This research was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) (#2017-05737) to AW and through the Jarislowsky Chair in Biotechnology. MP was supported by a NSERC-CREATE scholarship through the Integrated Training Program in Infectious Diseases, Food Safety and Public Policy (ITraP) at the University of Saskatchewan.
The authors would like to thank Dani Dale for filming and editing.

1. Flask culture cell growth
<ol>
	<li>From frozen stocks, streak Salmonella serovar Typhimurium strains on LB agar (20 mL solid media in 100 mm Petri plate) with the appropriate antibiotic and incubate at 37 &#176;C for 16&#8211;20 h to obtain isolated colonies.</li>
	<li>Inoculate 5 mL LB broth containing the appropriate antibiotic in a 25 mm x 150 mm borosilicate culture tube with 1&#8211;3 colonies from streak plates from step 1.1. Incubate at 37 &#176;C with shaking at 200 rpm for 7 h.</li>
	<li>Measure OD600 of broth culture using a spectrophotometer. Dilute an aliquot of culture 1 in 4 in PBS in a 2 mL cuvette and measure the absorbance at 600 nm. We have found the most critical factor at this step to be the time of growth. The OD values are used to normalize the cultures to deliver the desired number of cells in step 1.4.</li>
	<li>Add 1.0 OD600 volume equivalent of cell culture (i.e. ~109 cells) to an Erlenmeyer flask containing 100 mL of 1% tryptone with the appropriate antibiotic. Incubate at 28 &#176;C with shaking at 200 rpm for 13 h. 
	Note: Biofilm cells will appear as aggregated flakes and single planktonic cells are in the cloudy media.</li>
</ol>
2. Collecting cell-free conditioned 1% tryptone
<ol>
	<li>Collect flask culture for cell-free conditioned 1% tryptone. Pipette flask culture several times to mix before adding to a centrifuge tube.</li>
	<li>Centrifuge at 12,000 x g for 10 min at 10 &#176;C to pellet all cells.</li>
	<li>Decant supernatant into a 0.2 &#181;m filter unit, vacuum filter, and dispense into a new tube. 
	Note: Biofilm aggregates are adherent in conventional solutions (e.g. PBS) and will stick to the sides of tubes and pipette tips. Since it allows for easier manipulation, we use conditioned media (1% tryptone) to move biofilm initially and use warm PBS immediately before cross-linking (step 4.5) to remove any protein crosslinking substrates that could be present in the media.</li>
</ol>
3. Separating biofilm and planktonic cells from flask cultures2
<ol>
	<li>Using a sterile 25 mL pipette, aliquot flask culture into 15 mL tubes. Centrifuge at slow speed (210 x g) for 2 min, to separate the two cell types. 
	Note: Biofilm cells should form a loose pellet at the bottom of the tube</li>
	<li>Pipette the supernatant containing planktonic cells into two 40 mL centrifuge tubes for later (step 5). Remove all the remaining liquid, being careful not to disturb the pellet. Repeat until the cell types have been separated from all of the flask culture media.</li>
</ol>
4. Preparing biofilm aggregates
Note: Biofilm is harvested by wet weight to yield 25 &#181;g of DNA, the recommended minimum starting material for ChIP. The Biofilm Conversion Factor (BCF) of S. Typhimurium is 1.73 x 108 CFU/mg2. Researchers preparing other biofilm types will need to empirically define the number of cells per unit weight of biofilm, which is used to find the weight of biofilm required for 25 &#181;g of DNA starting material using this equation: 
<img alt="Equation 1" src="/files/ftp_upload/60736/60736eq1.jpg" />
<ol>
	<li>Weigh a 2 mL snap cap or screw cap tube accurately.</li>
	<li>Resuspend the biofilm pellet in 1 mL of conditioned tryptone. Move the resuspended biofilm into a pre-weighed 2 mL snap cap or screw cap tube.</li>
	<li>Centrifuge for 1 min at 11 000 x g at 20 &#176;C</li>
	<li>Carefully remove all supernatant from the tube and weigh the tube accurately. Subtract the tube weight from the weight of the tube with biofilm to find the weight of biofilm. It should be &#177;10% of the target biofilm weight.</li>
	<li>Wash cells to remove remaining conditioned tryptone. Add 1 mL of warm PBS to the tube and vortex gently to resuspend the pellet. Centrifuge for 3 min at 8 000 x g to pellet biofilm and remove the supernatant. Add 1 mL of PBS to the tube and vortex to resuspend the pellet.</li>
</ol>
5. Preparing planktonic cells
<ol>
	<li>Record the volume and the OD600 of the planktonic supernatant from slow speed centrifugation (step 3.2) using a spectrophotometer</li>
	<li>Calculate the required volume for a final OD600 of 6.0: 
	<img alt="Equation 2" src="/files/ftp_upload/60736/60736eq2.jpg" /></li>
	<li>Cool the centrifuge to 10 &#176;C and sediment planktonic cells by centrifugation (10,000 x g, 10 min at 10 &#176;C)</li>
	<li>Remove the supernatant and resuspend the cell pellet in the final volume of PBS calculated in step 5.2.</li>
	<li>Re-measure OD600 of the planktonic cells using a spectrophotometer and dispense the volume of 6.0 OD600 planktonic cells into a 2 mL snap cap or screw cap tube.</li>
	<li>Bring volume to 1 mL with PBS.</li>
</ol>
6. Homogenizing cells
<ol>
	<li>Add one sterilized 5 mm stainless steel bead to each of the tubes.</li>
	<li>Homogenize using a mixer mill for 5 min at 30 Hz. Observe aggregate tubes to confirm that biofilm has been broken apart.</li>
	<li>Transfer the homogenized cells to a new 1.5 mL tube, avoiding the metal bead. Bring the volume to 1 mL with PBS. 
	Note: Perform serial dilutions to enumerate input cells and check that the final cell number is close to the desired or predicted number.</li>
