This work was supported by NIH grants R01 AI125421, R01 AI127793, and U19 AI144182. The writing author would like to thank all lab members who contributed to proofreading and review of this method.

1. Preparation of Chelex-treated Defined Medium (CDM) Stock Solutions
<ol>
	<li>Stock solution I
	<ol>
		<li>Combine NaCl (233.8 g), K2SO4 (40.0 g), NH4Cl (8.8 g), K2HPO4 (13.9 g), and KH2PO4 (10.9 g) in deionized water to a final volume of 1 L.</li>
		<li>Filter sterilize the solution and aliquot into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution II
	<ol>
		<li>Combine thiamine HCl (0.2 g), thiamine pyrophosphate-Cl (0.05 g), calcium pantothenate (0.19 g), and biotin (0.3 g) in 50% (vol/vol) ethanol to a final volume of 1 L.</li>
		<li>Aliquot into 50 mL conical tubes and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution III
	<ol>
		<li>Combine L-aspartate (4.0 g), L-glutamate (10.4 g), L-arginine (1.2 g), glycine (0.2 g), L-serine (0.4 g), L-leucine (0.72 g), L-isoleucine (0.24 g), L-valine (0.48 g), L-tyrosine (0.56 g), L-proline (0.4 g), L-tryptophan (0.64 g), L-threonine (0.4 g), L-phenylalanine (0.2 g), L-asparagine-H2O (0.2 g), L-glutamine (0.4 g), L-histidine-HCl (0.2 g), L-methionine (0.12 g), L-alanine (0.8 g), L-lysine (0.4 g), and reduced glutathione (0.36 g) in 500 mL deionized water.</li>
		<li>Dissolve L-cysteine (0.44 g) and L-cystine (0.28 g) in a minimal volume (~1 mL) of 1 M HCl and add to the above amino acid solution.</li>
		<li>Adjust the pH of the solution with 10 N NaOH until all particulate is dissolved. The final pH will be 10.0&#8211;11.0.</li>
		<li>Bring the final volume to 1 L with deionized water.</li>
		<li>Filter sterilize the solution and aliquot into 250 mL volumes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution IV
	<ol>
		<li>Dissolve glucose (200 g) in deionized water to a final volume of 1 L. 
		NOTE: The solution may have to be heated to dissolve the glucose.</li>
		<li>Filter sterilize and aliquot the solution into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Stock solution V
	<ol>
		<li>Combine hypoxanthine (5.0 g), uracil (5.0 g), and NaOH (4.0 g) in deionized water to a final volume of 1 L.</li>
		<li>Filter sterilize and aliquot into 50 mL conical tubes.</li>
		<li>Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Solution VI
	<ol>
		<li>Prepare a 1 M CaCl2-2H2O (147 g/L) solution in deionized water. Filter sterilize and store at room temperature (RT).</li>
	</ol>
	</li>
	<li>Stock solution VII
	<ol>
		<li>Prepare a 1 M anhydrous MgSO4 solution in deionized water. Filter sterilize and store at RT.</li>
	</ol>
	</li>
	<li>Stock solution VIII
	<ol>
		<li>Prepare a 1 M NaHCO3 solution in deionized water. Filter sterilize and store at RT.</li>
	</ol>
	</li>
</ol>
2. Preparation of 4x Sterile Concentrate and 1x CDM
NOTE: This procedure is to be performed in either acid treated sterile glassware or plastic to prevent leaching of metals into the solutions.
<ol>
	<li>Combine stock solutions I (50 mL), II (20 mL), III (250 mL), IV (50 mL), and V (20 mL) with 20.0 g of HEPES and stir to mix.</li>
	<li>Adjust pH to 7.4, then bring the final volume to 500 mL with deionized water.</li>
	<li>Wash Chelex-100 resin in 1 L deionized water to remove preservatives prior to adding it to the 4x sterile concentrate. Do this by adding 50 g of resin to 1 L deionized water and stirring for at least 1 h. Remove the water by vacuum filtration and use this washed resin for step 2.4.</li>
	<li>Add 50 g of resin and slowly stir for exactly 90 min.</li>
	<li>Remove resin by filter sterilization and store the 4x sterile concentrate at 4 &#176;C.</li>
	<li>To prepare a 1x working concentration of CDM, first dilute the 4x concentrate with sterile deionized water, then add solution VI (125 &#181;L per 500 mL of 1x solution), solution VII (535 &#181;L per 500 mL), and solution VIII (10 mL per 500 mL). 
