We thank Alexander Horswill for the gift of the PsarAP1-tdTomato fusion, and Karen Creswell for help with flow cytometry analysis. We also thank Alyssa King for advice on statistical analysis. This work was funded in part by an NIH Exploratory/Developmental Research Award (grant AI123708) and faculty startup funds to SRB. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Georgetown University.
1. Generation of the Fluorescent Reporter Strain
<ol>
	<li>Digest the genome integrative pRB4 vector sequentially with EcoRI and SmaI restriction enzymes. Following the manufacturer&#39;s protocol, set up an overnight digestion with SmaI using 1 &#181;g of pRB4 at 25 &#176;C, and then proceed with the second digestion for 1h at 37 &#176;C by adding EcoRI to the reaction mixture. Inactivate the enzymes by incubating at 65 &#176;C for 20 min.</li>
	<li>PCR amplify the regulatory region of interest from genomic DNA (in this case, a ~350 base pair DNA fragment containing the thermonuclease [nuc] promoter region), incorporating a 5&#39; EcoRI restriction site and a 3&#39; SmaI restriction site. The SmaI recognition sequence must be in-frame with the translational start codon. In this study, the PCR was performed using a high-fidelity DNA Polymerase according to the manufacturer&#39;s recommendations with forward primer oRB015 (5&#39;-atcattgaattctccaaagtaaattataagttatac-3&#39;) and reverse primer oRB016 (5&#39;-gggcataactaacacctctttctttttag-3&#39;). The annealing temperature was 53 &#176;C. Purify the resulting fragment using a PCR clean-up kit following the manufacturer&#39;s instructions.</li>
	<li>Digest the resulting PCR product with EcoRI and SmaI as performed in step 1.1 and ligate the digested DNA fragment to the same sites of pRB4 using T4 DNA ligase as recommended by the manufacturer to generate the integrative construct. Introduce the ligation mixture into E. coli and confirm construct sequence.</li>
	<li>Electroporate the construct into S. aureus strain RN4220 as previously described34. In brief, use 1 &#181;g of plasmid with 70 &#181;L of electro-competent cells (100 &#937;, 25 &#181;F, and 2.3 kV) in B2 broth (1% [w/v] Casein hydrolysate, 2.5% [w/v] Yeast extract, 2.5% [w/v] NaCl, 0.1% [w/v] K2PO4, 0.5% [w/v] Glucose). Select for erythromycin resistance (Emr) at the permissive temperature (30 &#176;C) on tryptic soy agar (TSA) supplemented with erythromycin (5 &#181;g mL-1) and 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal; 80 &#181;g mL-1). The resulting transformants are blue due to plasmid-encoded &#946;-galactosidase activity. 
	NOTE: Transfer of DNA into clinical isolates is extremely difficult due to a strong restriction-modification barrier35. Thus, S. aureus RN4220 was used as an intermediate recipient of the fusion construct because it is restriction deficient and highly transformable.</li>
	<li>Transfer plasmid to S. aureus USA300 strain of interest using electroporation or phage-mediated transduction36 at the permissive temperature. In brief, propagate &#934;11 bacteriophage particles on the donor strain using TSA plates containing 5 mM CaCl2 overnight at 30 &#176;C, and then sterilize by filtration through a 0.45 &#181;M cellulose acetate syringe filter. Use lysates to transduce S. aureus strain LAC to Emr. 
	NOTE: LAC is a widely used clinical isolate that was previously made Em sensitive by serial passage in TSB medium to cure the strain of the native plasmid pUSA03 that confers Emr 37,38.</li>
	<li>Integrate the construct by two, successive homologous recombination events into the chromosome as described previously32. The recombinants are chloramphenicol-resistant (Cmr) on TSA plates containing 5 &#956;g mL-1 of chloramphenicol, erythromycin-susceptible (Erms), and no longer blue. 