</ol>
7. Cross-linking of proteins to DNA
<ol>
	<li>Dispense fresh formaldehyde into sample tubes to a final concentration of 1%. Incubate for 30 min at room temperature on a rotating wheel. 
	Caution: Formaldehyde is corrosive, a skin, eye, and respiratory irritant, and flammable. Dispense in a fume hood.</li>
	<li>Add 1 M glycine to a final concentration of 125 mM to stop crosslinking. Incubate for 5 min at room temperature on a rotating wheel.</li>
</ol>
8. Washing cells to remove excess crosslinker
<ol>
	<li>Sediment the cells at 8,000 x g for 3 min and remove the supernatant</li>
	<li>Resuspend the pellet in 20 &#181;L of 25x protease inhibitors and 500 &#181;L of filter sterilized PBS.</li>
	<li>Centrifuge tube for 3 min at 8000 x g and remove the supernatant.</li>
</ol>
9. Lysing cells
<ol>
	<li>Resuspend the pellet in 600 &#181;L Lysis buffer (refer to Table 1) and incubate on ice for 10 min.</li>
	<li>Add 1.4 mL of IP dilution buffer (refer to Table 1) to a sterile 15 mL tube and move lysed cells to the 15 mL tube. Keep the tube on ice for 1.5&#8211;2 h, and vortex gently and occasionally. 
	Note: Some resistant cell material may remain in tubes. A long incubation period on ice with occasional vortexing will break apart material. The remainder will be broken up through sonication.</li>
</ol>
10. Fragmenting DNA by sonication
<ol>
	<li>Place the 15 mL tube in a beaker of ice and place the 3 mm probe inside the tube.</li>
	<li>Perform 5 sonication rounds of 30 s on at 20&#8211;40% of 400 W with 2 min cooling on ice between bursts.</li>
	<li>Sediment precipitated material by centrifugation (8000 x g, 10 min at 4 &#176;C). Transfer the supernatant to a new tube.</li>
	<li>Optionally, perform decrosslinking at 65 &#176;C for 30&#8211;60 min and digest RNA and protein at 45 &#176;C for 30-60 min before running on a 2% agarose gel to check for proper size fragments. 
	Note: Immerse the probe into the cell lysate. Keep the solution on ice while sonicating and resting. Do not remove the tube while the sonicator probe is pulsing. The solution should appear cloudy pre-sonication and clear post-sonication.</li>
</ol>
11. Immunoprecipitating DNA-protein-antibody complexes
<ol>
	<li>Preparation
	<ol>
		<li>Dispense one 1.35 mL aliquot for immunoprecipitation and one 200 &#181;L aliquot as an input control. Store the input control at -80 &#176;C until decrosslinking and digesting steps are performed.</li>
	</ol>
	</li>
	<li>Immunoprecipitation with primary antibody
	<ol>
		<li>Add 10 &#181;g of purified protein-specific primary antibody to the 1.35 &#181;L aliquot. Incubate overnight at 4 &#176;C on a rotating wheel. 
		Note: The primary antibody can be a monoclonal antibody, high-quality polyclonal serum or commercial epitope antibody (i.e., anti-FLAG).</li>
		<li>Add 50 &#181;L of Protein G magnetic beads to the tubes from step 11.2.1 and incubate at 4 &#176;C for 3 h on a rotating wheel.</li>
		<li>Place IP wash buffer 1, IP wash buffer 2, and TE pH 8.0 (refer to Table 1) in an ice bucket. Warm elution buffer to 65 &#176;C. 
		Note: Do not put solution containing elution buffer on ice.</li>
		<li>Bind the Protein G magnetic beads to the side of tubes using a magnetic stand</li>
		<li>Perform washes: wash 2x with 750 &#181;L cold IP wash buffer 1, wash 1x with 750 &#181;L cold IP wash buffer 2, and wash 2x with 750 &#181;L cold TE at pH 8.0. Keep the tubes on the magnetic stand during washes.</li>
		<li>Add 450 &#181;L of IP Elution buffer (refer to Table 1) to each tube and incubate at 65 &#176;C for 30 min with gentle vortexing every 5 min.</li>
		<li>Bind beads to the side of tubes using a magnetic stand. Wait at least 2 min until the solution is clear.</li>
		<li>Dispense the cleared supernatant to a new 1.5 mL tube, being careful to not disturb the magnetic bead pellet.</li>
	</ol>
	</li>
</ol>
12. Decrosslinking DNA-protein complexes and digesting co-purifying RNA and protein
<ol>
	<li>Add 2 &#181;g RNase A and NaCl to a final concentration of 0.3 M to each tube.</li>
	<li>Incubate at 65 &#176;C, for &#8805;6 h, or overnight.</li>
	<li>Complete protein digestion by adding 180 &#181;g Proteinase K to each tube, then incubating at 45 &#176;C for 3&#8211;5 h.</li>
</ol>
13. Library preparation and sequencing
<ol>
	<li>Purify DNA using magnetic beads 
	Note: Magnetic beads are preferred for isolating the small amounts of DNA usually recovered during ChIP with bacterial cells.</li>
	<li>Prepare libraries using a kit that is compatible with the selected sequencing platform.</li>
	<li>Check library concentration with a fluorometer and a broad range dsDNA kit or a qRT-PCR library quantification kit. 
	Note: In our experience, fluorometer measurements may overestimate DNA concentration.</li>
	<li>Pool libraries, accounting for adequate coverage for each library sample. For Illumina sequencing with Salmonella, we pooled 10&#8211;12 libraries. Add Tris-HCl pH 8.0 to a final concentration of 1 mM.</li>
	<li>Sequence according to the selected platform&#39;s specifications.</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol outlines a technique for performing ChIP-seq with Salmonella enterica serovar Typhimurium biofilm and planktonic cells from pure culture, to find regulatory targets of the master biofilm regulator, CsgD. We expect differential binding information due to the bi-stability of CsgD in the two cell types, with high levels in biofilm aggregates and low levels in planktonic cells2,3. However, this technique can also be applied to other bacterial transcription factors, cell types, or growth conditions. In particular, this technique can be adapted to other bacterial biofilms using an experimentally determined conversion factor for CFU per unit weight of biofilm cells.