	NOTE: Although all stock solutions have already been sterilized, it is recommended to filter sterilize the 1x solution again after preparation.</li>
</ol>
3. Preparation of CDM Plates
NOTE: The recipe below makes 1 L media for plates, but it is best to prepare these in smaller volumes. Everything scales down proportionally.
<ol>
	<li>Washed agarose
	<ol>
		<li>Dissolve 50 g of agarose in 1 L deionized water, stirring for 1 h.</li>
		<li>Transfer the solution to a centrifuge bottle and centrifuge at 1,200 x g for 15 min at RT. Carefully pour off the supernatant and discard.</li>
		<li>Add sufficient deionized water to resuspend the agarose pellet, then transfer to a 1 L flask. Bring to a final volume of 1 L with deionized water and then stir and centrifuge as in step 3.1.2. Discard the supernatant.</li>
		<li>Add sufficient 100% ethanol to resuspend the pellet, then transfer to a new 1 L flask. Bring to a final volume of 1 L with ethanol. Stir and centrifuge as in step 3.1.2.</li>
		<li>Repeat step 3.1.4.</li>
		<li>Add methanol to the agarose pellet to resuspend, then transfer to a 1 L flask. Bring to a final volume of 1 L with methanol. Stir and centrifuge as in step 3.1.2.</li>
		<li>Repeat step 3.1.6.</li>
		<li>Transfer washed agarose to a tray lined with aluminum foil and allow to dry in a fume hood. When dry, transfer to a metal-free container for long-term storage.</li>
	</ol>
	</li>
	<li>Add 10 g of washed agarose and 5 g of potato starch to 750 mL of deionized water.</li>
	<li>Autoclave for 30 min at 121 &#176;C, 100 kPa above atmospheric pressure.</li>
	<li>Allow media to cool to ~65 &#176;C, then add 250 mL of 4X CDM, 250 &#181;L of solution VI, 1.07 mL of solution VII, and 20 mL of solution VIII.</li>
	<li>If desired, add the metals of choice before pouring plates. The addition of chelators is not necessary to maintain metal-free conditions.</li>
	<li>Pour into Petri dishes and allow to solidify.</li>
</ol>
4. Metal Limited Growth of Neisseria gonorrhoeae
NOTE: For most applications, it is not necessary to metal stress the bacteria prior to inoculation of CDM. The initial doubling step in CDM and the subsequent dilution is sufficient to deplete the gonococci of their internal iron and zinc stores. As such, the first two steps of the following procedure are conducted using agar plates made from GC medium base that have been supplemented with Kellogg&#39;s supplement I14 and 12.5 &#956;M Fe(NO3)3. If early metal stress is desired, we recommend preparing GC medium base plates without Fe(NO3)3 and with 5 &#956;M TPEN (N,N,N&#39;,N&#39;-tetrakis (2-pyridylmethyl) ethylenediamine) for zinc chelation or 10 &#956;M deferoxamine for iron chelation. All incubation is conducted at 37 &#176;C with 5% CO2.
<ol>
	<li>Two days prior to the experiment, on day -2, streak gonococci from freezer stocks onto GC medium plates and incubate for no more than 24 h.</li>
	<li>On day -1, streak single colonies onto fresh GC medium plates. Try to do this 14&#8211;16 h prior to the growth experiment.</li>
	<li>On the day of the experiment, add 5&#8211;10 mL 1x CDM to an acid washed 125 mL baffled sidearm flask (Supplemental Figure 1) and use this to blank a Klett colorimeter.</li>
	<li>Use a sterile, cotton-tipped swab to inoculate CDM from healthy, single colonies. Aim for 20 Klett units. 