	NOTE: When possible, re-introduce the marked fusion into USA300 via phage-mediated transduction to avoid the accumulation of mutations associated with in vitro strain passage39 and temperature shifts.</li>
</ol>
2. Animal Infection: Preparation of the Inoculum, Systemic Infection, and Tissue Processing
<ol>
	<li>Preparation of bacterial inoculum
	<ol>
		<li>Streak S. aureus strains of interest for isolation on blood agar plates from a frozen glycerol stock. Incubate at 37 &#176;C for 16&#8211;24 h to confirm hemolytic phenotypes based on their ability to lyse red blood cells (RBCs) and form clear transparent zones.</li>
		<li>Initiate overnight cultures by inoculating single colonies of each strain to 4 mL of tryptic soy broth (TSB) in sterile glass tubes. Rotate the tubes at 37 &#176;C for 16&#8211;24 h in a tube roller set at approximately a 70&#176; angle and 70 rotations per minute [RPM].</li>
		<li>Use a spectrophotometer to measure the optical density at 600 nm (OD600) of the cultures from step 2.1.2. Use sterile medium as an optical reference (blank). Dilute these cells to an OD600 of 0.05 in 25 mL of sterile Tryptic Soy Broth (TSB) in separate, 125 mL DeLong flasks (5:1 flask to volume ratio) and incubate cultures in a water bath (for proper heat transfer to the cultures), shaking at 280 RPM and set to 37 &#176;C. 
		NOTE: Growth of the inoculum can be performed in various ways; one method for growing cells to exponential phase is presented. Regardless, it is important to consistently use the same method for preparing the cells for inoculation.</li>
		<li>At an OD600 of ~1, re-dilute the cells into fresh medium to a starting OD600 of 0.05 to ensure the cells achieve exponential phase and that factors that accumulate during overnight incubation have been reduced to exponential phase levels.</li>
		<li>Harvest the cells during exponential phase (OD600 ~0.6&#8211;0.8) by centrifuging at 3,000 x g for 10 min at room temperature.</li>
		<li>Wash the pellets twice in equivalent volume of sterile 1x phosphate-buffered saline (PBS; pH 7.4).</li>
		<li>Suspend the cells in 1x PBS (pH 7.4) to a concentration of 1 x 108 colony forming units (CFUs) mL-1 or as appropriate for the desired experiment. 
		NOTE: it is important to determine the relationship between OD600 and CFU mL-1 for each strain of interest because strain-dependent alterations of cell size and shape can affect light scattering properties and ultimately, the infection dose.</li>
	</ol>
	</li>
	<li>Preparation of the animals and bacterial infection
	<ol>
		<li>Acclimate mice for 7 days on purified diet AIN-93. The diet is formulated to provide adequate nutrition while reducing tissue auto-fluorescence associated with the consumption of plant-based ingredients used in standard mouse chow40. 
		NOTE: Female C57/BL6 mice (6-8 weeks old) are used here, but the mouse strain and sex will depend on the study.</li>
		<li>Before infection, dilate mouse tail veins with lukewarm water.</li>
		<li>Infect animals by injecting 100 &#181;L of the bacterial inoculum via tail-veins to produce a systemic infection. Save an aliquot of the initial inoculum if performing flow cytometry (see Section 4).</li>
		<li>Monitor animals daily and evaluate their health status using a monitoring system reviewed and approved by one&#39;s Institutional Animal Care and Use Committee.</li>
		<li>Allow the infection to progress for the desired duration. Here, the experiments are terminated 3 days post-infection. 
		NOTE: Under these conditions, abscesses consist of a staphylococcal abscess community of bacteria, enclosed by fibrin deposits, and surrounded by concentric layers of immune cells5,41.</li>
	</ol>
	</li>
	<li>Harvesting the organs and tissue processing
	<ol>
		<li>Euthanize the animals by CO2 inhalation and cervical dislocation as a secondary method and perform necropsy.</li>
		<li>Harvest kidney (right) and other vital organs (heart, liver, lungs, spleen) and transfer into 15 mL polypropylene tubes containing 10% [v/v] buffered formalin. Proceed with these organs to step 2.3.4 below.</li>
		<li>Transfer the left kidney to a sterile 2 mL impact-resistant tube containing ~500 &#181;L of 2 mm silica beads and 1 mL sterile 1x PBS (pH 7.4). Proceed with this organ to step 4.2.</li>
		<li>Allow organs from step 2.3.2 to fix in the dark at room temperature with gentle shaking or rotation for at least 24 hours but no more than 48 hours.</li>
		<li>Embed organs in clear tissue freezing medium and store the tissues at -80 &#176;C.</li>
		<li>Using a cryostat, section tissue into slices of 10 &#181;m thickness.</li>
		<li>Dry the sections on a pre-cleaned, charged glass slide for 20 min in darkness, apply hard mounting medium with 4&#39;,6-diamidino-2-phenylindole (DAPI) stain, and apply coverslip. Cure mounted slides at room temperature overnight, and transfer to 4 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Laser Scanning Confocal Microscopy and Image Processing
<ol>
	<li>Examine mounted slides to locate lesions using lasers appropriate for the fluorescent reporter being used. In this case, the green (GFP), red (tdTomato), and blue (DAPI) fluorescence signals are used.</li>
	<li>Acquire the image using the appropriate objective for visualizing individual cells (e.g., typically 20x or 40x objectives are used).</li>
	<li>Measure the fluorescence intensities in the confocal images.