ChIP-seq can be prone to amplification of technical error through sequencing; however, confirmation at critical steps can prevent this27. Primary antibody specificity is important for selecting only the target transcription factor and the DNA it controls during immunoprecipitation7. In this experiment, csgD was fused to a plasmid-encoded 3xFLAG tag at the 3&#39; end of the gene, corresponding to the C-terminus of CsgD22. The p3xFLAG plasmid without csgD was used as a &#34;control&#34; (S. Typhimurium 14028 &#8710;csgD + p3xFLAG). ENCODE guidelines recommend performing immunoblots with primary antibody against the protein of interest, and lysates of ChIP strains and deletion strains21. It is important to ensure that growth conditions are consistent across replicates to reduce variability in transcription factor binding and downstream sequencing results. If one is careful to ensure inoculum sizes and pre-inoculum growth conditions are consistent, biofilm growth in flask culture is consistent4. It is important to confirm that all biofilm has been broken apart after homogenization, to ensure equal access of reagents, particularly formaldehyde cross-linker, to all cells.
Several modifications may enable researchers to use this protocol for other DNA-binding proteins, cell types, strains, growth conditions, or lab equipment. If it is used for biofilm-forming species other than S. Typhimurium, the conversion factor of colony forming units per unit weight of biofilm needs to be determined experimentally by harvesting different amounts of biofilm, homogenizing the biofilm thoroughly, and performing serial dilutions and plate counting to create a standard curve, which may not be accurate at lower biofilm weights2. Sonicator energy transfer and cell type resistance may differ; therefore, a sonication assay is recommended to find the number of sonication bursts that will produce the fragment size range required for downstream library preparation and sequencing11. Chromatin fragmentation by micrococcal nuclease is an alternative to sonication that produces high resolution of occupancy sites; however, this method may introduce fragmentation bias7,28. The DNA size range may be altered depending on downstream library preparation kits and sequencing platforms. Other methods of homogenization may be used in lieu of the mixer mill. For example, a glass tissue homogenizer may be substituted, but it may aerosolize or incompletely break up biofilm. In terms of controls, pre-immunoprecipitate DNA can be normalized in post-sequencing processing and analysis. A mock-IP control with species-matched normal antibody serum can be used as a control as well13.
Researchers must consider the limitations to this method when considering a ChIP-seq experiment. Significant modifications may be required to adapt it for other biofilm types. For example, with Salmonella Typhimurium immediate resuspension of biofilm in PBS leads to measurable loss of biofilm aggregates. Immunoprecipitation targeting regulatory targets of transcription factors in bacteria may result in very small amounts of DNA, which is a challenge for purification and detection at later steps. Bias can be introduced during shearing and downstream manipulation during library detection9. In addition, this method does not reveal in vivo expression levels in response to transcription factor binding. Researchers who have little experience in bioinformatics may be challenged by the analysis pipeline. This method can be time-intensive and expensive; however, the cost of sequencing is often lower than a microarray and is decreasing over time as technological improvements continue to be made.
Few methods in the literature describe ChIP with bacteria, and most bacterial experiments begin with a simple growth condition13,14,15,17,18. ChIP sample preparation with single cells is straightforward and presents fewer challenges than ChIP sample preparation with biofilm aggregates. As for ChIP-seq, previous methods of studying cis-regulatory circuitry, including systematic evolution of ligands by exponential enrichment (SELEX), electrophoretic mobility shift assays (EMSA), ChIP-chip, promoter deletion and reporter analysis have been complemented or replaced by ChIP-seq29. ChIP-seq is replacing ChIP-chip due to advancing technologies and availability of sequencing. Deep sequencing provides researchers with massive amounts of data that can identify sites with lower binding affinity and higher base pair resolution with less starting material than ChIP-chip11,30. Analysis of ChIP data from organisms with high quality reference genomes is generally fast and specific. Additionally, ChIP-seq is a thorough method for discovering in silico predicted binding sites that may not be occupied in all cell types, growth phase, or environmental conditions31.
In the future, this protocol can be used to facilitate the understanding of bacterial pathogenesis, particularly biofilm-forming organisms. Broad adoption of this technique and availability of new datasets can lead to data integration to identify groups of proteins that co-localize to control cellular processes in biofilm-forming microorganisms32. In addition, ChIP-seq and RNA-seq data can be integrated to build regulatory system models33.