	NOTE: If a Klett colorimeter is not available, a spectrophotometer is suitable as well. There is no universal conversion formula for Klett units to OD, but a rough guide is available (<a href="https://support.hunterlab.com/hc/en-us/articles/214490283-Klett-Color-Scales" target="_blank">https://support.hunterlab.com/hc/en-us/articles/214490283-Klett-Color-Scales</a>).</li>
	<li>Incubate with shaking at 250 rpm until approximately one mass doubling (40 Klett units). This should take between 1&#8211;2 h.</li>
	<li>At this point, the cultures are back diluted by addition of a sufficient volume of CDM to reach half of the initial culture density (e.g., if 5 mL of culture has gone from 20 to 40 Klett units, 15 mL of CDM will bring it back down to 10 Klett units) and growth will continue as in step 4.5. The specific amount of back dilution, metal treatments, etc. depend on downstream applications. We give three examples below (sections 5, 6, or 7).</li>
</ol>
5. Western Analysis of Metal Responsive Gene Products
<ol>
	<li>Beginning at step 4.6, back dilute the cultures with three volumes of CDM (e.g., 15 mL of CDM if starting with 5 mL). At this point, add metal treatments if desired.
	<ol>
		<li>Iron-sensing genes can be derepressed with 12.5 &#956;M Fe(NO3)3. Zinc-responsive genes may be derepressed with 10 &#956;M ZnSO4.</li>
		<li>Additional iron or zinc stress is not necessary, as the media is already depleted, so responsive genes will already be expressed. If further stress is desired, we recommend no more than 1&#8211;2 &#956;M of deferoxamine or TPEN, as excessive stress will prevent cultures from growing.</li>
	</ol>
	</li>
	<li>Grow cultures as in step 4.5 for 4 h and record the final cell density of the samples. Less metal-stressed cultures will grow to higher final densities.</li>
	<li>Standardize cultures to a suitable density and prepare lysates.
	<ol>
		<li>Standardize whole-cell lysates to a density equivalent to 100 Klett units in 1 mL of culture. To accomplish this, divide 100 by the Klett unit density of your sample. The number you get is the volume, in mL, of culture that will be used to make the cell pellet.</li>
	</ol>
	</li>
	<li>Follow standard SDS-PAGE and Western blotting procedures15 to probe for proteins of interest.</li>
</ol>
6. Metal-limited Growth Assays
NOTE: These assays describe premade growth premixes. The preparation of these mixes is described in section 8.
<ol>
	<li>During the mass doubling in step 4.5, pretreat the wells of a 96 well microplate with 10x premixes. Additionally, designate three wells to serve as blanks. To these wells, add 10 &#181;L of 10x premix and 90 &#181;L of CDM.</li>
	<li>Once the cultures in the sidearm flasks have doubled, add 100 &#181;L of each culture to an unused well in the microplate and measure the optical density at 600 nm (OD600). This may be done with cuvettes in a spectrophotometer, but with larger numbers of strains within an assay this can become cumbersome and is generally not advised unless your spectrophotometer can measure directly from the arm of the side arm flask (Supplemental Figure 2).