	<ol>
		<li>Open the confocal image in Image J and adjust brightness/contrast to properly visualize the fluorescence signal of the lesion.</li>
		<li>Define the region of interest, and by using the Thresholding option, set the lower and upper fluorescence limits as necessary.</li>
		<li>Define the centroid by selecting Area &#8594; Mean gray value &#8594; Centroid &#8594; Limit to Threshold under the Analyze tab in Image J.&#160;</li>
		<li>Extract centroid fluorescence intensity value or measure the mean fluorescence intensity in a given lesion per unit area (mean fluorescence intensity, MFI) for GFP and tdTomato. Here, areas of one &#181;m2 were used. 
		NOTE: A blinded second analysis minimizes bias when the identity of the sample is known.</li>
		<li>Plot the data and perform the appropriate statistical analyses for each comparison.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry Analysis
<ol>
	<li>(Optional) Determine fusion activity of the inoculum on the day of the infection (from section 2.2.3)
	<ol>
		<li>To 899 &#181;L of 1x sterile PBS (pH 7.4) add 100 &#181;L of each inoculum and 1 &#181;L of membrane permeant nucleic acid stain (see Table of Materials). As a control, be sure to include a sample lacking nucleic acid stain.</li>
		<li>Perform flow cytometry on all samples. 
		NOTE: For the very first experiment, it is useful to include a vector only control for the fusion of interest to determine the proper voltage to use. A clear separation between positive and negative samples is essential for data analysis, indicating the reporter is well above background signal.</li>
		<li>To identify the bacterial population, plot events using nucleic acid stain (y-axis) and forward scatter (x-axis).</li>
		<li>Draw an inclusion gate around events that are both nucleic acid stain positive and the correct size of the bacterium based on the forward scatter (between 0.5 to 2.0 &#181;m for S. aureus). 
		NOTE: Be rigorous when identifying this population as it is critical to include events that are clearly within these parameters. Be sure this gate excludes all events for the negative controls under other conditions. In this study, roughly 70% the cell population met these requirements in the analysis.</li>
	</ol>
	</li>
	<li>Analysis of tissue samples after sacrifice:
	<ol>
		<li>Euthanize the animals, harvest the left kidneys (see step 2.3), and transfer into bead-beating tubes containing 1 mL of 1x sterile PBS (pH 7.4) and 250 &#181;L of 2 mm borosilicate beads.</li>
		<li>Disrupt tissues to release bacterial cells. For kidneys, use a homogenizer to disrupt cells. Here, three 30 s bursts at 6800 rpm with 1 min cooling periods on wet ice between cycles were used to minimize heating samples.</li>
		<li>Pellet the larger tissue debris by centrifuging at 250 x g for 3 min in a tabletop microcentrifuge cooled to 4 &#176;C. 
		NOTE: Typically, there is a cell pellet at the bottom of the tube and a layer of floating debris on the top. The middle aqueous layer contains the bacterial cells.</li>
		<li>Transfer 10 &#181;L from the aqueous layer into a clean 1.5 mL microcentrifuge tube containing 1 &#181;L of nucleic acid stain in 989 &#181;L of PBS (total volume 1 mL).</li>
		<li>Perform flow cytometry using the same data acquisition parameters and gating as for the initial inocula. 