Bacterial biofilms, structural aggregates of cells embedded in a self-produced matrix, are resistant to desiccation, antibiotics, and host immune responses1. As such, they cause persistent and chronic infections and present unique challenges to researchers due to their solutions to survival and transmission. Salmonella enterica serovar Typhimurium (S. Typhimurium) has been found to establish phenotypically heterogeneous populations of biofilm aggregates that are resistant to desiccation and antibiotics, and planktonic cells that express virulence factors in pure culture when exposed to environmental stress (28 &#176;C, limiting nutrients)2. These two cell types arise due to bistable expression of the master biofilm regulator, CsgD3. We have hypothesized that this may be a bet-hedging strategy for Salmonella, where the planktonic cells participate in short-term transmission and biofilm cells are able to persist long-term in the environment until an opportunity to infect a new host arises2,4. Many of the regulatory elements that control csg operon expression are well characterized5. However, apart from adrA and the csg operon6, few regulatory targets of CsgD are known. Identifying the CsgD regulatory network would help us to better understand the Salmonella lifecycle and the role that biofilms play in transmission.
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is a powerful in vivo technique for genome-wide identification of protein-DNA interactions and provides information on regulatory processes involving transcription factors, histone modifications, or nucleosomes7. Newer studies have used this technique to assess differential protein binding between growth conditions and cell types8. In chromatin immunoprecipitation (ChIP), DNA fragments with interacting proteins are enriched through antibody selection of the target proteins. Cells are harvested and DNA-binding proteins are crosslinked to DNA in vivo through formaldehyde cross-linker treatment. Cells are lysed, releasing the cell contents, and chromatin is sheared into approximately 200&#8211;600 bp fragments7. DNA fragments bound to the target protein are immunoprecipitated using antibody, and isolated by de-crosslinking followed by protein digestion9. These DNA fragments represent protein binding sites matched by hybridization to a microarray (ChIP-chip) or by deep sequencing and mapping to the genome sequence10,11. Although ChIP-chip experiments yield adequate regulatory data, they are limited due to the use of expensive probes, as well as hybridization and array bias12. ChIP-seq has a larger dynamic range with increased nucleotide resolution and coverage, improved signal-to-noise ratio, and fewer artifacts when compared to ChIP-chip7.
ChIP has been used to analyze Sfh and OmpR regulation in Salmonella serovar Typhimurium13,14, quorum-sensing AphA regulation in Vibrio alginolyticus15, RpoS regulation in Escherichia coli16, virulence regulator EspR regulation in Mycobacterium tuberculosis17, potential diguanylate cyclases through AmrZ regulation in Pseudomonas aeruginosa18, as well as VraSR regulation in Staphylococcus aureus19. The bacterial ChIP-seq experiments that have been described begin with growing cells in liquid media and harvesting in single cell form. However, analysis of biofilms and corresponding gene regulation represents a new set of challenges to generate a successful ChIP-seq protocol. In this protocol, ChIP-seq is used to find differential regulatory targets of CsgD, the S. Typhimurium master biofilm regulator in biofilm and planktonic cell types. This protocol describes techniques used to determine the appropriate amounts of biofilm for ChIP-seq, harvest biofilm from flask culture, normalize biofilm and single cell control samples, homogenize biofilm, and complete immunoprecipitation. This will be useful for studying the regulatory targets in bacterial biofilms and can be adapted for many species.