	<ol>
		<li>While measuring the OD, place the flasks back in the incubator to ensure the gonococci remain viable.</li>
	</ol>
	</li>
	<li>Calculate the correct amount of dilution required to bring the cultures to OD600 = 0.02. The 10x premixes have a negligible effect on OD and can be omitted from calculation.</li>
	<li>Dilute cultures with CDM in small culture tubes and add sufficient volume to dilute the 10x premixes to 1x in the plate. If a plate warmer is available, keep the plate at 37 &#176;C while working.</li>
	<li>Incubate the plate for 8&#8211;12 h with shaking in a plate reader, taking OD600 measurements at desired intervals.</li>
</ol>
7. Detection of Ligand Binding by Outer Membrane Metal Transporters
<ol>
	<li>Treat cultures as desired as in step 5.1, then incubate with shaking for 4 h.</li>
	<li>Shortly before the 4 h mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot blot apparatus (Supplemental Figure 3). Presoak the nitrocellulose in deionized water, then assemble the apparatus with filter paper below the nitrocellulose.</li>
	<li>At 4 h, record the cell densities and standardize to an appropriate final density. It is recommended to use ~10% of the density used for the preparation of the cell lysates in step 5. For example, if using 100 KU in 1 mL for the lysates, this means ~10 KU in 1 mL for these blots. The calculation is otherwise the same as described in 5.3.1, and cultures are added directly to the nitrocellulose in the calculated volumes rather than made into lysates.</li>
	<li>Pipet cell cultures onto the nitrocellulose and allow sufficient time for the filter paper to absorb all the liquid.</li>
	<li>Disassemble the apparatus, allow the blot to dry, and block the nitrocellulose membrane for 1 h in 5% bovine serum albumin or nonfat milk (w/v) in Tris-buffered saline.</li>
	<li>Reassemble the dot blot apparatus, replacing the filter paper with paraffin film to create a leak-proof seal under the nitrocellulose.</li>
	<li>Dilute the metal-binding ligand of interest to 0.2 &#956;M in blocker and probe cells for 1 h.</li>
	<li>Siphon off the liquid with a vacuum, wash the blot, then follow standard immunological procedures to develop the signal16. The wash steps may be done in or out of the apparatus.</li>
</ol>
8. Metal Loading of Transferrin, S100A7, and Calprotectin, and Preparation of 10x Premixes
NOTE: As with CDM preparation, use acid washed glass or plastic for solution preparation.
<ol>
	<li>Dissolve human transferrin at 10 mg/mL (125 &#956;M) in initial buffer (100 mM Tris, 150 mM NaCl, 20 mM NaHCO3, pH = 8.4). S100A7 and calprotectin are suspended in buffer consisting of 20 mM Tris, 100 mM NaCl, 10 mM 2-Mercaptoethanol, and 1 mM CaCl2, pH = 8.0).
	<ol>
		<li>Add ferration solution (100 mM sodium citrate, 100 mM NaHCO3, 5 mM FeCl3-6H2O, pH = 8.4) to the transferrin solution to achieve 30% iron saturation (e.g., 75 &#181;L of ferration solution is suitable for 5 mL transferrin if made at 10 mg/mL).</li>
		<li>Add ZnSO4 to S100A7 or calprotectin at a 50% molar ratio to the protein to create 25% saturation (each protein molecule has two metal binding sites). Preparations of 100 &#956;M S100A7 or calprotectin with 50 &#956;M ZnSO4 can be used.</li>
		<li>For both cases, allow end-over-end mixing for at least 1 h for metal loading.</li>
	</ol>
	</li>
	<li>Prepare 4 L of dialysis buffer (40 mM Tris, 150 mM NaCl, 20 mM NaHCO3, pH = 7.4). Split this into two separate 2 L volumes and place one at 4 &#176;C.</li>
	<li>Add the metal loaded proteins to a dialysis cassette using a syringe and dialyze against the first buffer volume for 4 h at RT.</li>
	<li>Move the cassette to the second buffer volume and dialyze overnight at 4 &#176;C. After these steps, any unbound metals should be removed. 
	NOTE: We advise a bicinchoninic acid assay to determine protein concentrations after dialysis.</li>
	<li>Use a 10x transferrin premix for transferrin utilization as a sole iron source.
	<ol>
		<li>Prepare this premix by diluting 30% human Fe-transferrin and bovine apo-transferrin (prepared at 125 &#956;M as described for human transferrin, without the iron loading step) to 75 &#956;M and 30 &#956;M, respectively, in PBS. A positive control premix replaces 30% Fe-transferrin with 75 &#956;M Fe(NO3)3, and a negative control premix omits any added iron, retaining only the bovine apo-transferrin. In the growth assay, dilute 10 &#181;L of these concentrates with 90 &#181;L of culture. Final concentrations are 7.5 &#956;M 30% human Fe-transferrin and 3 &#956;M bovine apo-transferrin.</li>
	</ol>
	</li>
	<li>Use a modified version of the transferrin 10x premix for S100A7 or calprotectin utilization as a sole zinc source.