		NOTE: Bacterial cell counts will be very low because the sample mainly contains tissue debris. The &#34;Live gate&#34; option can also be used if the cells are nucleic acid stain positive and size-gated when data are loaded into the application. This will also help to analyze more of the target events and reduce file size. Nearly one million events per sample are counted, but more event counts may be necessary depending on the severity of the infection and size of tissue analyzed.</li>
		<li>Data Analysis: Determine mean fluorescent intensity (MFI) for each fluorophore in a given sample using the inclusion gates determined in section 4.1.4. Normalize samples in flow analysis software to event counts. 10,000 counts are usually sufficient in the analysis. 
		NOTE: It is not uncommon to find samples that contain less than this number of events since this is dependent on abscess formation and infection severity. Using this approach, 500-40,000 events are typically found in infected tissue.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

Bacterial infectious diseases are an increasing health problem worldwide due to the acquisition of antibiotic resistance determinants46. Because adaptation to host environments is essential for growth and survival during infection, strategies targeting gene expression programs that increase pathogen fitness may prove useful therapeutically. One such program is the set of genes controlled by the SaeR/S two component system (TCS), shown previously to play an essential role in immune evasion<sup clas...

Bacteria respond to changing physiological conditions and alterations in the nutritional state of their environment by differentially expressing genes required for adaptation and survival. For instance, opportunistic pathogens colonize body surfaces at relatively low densities, and are often harmless. However, once the bacterium has penetrated physical and chemical barriers, it must contend with host immune cell counter-defenses and restricted nutrient availability1. As an example, Staphylococcus aureus colonizes approximately one third of the population asymptomatically but is also the cause of devastating skin and soft tissue infections, osteomyelitis, endocarditis, and bacteremia2. The success of S. aureus as a pathogen is often attributed to its metabolic flexibility as well as an arsenal of surface-associated and secreted virulence factors that enable the bacterium to escape the bloodstream and replicate in peripheral tissues3,4,5. Because host death due to staphylococcal disease is an evolutionary dead end and limits transmission to new hosts6, the commitment to virulence factor production must be carefully controlled.
A complex regulatory network of proteins and non-coding RNAs responds to a variety of environmental stimuli, including cell density, growth phase, neutrophil-associated factors, and nutrient availability, to ensure that virulence genes are expressed at the precise time and location within host tissues7,8,9,10,11,12,13. For instance, the SaeR/S two component system (TCS) regulates expression of several virulence factors via the sensor kinase (SaeS) and the response regulator (SaeR)14. SaeS is autophosphorylated on a conserved histidine residue in response to host signals (e.g., human neutrophil peptides [HNPs], calprotectin)8,15,16. The phosphoryl group is then transferred to an aspartate residue on SaeR, activating it as a DNA-binding protein (SaeR~P)17. The SaeR/S TCS regulates over 20 genes that contribute to pathogenesis including fibronectin binding proteins (FnBPs), leukocidins, and coagulase14,18,19,20. Targets can be classified into high-affinity and low-affinity gene targets, which are likely induced as the level of SaeR~P rises when exposed to its cues21. The SaeR/S activity is controlled by other regulators of gene expression such as the Agr quorum sensing system, repressor of toxins protein (Rot), and the alternative sigma factor B (SigB)22,23,24.
nuc is an Sae-dependent virulence gene in Staphylococcus aureus and encodes thermonuclease (Nuc), which is essential for escaping from neutrophilic extracellular traps (NETs) and for dissemination during the course of infection25,26. The expression of nuc is also strongly indirectly repressed by CodY in the presence of branched-chain amino acids and GTP27, and directly repressed by the staphylococcal accessory regulator protein SarA28,29, whose activity is influenced by oxygen (redox state) and pH30. Given that sae and nuc mutants are attenuated in mouse models of infection, there is interest in developing chemical interventions that inhibit their corresponding activities26,31. Despite this, there is no information regarding their regulation during infection.
Fluorescent reporters have been used to monitor and quantify gene expression on the single cell level. Herein, we present a method for quantifying S. aureus gene expression during infection that, when paired with in vitro transcriptome analysis and powerful imaging techniques like magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS), can reveal how bacterial physiology is regulated in vivo and the relative abundances of nutrients in certain niches. The method can be applied to any bacterial pathogen with a tractable genetic system.