<table><tbody><tr><td>Agarose powder</td><td>FroggaBio</td><td>A87-500G</td><td></td></tr><tr><td>Balance (Explorer pro)</td><td>Ohaus</td><td>EP214</td><td></td></tr><tr><td>Benchtop Centrifuge (8510R)</td><td>Eppendorf</td><td>022627023</td><td></td></tr><tr><td>Bioanalyzer 2100</td><td>Agilent</td><td>G2939BA</td><td></td></tr><tr><td>BR dsDNA kit</td><td>ThermoFisher Scientific</td><td>Q32850</td><td></td></tr><tr><td>ChIP-Grade Protein G Magnetic Beads</td><td>Cell Signaling Technology</td><td>9006</td><td></td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>COEDTAF-RO ROCHE</td><td></td></tr><tr><td>Conical tube 15 mL</td><td>FroggaBio</td><td>TB15-500</td><td></td></tr><tr><td>Conical tube 50 mL</td><td>FroggaBio</td><td>TB50-500</td><td></td></tr><tr><td>DC Protein Assay Kit II</td><td>BioRad</td><td>5000112</td><td></td></tr><tr><td>Disposable Cuvette, 1.5 mL</td><td>Fisher Scientific</td><td>14-955-127</td><td></td></tr><tr><td>Electrophoresis equipment (mini-PROTEAN)</td><td>Bio-Rad</td><td>1658000</td><td></td></tr><tr><td>Floor centrifuge (Sorvall Evolution RC)</td><td>Thermo Scientific</td><td>728211</td><td></td></tr><tr><td>Fluorometer (Qubit 3.0)</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>GelRed&amp;reg; Nucleic Acid Gel Stain 10 000x in water</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>High Sensitivity DNA kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Incubator (shaking)</td><td>VWR</td><td>1570-ZZMFG</td><td></td></tr><tr><td>Incubator (Water bath shaking)</td><td>New Brunswick</td><td>G76D</td><td></td></tr><tr><td>LB media</td><td>VWR</td><td>90000-808</td><td></td></tr><tr><td>Magnetic stand (DynaMag)</td><td>Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Microcentrifuge (refrigerated)</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Microcentrifuge Tubes, 1.5mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>MiSeq Reagent Kit v3 (150 cycle)</td><td>Illumina</td><td>MS-102-3001</td><td></td></tr><tr><td>Mixer mill</td><td>Retsch</td><td>MM400</td><td></td></tr><tr><td>Nalgene 115 mL Filter Units, Sterile, 0.2 &amp;micro;m pore size</td><td>Thermo Scientific</td><td>73520-980</td><td></td></tr><tr><td>NEBNext Ultra DNA Library Prep Kit for Illumina</td><td>New England BioLabs</td><td>E7370S</td><td></td></tr><tr><td>NGS Cleanup and Size Select magnetic beads</td><td>Machery-Nagel</td><td>744970.5</td><td></td></tr><tr><td>Normal mouse serum</td><td>AbCam</td><td>ab188776</td><td></td></tr><tr><td>Petri Dishes with Clear Lid (100 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Pipette controller</td><td>FroggaBio</td><td>MP001</td><td></td></tr><tr><td>Proteinase K</td><td>ThermoFisher Scientific</td><td>AM2542</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Rabbit Anti-FLAG Polyclonal Antibody</td><td>Sigma-Aldrich</td><td>F7425</td><td></td></tr><tr><td>RNase A (10 mg/mL; DNase and protease-free)</td><td>ThermoFisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Rotating wheel</td><td>Crystal Technologies &amp;amp; Industries</td><td>TR 01A</td><td></td></tr><tr><td>Safe-Lock Tubes (2.0 mL, colorless)</td><td>Eppendorf</td><td>22363344</td><td></td></tr><tr><td>Serological pipette 25 mL</td><td>FroggaBio</td><td>94024</td><td></td></tr><tr><td>Spectrophotometer</td><td>Montreal Biotech Inc.</td><td>Libra S22</td><td></td></tr><tr><td>Stainless Steel Beads, 5 mm</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Tapered microtip 1/8&quot; (3mm) Sonicator probe</td><td>Sonics &amp;amp; Materials, Inc.</td><td>630-0418</td><td></td></tr><tr><td>Tryptone media</td><td>VWR</td><td>90000-286</td><td></td></tr><tr><td>Ultrasonic Liquid Processor (Sonicator)</td><td>Sonics &amp;amp; Materials, Inc.</td><td>VC300</td><td></td></tr><tr><td>Vacuum equipment (Vacufuge plus)</td><td>Eppendorf</td><td>022820001</td><td></td></tr><tr><td>Water bath 1 (Lab-line aquabath)</td><td>Thermo Scientific</td><td>18000-1</td><td></td></tr><tr><td>Water bath 2 (Precision)</td><td>Thermo Scientific</td><td>51221044</td><td></td></tr></tbody></table>