	<ol>
		<li>For a positive control premix, incorporate 50 &#956;M ZnSO4 into the transferrin premix. For a negative control, incorporate 50 &#956;M TPEN and omit the zinc. Then, take 10 &#181;L of each of these for every sample well needed, and move that volume to a new tube. Add to this half as much volume of sterile PBS. For example, if 10 wells will receive the positive control premix, take 100 &#181;L of premix, move it to a new tube, and add 50 &#181;L of PBS. Do the same for the negative control.</li>
		<li>To make the S100A7 or calprotectin premix, take 10 &#181;L of the negative control premix per well needed, move to a new tube, and add half that volume of the 25% Zn-S100A7 or calprotectin. In the growth assay, use 15 &#181;L of premix and dilute with 85 &#181;L of culture. Final concentrations for the transferrins remain the same as in step 8.5, with an added 5 &#956;M of Zn, TPEN, or S100A7/calprotectin.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+requirement+of+Neisseria+gonorrhoeae+growing+on+a+solid+medium.">Carbon dioxide requirement of Neisseria gonorrhoeae growing on a solid medium.</a> Journal of Clinical Microbiology. 4 (2), 129-132 (1976).</li><li>Grim, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Metallophore+Staphylopine+Enables+Staphylococcus+aureus+To+Compete+with+the+Host+for+Zinc+and+Overcome+Nutritional+Immunity.">The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity.</a> MBio. 8 (5), 01281-01317 (2017).</li><li>Helbig, K., Bleuel, C., Krauss, G. J., Nies, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+transition-metal+homeostasis+in+Escherichia+coli.">Glutathione and transition-metal homeostasis in Escherichia coli.</a> Journal of Bacteriology. 190 (15), 5431-5438 (2008).</li><li>Calmettes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+Zinc+acquisition+by+the+neisserial+outer-membrane+transporter+ZnuD.">The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD.</a> Nature Communications. 6, 7996 (2015).</li><li>Hubert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZnuD,+a+potential+candidate+for+a+simple+and+universal+Neisseria+meningitidis+vaccine.">ZnuD, a potential candidate for a simple and universal Neisseria meningitidis vaccine.</a> Infection and Immunity. 81 (6), 1915-1927 (2013).</li><li>Kumar, P., Sannigrahi, S., Tzeng, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neisseria+meningitidis+ZnuD+zinc+receptor+contributes+to+interactions+with+epithelial+cells+and+supports+heme+utilization+when+expressed+in+Escherichia+coli.">The Neisseria meningitidis ZnuD zinc receptor contributes to interactions with epithelial cells and supports heme utilization when expressed in Escherichia coli.</a> Infection and Immunity. 80 (2), 657-667 (2012).</li><li>Stork, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+outer+membrane+receptor+of+Neisseria+meningitidis+involved+in+zinc+acquisition+with+vaccine+potential.">An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.</a> PLoS Pathogens. 6, 1000969 (2010).</li><li>Rosadini, C. V., Gawronski, J. D., Raimunda, D., Arg&#252;ello, J. M., Akerley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+zinc+binding+system,+ZevAB,+is+critical+for+survival+of+nontypeable+Haemophilus+influenzae+in+a+murine+lung+infection+model.">A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.</a> Infection and Immunity. 79 (8), 3366-3376 (2011).</li><li>Ammendola, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+Zn2++uptake+system+ZnuABC+is+required+for+bacterial+zinc+homeostasis+in+intracellular+environments+and+contributes+to+the+virulence+of+Salmonella+enterica.">High-affinity Zn2+ uptake system ZnuABC is required for bacterial zinc homeostasis in intracellular environments and contributes to the virulence of Salmonella enterica.</a> Infection and Immunity. 75 (12), 5867-5876 (2007).</li><li>Gabbianelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ZnuABC+and+ZinT+in+Escherichia+coli+O157:H7+zinc+acquisition+and+interaction+with+epithelial+cells.">Role of ZnuABC and ZinT in Escherichia coli O157:H7 zinc acquisition and interaction with epithelial cells.</a> BMC Microbiology. 11, 36 (2011).</li><li>Biswas, G. D., Anderson, J. E., Chen, C. J., Cornelissen, C. N., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+functional+characterization+of+the+Neisseria+gonorrhoeae+lbpB+gene+product.">Identification and functional characterization of the Neisseria gonorrhoeae lbpB gene product.