0.6, and the optical density is measured using a microplate reader to assess the toxicity of the drugs towards yeast cells. Another optically clear glass bottom 96-well plate was pre-coated with concanavalin A, for adhering yeast cells. This 96-well plate was used for visualization and image acquisition with an epifluorescence microscope. The images obtained were then analyzed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/66657fig01large.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/66657/66657fig02.jpg" /> 
Figure 2: Schematic of the image acquisition&#160;software for automated imaging of a 96-well plate. Using the navigator in image acquisition&#160;software, a 96-well plate was aligned by marking the edges. Well-coverage parameters like the number of images which need to be taken from each well and its location (random or from the center of the well) were selected as desired. Finally, image parameters such as the filter set to be used (DIC and FITC-filter; Ex 475/28 nm and Em 525/48 nm), percentage intensity of illumination light, autofocus and exposure time for image capture were set. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/66657fig02large.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/66657/66657fig03v2.jpg" /> 
Figure 3: Visualization of the effect of PC190723 and A22 drugs on SaFtsZ-GFP expressed in S. pombe. S. pombe culture expressing SaFtsZ-GFP was grown in the absence of thiamine for 8 - 9 h at 30 &#176;C before DMSO and drug treatment. The culture was further grown in a 96-well plate for 7 - 9 h at 30 &#176;C in the presence or the absence of the drugs. (A) In DMSO control, where an equivalent amount of DMSO was added, cells exhibited a few polymeric structures. (B) In the presence of PC190723 (56.2 &#181;M), SaFtsZ expresses yeast cells showing a considerable increase in the polymeric structures than that of the DMSO control. (C) The MreB inhibitor, A22 (72.6 &#181;M), did not affect SaFtsZ-GFP structures. (D) Longer hours of protein expression were necessary for SaFtsZ-GFP assembly in the absence of PC190723, and therefore, control cultures grown at 30 &#176;C for 12 - 15 h after sub-culturing in 96-well plate containing DMSO also exhibit FtsZ polymers. Scale bar is 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/66657fig03large.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/66657/66657fig04.jpg" /> 
Figure 4: Visualization of the effect of PC190723 and A22 drugs on GFP-EcMreB expressed in S. pombe. S. pombe culture expressing GFP-EcMreB was grown in the absence of thiamine for 9 - 11 h at 30 &#176;C prior to adding DMSO or the drugs (A22 or PC190723). The culture was further grown in a 96-well plate for 8 - 10 h at 30 &#176;C with and without drugs as a control. (A) In cultures where an equivalent amount of DMSO was added as a control, cells exhibited linear filaments of EcMreB oriented along the longitudinal axis of the yeast cells. (B) Assembly of GFP-EcMreB was not affected in cultures treated with PC190723 (56.2 &#181;M). (C) The small molecule A22 (72.6 &#181;M), a known inhibitor of MreB polymerization, prevented the assembly of GFP-EcMreB in fission yeast cells. Scale bar is 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/66657fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Strains and plasmids used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/Table 1 _RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Composition of media and buffers used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66657/Table 2_RE.xlsx" target="_blank">Please click here to download this Table.</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays at JoVE.com

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

fission-yeast-as-platform-for-antibacterial-drug-screens-targeting

Nanyang Technological University

Research

JoVE Journal

Biology

732 Views.  National Institute of Science Education and Research. Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Video: Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetics

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunology and Infection

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

An Introduction to Saccharomyces cerevisiae

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans).
This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer&rsquo;s and Parkinson&rsquo;s diseases.

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

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Mating-based Overexpression Library Screening in Yeast

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

High-throughput Yeast Plasmid Overexpression Screen

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Microscopy of Fission Yeast Sexual Lifecycle

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

An Introduction to Saccharomyces cerevisiae