discovering csgd regulatory targets in salmonella biofilm using chromatin immunoprecipitation and high-throughput sequencing (chip-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Preparation of chromatin immunoprecipitation samples from bacterial biofilm involves several steps, as shown in Figure 1. These include growing biofilm in flask culture, collecting conditioned media, collecting an adequate amount of biofilm and planktonic cells as control, washing cells in PBS, homogenization, crosslinking proteins to DNA, lysing cells, fragmenting DNA by sonication, immunoprecipitating DNA with the appropriate antibodies and protein G beads, washing and eluting immunoprecipitated DNA, and purifying DNA for library preparation and sequencing.
S. Typhimurium cells grown in liquid culture under biofilm-inducing conditions undergo phenotype switching to form multicellular biofilm aggregates and planktonic cells. This population will contain approximately 40% biofilm aggregates, which express higher levels of diguanylate cyclases (DGCs) and biofilm regulators, and 60% planktonic cells, which express higher levels of virulence-associated genes2,4 (Figure 2A). In this protocol, we describe a method to harvest both cell types to compare transcription sites through ChIP-seq; however, this protocol can be adapted for other types of biofilms formed by other bacterial species. Biofilm aggregates and planktonic cells are separated by slow speed centrifugation, which pellets biofilm and leaves planktonic cells in the supernatant2 (Figure 2B). The cell types are then treated separately for subsequent steps in the protocol.
The recommended amount of DNA input for ChIP-seq is 10-25 &#181;g20. In S. Typhimurium flask culture, 30 mg of biofilm yields approximately 25 &#181;g DNA. Since biofilm aggregates have an abundance of proteinaceous extracellular material, we hypothesized that this would interfere with the efficiency of cross-linking. If we were simply to normalize the different cell types by cell number, treatment of planktonic cells may result in an unequal amount of crosslinked product presented for immunoprecipitation. A protein assay was performed to determine the equivalent amount of planktonic cell material to match 30 mg of biofilm (Figure 3). 6.0 OD600 of planktonic cells were harvested to match 30 mg biofilm.
Biofilm aggregates must first be broken apart to allow equal access of cross-linker to all cells. We found that the most uniform and high-throughput way to homogenize the cells was using a mixer mill and 5 mm stainless steel bead (Figure 4A). Planktonic cells are treated the same way to reduce variables in the ChIP-seq experiment (Figure 4B).
Once the DNA has been fragmented by sonication, crosslinked, and treated with RNase and Proteinase K, it can be visualized on a 2% agarose gel. Sonicated DNA is broken down into a collection of fragments that appear as a smear on the agarose gel. The average fragment size decreases with an increasing number of sonication bursts (Figure 5). Energy transfer will vary for different sonicator units, so a sonication assay is recommended to determine the number of sonication bursts required to fragment DNA to an average of less than 1 kb. For our ChIP-seq experiment, we chose 5 bursts.
ENCODE guidelines recommend a confirmation of antibody target-binding activity with an immunoblot21. In this case we measured our antibody&#39;s specificity and strength of binding by immunoblot on whole cell lysates prepared for ChIP (Figure 6). We confirmed that the antibody binds to CsgD-3xFLAG; anti-FLAG and anti-CsgD signals colocalize at the expected molecular weight (28 kDa)22.
ChIP procedures often yield low concentrations of DNA. Therefore, a purification method that removes contaminants and retains a high proportion of the DNA was chosen. Magnetic bead purification was chosen over column-based purification because it retained low concentrations of input ChIP DNA better (Figure 7) and removed contaminants (data not shown).
Due to reduced starting amounts of DNA, library preparation of ChIP DNA may require diluted adapters and PCR enrichment. Prior to sequencing, libraries can be visualized by Bioanalyzer trace to confirm correct size distribution before and after PCR enrichment (Figure 8). Additionally, if there is a known regulatory target of the transcription factor, qPCR should be used to confirm enrichment of binding regions by comparing fold change (&#8710;&#8710;Ct) between known target and reference gene sequence abundance in immunoprecipitated and sonicated input DNA.
ChIP-sequencing data should be analyzed by assigning quality scores, trimming low quality bases, aligning forward and reverse reads (in paired end sequencing), assembling to a high-quality reference genome, finding peaks above background, and performing downstream analysis (Figure 9A). Downstream analysis should include visualization and annotation of peaks, consistency with biological replicate samples, and motif analysis, and could include differential binding analysis between conditions or cell types, and data integration with gene expression from known pathways. For ChIP-seq data such as ours, peaks should be identified as significantly above background (Figure 9B,C, red bars) with a p value determination, not simply by fold-change. In the final analysis, the mapped reads are visualized against the annotation of the reference genome (Figure 9D). A poor ChIP-seq result would appear like input-seq without any significant peaks above background.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60736/60736fig1.jpg" /> 