</a> Infection and Immunity. 67 (1), 455-459 (1999).</li><li>Biswas, G. D., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lbpA,+the+structural+gene+for+a+lactoferrin+receptor+in+Neisseria+gonorrhoeae.">Characterization of lbpA, the structural gene for a lactoferrin receptor in Neisseria gonorrhoeae.</a> Infection and Immunity. 63 (8), 2958-2967 (1995).</li><li>Chen, C. J., Sparling, P. F., Lewis, L. A., Dyer, D. W., Elkins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+purification+of+a+hemoglobin-binding+outer+membrane+protein+from+Neisseria+gonorrhoeae.">Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.</a> Infection and Immunity. 64 (12), 5008-5014 (1996).</li><li>Wong, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+haemoglobin+binding+by+HpuA+from+the+Neisseriaceae+family.">Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family.</a> Nature Communications. 6, 10172 (2015).</li><li>Carson, S. D., Klebba, P. E., Newton, S. M., Sparling, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+enterobactin+binding+and+utilization+by+Neisseria+gonorrhoeae.">Ferric enterobactin binding and utilization by Neisseria gonorrhoeae.</a> Journal of Bacteriology. 181 (9), 2895-2901 (1999).</li><li>Tseng, H. J., Srikhanta, Y., McEwan, A. G., Jennings, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+manganese+in+Neisseria+gonorrhoeae+correlates+with+resistance+to+oxidative+killing+by+superoxide+anion+and+is+independent+of+superoxide+dismutase+activity.">Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity.</a> Molecular Microbiology. 40 (5), 1175-1186 (2001).</li></ol>

The authors have nothing to declare.

Growth media serves a variety of roles in microbiological research. Specialized media are used for selection, enrichment, and various other applications for many unique types of study. One such application is the induction of metal-responsive genes, which is typically accomplished by addition of a specific chelator that targets a particular metal ion. This method is limited, as the amount of chelation necessary for various trace metals is likely to be variable due to different water sources containing unique metal profil...

Neisseria gonorrhoeae causes the common sexually-transmitted infection gonorrhea. During infection, pathogenic Neisseria express a repertoire of metal-responsive genes that allow the bacteria to overcome metal restriction efforts by the human host1,2,3. Trace metals like iron and zinc play key roles in many cellular processes, such as binding to enzymes in catalytic sites, participation in redox reactions, and as structural factors in various proteins4,5. In metal-limited conditions, metal-responsive loci are derepressed and their resultant proteins can aid the acquisition of these nutrients. Characterization of these genes and proteins presents a unique technical challenge for the investigator. Metal ions must be withheld from bacteria in order to induce transcription of these genes from their native loci, but effective chelation of these ions from metal-laden media can be difficult to optimize. The different metal profiles of source water and inherent lot-to-lot variation6 of powdered ingredients means that the amount of chelator required to remove a specific metal from a rich medium will vary between different locations, ingredient vendors, and even over time within a single laboratory as chemical inventory is replaced.
To circumvent this challenge, we describe the preparation of a defined medium that is treated with Chelex-100 resin during preparation to remove trace metals from the solution. This medium is sufficiently nutrient dense to allow for the growth of gonococcus, which is difficult to culture outside of the human host, and allows the investigator to introduce a specific metal profile by addition of their own defined sources and concentrations of metals. The method of controlled add-back of desired metals to depleted medium increases experimental consistency and allows for robust, replicable experiments regardless of factors such as water source and chemical lot numbers. Moreover, this media can be deployed as either a liquid or solid with only minor modifications, making it quite versatile.