Overview of the genome integrative vector. 
The genome integrative vector pRB4 contains 500 base pairs each from the upstream and downstream regions of the S. aureus USA300 SAUSA300_0087 pseudogene to facilitate homologous recombination. pRB4 is derived from the temperature-sensitive pMAD vector backbone containing the erythromycin resistance cassette (ermC) and thermostable beta-galactosidase gene bgaB for blue/white screening of recombinants32. The engineered reporter construct also contains a chloramphenicol resistance marker (cat) for selection after genome integration and plasmid elimination, as well as EcoRI and SmaI sites to fuse the regulatory region of interest to superfolder green fluorescent protein (sGFP) (Figure 1). It is known that the choice of ribosome binding site (RBS) influences the activity of the reporter, and often requires empirical optimization33. Thus, an RBS is not supplied. Here, the native ribosome binding site is used to provide for a more natural pattern of gene expression, but other sites may be used.

<table border="0" cellpadding="0" cellspacing="0" style="width:1015px;" width="1014">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">5% sheep blood</td>
			<td style="width:343px;">Hardy Diagnostics (Santa Maria, CA)</td>
			<td style="width:139px;">A10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>ThermoScientific</td>
			<td>R0402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ampicillin</td>
			<td style="width:343px;">Fisher Scientific</td>
			<td style="width:139px;">BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">C57Bl/6 Mice</td>
			<td style="width:343px;">Charles River</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Chloramphenicol</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">C-0378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryostat microtome</td>
			<td>Thermo Scientific</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture, Cap</td>
			<td>VWR International</td>
			<td>2005-02512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">D+2:25NA Oligo</td>
			<td>Integrated DNA Technologies (Coralville, Iowa)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Erythromycin</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">E5389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Analyzer</td>
			<td>Becton Dickinson</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass beads</td>
			<td>Sigma</td>
			<td>Z273627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miniprep, plasmid</td>
			<td>Promega</td>
			<td>A1220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaking water bath</td>
			<td>New Brunswick Innova</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR purification</td>
			<td>QIagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffer Saline (PBS)</td>
			<td style="width:343px;">Cellgrow</td>
			<td style="width:139px;">46-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:343px;">Tecan</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precellys 24 homogenizer</td>
			<td>Bertin Laboratories</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pUC57-Kan</td>
			<td>GenScript (Piscataway, NJ)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Q5 Taq DNA Polymerase</td>
			<td>New England Biolabs (Ipswich, MA)</td>
			<td style="width:139px;">M0491S</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:367px;">Restriction Enzymes</td>
			<td>New England Biolabs (Ipswich, MA)</td>
			<td style="width:139px;">R0150S (PvuI), R3136S (BamHI), R0144S (BglII), R3131S (NheI), R0101S (EcoRI), R0141S (SmaI).</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse transcriptase</td>
			<td>New England BioLabs</td>
			<td>E6300L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sanger Sequencing</td>
			<td>Genewiz (Germantown, MD)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sub Xero clear tissue freezing medium</td>
			<td>Mercedes Medical</td>
			<td style="width:139px;">MER5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides</td>
			<td>Fisher Scientific</td>
			<td style="width:139px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">superloop</td>
			<td>GE Lifesciences</td>
			<td>18111382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, Filter</td>
			<td>VWR International</td>
			<td>28145-481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syto 40</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:139px;">S11351</td>
			<td>Membrane permeant nucleic acid stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tetracycline</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">T7660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tryptic Soy Broth</td>
			<td style="width:343px;">VWR</td>
			<td style="width:139px;">90000-376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-visible spectrophotometer</td>
			<td>Beckman Coulter-DU350</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield Antifade Mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td style="width:139px;">H-1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fluorescence-based method to study bacterial gene regulation in infected tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

We developed a plasmid derived from pMAD32 that can deliver any reporter fusion construct into the chromosome by double crossover homologous recombination (Figure 1). This construct allows for quantitative analysis of any regulatory region that supports the production of GFP protein and fluorescent signal above background. The plasmid confers ampicillin resistance (Apr) for maintenance and propagation in E. coli and...

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A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

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Genetics

8.8K Views.  Georgetown University. Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Video: A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...