Figure 1: Illustration of steps in ChIP-seq with bacterial biofilms. In the described procedure, S. Typhimurium cells are grown in 1% Tryptone flask culture at 28 &#176;C with shaking (1), cell-free conditioned media is collected for handling biofilm cell types (2), which is used to collect an adequate amount of biofilm and planktonic cells (3). These cells are washed with PBS and homogenized to break apart biofilm (4). Proteins are crosslinked to DNA with a formaldehyde crosslinker, after which cells are lysed to release cell contents (5). DNA in the cell lysate is fragmented by sonication (6). DNA crosslinked to the target protein is selected through immunoprecipitation with an antibody that binds to the target protein and Protein G magnetic beads (7). Selected DNA is washed and eluted from Protein G magnetic beads (8) and purified for library preparation and sequencing (9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig1large.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/60736/60736fig2.jpg" /> 
Figure 2: In vitro flask model for studying S. Typhimurium biofilm development. (A) S. Typhimurium cells grown in 1% tryptone for 13 h at 28 &#176;C undergo phenotype switching to form biofilm aggregates and planktonic cells in the liquid phase of the flask culture. (B) Biofilm aggregates and planktonic cells from the flask culture in (A) were separated through centrifugation at 210 x g for 2 min. The supernatant was harvested for planktonic cell preparations and the pellet was harvested for biofilm cell preparations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig2large.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/60736/60736fig3a.jpg" /> 
Figure 3: Total protein concentrations for planktonic cells and biofilm cell samples harvested from S. Typhimurium flask culture. Colorimetric protein assays were performed and protein amounts relative to a BSA standard curve were measured at 750 nm. Protein content of lysed planktonic cell samples at 4.0, 6.0, and 8.0 OD600 were compared to 30 mg of biofilm aggregates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig3large.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/60736/60736fig4.jpg" /> 
Figure 4: Homogenization of cell samples from S. Typhimurium biofilm flask cultures. (A) Biofilm aggregates from flask culture resuspended in PBS (left) were homogenized using a mixer mill at 30 Hz for 5 min (right). (B) Planktonic cells from flask culture resuspended in PBS were processed by mixer mill at 30 Hz for 5 min to ensure consistency between cell type samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig4large.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/60736/60736fig5a.jpg" /> 
Figure 5: Sonication assay with pre-ChIP cell lysates. Biofilm aggregate and planktonic cell samples were separated, homogenized, crosslinked, and lysed. DNA-protein complexes were sonicated up to 8 times at 30 s on and 2 min off on ice to break DNA into smaller fragments. A portion of the sonicated lysate was separated on a 2% agarose gel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig5large.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/60736/60736fig6a.jpg" /> 
Figure 6: Target-binding specificity of anti-FLAG antibody used in ChIP-seq. Antibody specificity was assessed by immunoblotting against whole cell lysates prepared from flask cultures of the S. Typhimurium strains as shown. Strain &#8710;csgD + p3xFLAG/csgD and WT biofilm samples represent biofilm aggregates harvested from flasks, whereas strain &#8710;csgD &#177; p3xFLAG and WT planktonic cells represent planktonic cells. Purified His-tagged CsgD was loaded as a control. Whole cell lysates were normalized so that 30 &#181;g of total protein was analyzed in each lane. Numbers on the left refer to the size (in kDa) of the prestained molecular weight markers. Primary antibody used for the upper blot was rabbit-anti-FLAG polyclonal antibody (Sigma-Aldrich #F7425), whereas the lower blot was incubated with rabbit-anti-FLAG together with a CsgD-specific monoclonal antibody2. Goat anti-rabbit IgG (LiCor IRDye 680RD, 925-68071) and goat anti-mouse IgG (LiCor IRDye 800CW, 925-32210) were used as secondary antibodies. Fluorescent signals were visualized using the Odyssey CLx imaging system and Image Studio 4.0 software package (Li-Cor Biosciences). Representative images are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60736/60736fig7a.jpg" /> 
Figure 7: Column- or magnetic bead-based purification strategies for small amounts of DNA resulting from ChIP-seq experiments. Samples of known quantities of DNA were purified using column-based kits or magnetic beads and the concentration of the eluate was measured post-purification. Magnetic beads showed better recovery at low DNA levels, similar to the low amounts of DNA typically encountered at the end of ChIP-seq protocol, but prior to NGS library preparation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60736/60736fig8.jpg" /> 