In order to demonstrate the utility of the this medium, we outline a protocol for its use for gonococcal growth and describe three successful experiments to characterize metal-responsive Neisseria genes. First, we prepare gonococcal whole-cell lysates from metal-depleted or supplemented cultures and demonstrate variable levels of protein production from metal-responsive loci. We then outline a zinc-restricted growth assay in which gonococcal growth is controlled by supplementation of specific, useable zinc sources. Finally, we show binding assays that demonstrate whole gonococcal cells expressing metal-responsive surface receptors binding to their respective metal-containing ligands. Successful surface presentation of these receptors requires growth in metal-depleted medium.
The present protocol was optimized specifically for Neisseria gonorrhoeae, but numerous other bacterial pathogens employ metal acquisition strategies during infection7, so this protocol may be adapted for the study of metal homeostasis in other bacteria. Optimizing this media and these experimental protocols for use in other bacteria will likely require slight modification of metal chelator concentrations and/or treatment time with Chelex-100, as other bacteria may have slightly different metal requirements than gonococcus. Iron and zinc are the primary metals of concern for the described investigations, but other metals (e.g., manganese) have been demonstrated as critical for bacteria, including Neisseria8,9,10,11,12. Furthermore, similar methods have been described for metal characterizations in eukaryotic cell culture work, which may also be considered.13

<table><tbody><tr><td>125 mL sidearm flasks</td><td>Bellco</td><td>2578-S0030</td><td>Must be custom ordered</td></tr><tr><td>2-Mercaptoethanol</td><td>VWR</td><td>M131</td><td>Open in fume hood</td></tr><tr><td>3MM Paper</td><td>GE Health</td><td>3030-6461</td><td>Called &quot;filter paper&quot; in text</td></tr><tr><td>Agarose</td><td>Biolone</td><td>BIO-41025</td><td>Powder</td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>A9434</td><td>Powder</td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>B4501</td><td>Powder</td></tr><tr><td>Blotting grade blocker</td><td>Bio-Rad</td><td>170-6404</td><td>Nonfat dry milk</td></tr><tr><td>Bovine serum albumin</td><td>Roche</td><td>3116964001</td><td>Powder</td></tr><tr><td>Bovine transferrin</td><td>Sigma-Aldrich</td><td>T1428</td><td>Powder</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C5080</td><td>Powder</td></tr><tr><td>Calcium pantothenate</td><td>Sigma-Aldrich</td><td>C8731</td><td>Powder</td></tr><tr><td>Calprotectin</td><td>N/A</td><td>N/A</td><td>We are supplied with this by a collaborator</td></tr><tr><td>Chelex-100 Resin</td><td>Bio-Rad</td><td>142-2832</td><td>Wash with deionized water prior to use</td></tr><tr><td>Cotton-tipped sterile swab</td><td>Puritan</td><td>25-806</td><td>Cotton is better than polyester for this application</td></tr><tr><td>Deferoxamine</td><td>Sigma-Aldrich</td><td>D9533</td><td>Powder</td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>Powder</td></tr><tr><td>Dialysis cassette</td><td>Thermo</td><td>66380</td><td>Presoak in buffer prior to use</td></tr><tr><td>Dot blot apparatus</td><td>Schleicher &amp;amp; Schwell</td><td>10484138</td><td>Lock down lid as tightly as possible before sample loading</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1016</td><td>Flammable liquid, store in flammables cabinet</td></tr><tr><td>Ferric