Figure 8: ChIP DNA preparations and subsequent libraries visualized by Bioanalyzer. (A) Average fragment size of genomic DNA after cell lysis and sonication was &#60;1000 bp, with a majority of fragments in the 200&#8211;500 bp range. (B) Starting material for library preparation was below detection. (C) Adapter ligation and PCR enrichment allows for amplification of library fragments. (D) A poor library preparation has low DNA peaks, odd shaped or high molecular weight peaks, or abundant adapter peaks at approximately 100 bp. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60736/60736fig9a.jpg" /> 
Figure 9: ChIP-seq data is analyzed using bioinformatic tools and visualized on a genome browser. A. Flowchart for typical bioinformatic analysis of ChIP-seq data. Raw reads for biofilm (&#8710;csgD strain + p3xFLAG/csgD) and planktonic cells (&#8710;csgD + p3xFLAG) were cleaned for low-quality reads and adapters, and mapped to S. Typhimurium 14028s genomes (NC_016856.1 for the chromosome and NC_016855.1 for the plasmid) by Bowtie2 (v2.3.3.1) with default parameters23. MACS2 (v2.1.2) was used to call the peaks with parameters of &#39;-q 0.01 -- nomodel&#39;, taking biofilm replicates as testing datasets and planktonic ones as control24. The MACS2 bdgcmp module was further used to generate fold-enrichment track with parameters of &#39;-m FE&#39;. The significant peak file with fold-enrichment track was transformed to a wig file using bedtools/bedClip/bedGraphToBigWig25 and visualized on the reference genome using Integrative Genomics Viewer v2.5.126. Representative peaks are shown (red bars). (B) Large region of ~40 kbp, which contains multiple peaks, is an atypical result but still potentially valuable. (C) This is a more typical result, showing two significant peak regions. (D) Zoom in on left peak in C, showing reads mapping to the region of the S. Typhimurium 14028s genome containing divergent promoters for uspA and uspB. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60736/60736fig9alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cell lysis buffers</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>50 mM Tris-HCl pH 8.1</td>
		</tr>
		<tr>
			<td>10 mM EDTA</td>
		</tr>
		<tr>
			<td>1% SDS</td>
		</tr>
		<tr>
			<td>protease inhibitors</td>
		</tr>
		<tr>
			<td>IP dilution buffer</td>
		</tr>
		<tr>
			<td>20 mM Tris-HCl pH 8.1</td>
		</tr>
		<tr>
			<td>2 mM EDTA</td>
		</tr>
		<tr>
			<td>150 mM NaCl</td>
		</tr>
		<tr>
			<td>1% Triton X-100</td>
		</tr>
		<tr>
			<td>0.01% SDS</td>
		</tr>
		<tr>
			<td>Immunoprecipitation buffers</td>
		</tr>
		<tr>
			<td>IP wash buffer 1</td>
		</tr>
		<tr>
			<td>20 mM Tris-HCl pH 8.1</td>
		</tr>
		<tr>
			<td>2 mM EDTA</td>
		</tr>
		<tr>
			<td>50 mM NaCl</td>
		</tr>
		<tr>
			<td>1% Triton X-100</td>
		</tr>
		<tr>
			<td>0.1% SDS</td>
		</tr>
		<tr>
			<td>IP wash buffer 2</td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 8.1</td>
		</tr>
		<tr>
			<td>250 mM LiCl</td>
		</tr>
		<tr>
			<td>1 mM EDTA</td>
		</tr>
		<tr>
			<td>1% NP-40</td>
		</tr>
		<tr>
			<td>1% deoxycholic acid</td>
		</tr>
		<tr>
			<td>TE (T10E1) pH 8.0</td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl</td>
		</tr>
		<tr>
			<td>1 mM EDTA</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
		</tr>
		<tr>
			<td>1% SDS</td>
		</tr>
		<tr>
			<td>100 mM NaHCO3</td>
		</tr>
	</tbody>
</table>
Table 1. Buffer recipes.

Watch this Scientific Journal Video about Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq at JoVE.com

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

discovering-csgd-regulatory-targets-salmonella-biofilm-using

Nanyang Technological University

Research

JoVE Journal

Genetics

7.5K Views.  Vaccine and Infectious Disease Organization-International Vaccine Centre. Chromatine immunoprecipitatie gevolgd door sequencing, of ChIP-seq, is een techniek die kan worden gebruikt om de regelgevende doelstellingen van transcriptiefactoren, histone-modificaties en andere DNA-geassocieerde eiwitten te ontdekken. In ChIP-seq worden cellen geoogst en DNA-bindend eiwit. is gekruist-gekoppeld aan DNA in vivo met formaldehyde.Cellen worden gelysed, het vrijgeven van de celinhoud en chromatine wordt scherven in fragmenten van minder dan 1000 basisparen, meestal tu...

Video: Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

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 ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunology and Infection

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&amp;RUN Method and Data Analysis Workflow

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&amp;RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&amp;RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&amp;RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&amp;RUN data analysis.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&amp;RUN) in the human fungal pathogen Candida albicans.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

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