chloride</td><td>Sigma-Aldrich</td><td>F7134</td><td>Irritant, do not inhale</td></tr><tr><td>Ferric nitrate nonahydrate</td><td>Sigma-Aldrich</td><td>F1143</td><td>Irritant, do not inhale</td></tr><tr><td>GC medium base</td><td>Difco</td><td>228950</td><td>Powder, already contains agar</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td>Powder</td></tr><tr><td>HEPES</td><td>Fisher</td><td>L-15694</td><td>Powder</td></tr><tr><td>Human transferrin</td><td>Sigma-Aldrich</td><td>T2030</td><td>Powder</td></tr><tr><td>Hypoxanthine</td><td>Sigma-Aldrich</td><td>H9377</td><td>Powder</td></tr><tr><td>Klett colorimeter</td><td>Manostat</td><td>37012-0000</td><td>Uses color transmission to assess culture density</td></tr><tr><td>L-alanine</td><td>Sigma-Aldrich</td><td>A7627</td><td>Powder</td></tr><tr><td>L-arginine</td><td>Sigma-Aldrich</td><td>A5006</td><td>Powder</td></tr><tr><td>L-asparagine monohydrate</td><td>Sigma-Aldrich</td><td>A8381</td><td>Powder</td></tr><tr><td>L-aspartate</td><td>Sigma-Aldrich</td><td>A9256</td><td>Powder</td></tr><tr><td>L-cysteine hydrochloride</td><td>Sigma-Aldrich</td><td>C1276</td><td>Powder</td></tr><tr><td>L-cystine</td><td>Sigma-Aldrich</td><td>C8755</td><td>Powder</td></tr><tr><td>L-glutamate</td><td>Sigma-Aldrich</td><td>G1251</td><td>Powder</td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G3126</td><td>Powder</td></tr><tr><td>L-histidine monohydrochloride</td><td>Sigma-Aldrich</td><td>H8125</td><td>Powder</td></tr><tr><td>L-isoleucine</td><td>Sigma-Aldrich</td><td>I2752</td><td>Powder</td></tr><tr><td>L-leucine</td><td>Sigma-Aldrich</td><td>L8000</td><td>Powder</td></tr><tr><td>L-lysine</td><td>Sigma-Aldrich</td><td>L5501</td><td>Powder</td></tr><tr><td>L-methionine</td><td>Sigma-Aldrich</td><td>M9625</td><td>Powder</td></tr><tr><td>L-phenylalanine</td><td>Sigma-Aldrich</td><td>P2126</td><td>Powder</td></tr><tr><td>L-proline</td><td>Sigma-Aldrich</td><td>P0380</td><td>Powder</td></tr><tr><td>L-serine</td><td>Sigma-Aldrich</td><td>S4500</td><td>Powder</td></tr><tr><td>L-threonine</td><td>Sigma-Aldrich</td><td>T8625</td><td>Powder</td></tr><tr><td>L-tryptophan</td><td>Sigma-Aldrich</td><td>T0254</td><td>Powder</td></tr><tr><td>L-tyrosine</td><td>Sigma-Aldrich</td><td>T3754</td><td>Powder</td></tr><tr><td>L-valine</td><td>Sigma-Aldrich</td><td>V0500</td><td>Powder</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td>Powder</td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td>Flammable liquid, store in flammables cabinet</td></tr><tr><td>Nitrocellulose</td><td>GE Health</td><td>10600002</td><td>Keep in protective sheath until use</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma-Aldrich</td><td>60356</td><td>Powder</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td>Powder</td></tr><tr><td>Potassium sulfate</td><td>Sigma-Aldrich</td><td>P0772</td><td>Powder</td></tr><tr><td>Potato starch</td><td>Sigma-Aldrich</td><td>S4251</td><td>Powder</td></tr><tr><td>Reduced glutathione</td><td>Sigma-Aldrich</td><td>G4251</td><td>Handle carefully. Can oxidize easily.</td></tr><tr><td>S100A7</td><td>N/A</td><td>N/A</td><td>We are supplied with this by a collaborator</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>Powder</td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>470302</td><td>Powder</td></tr><tr><td>Sodium citrate</td><td>Fisher</td><td>S279</td><td>Powder</td></tr><tr><td>Sodium hydroxide</td><td>Acros Organics</td><td>383040010</td><td>Highly hygroscopic</td></tr><tr><td>Thiamine hydrochloride</td><td>Sigma-Aldrich</td><td>T4625</td><td>Powder</td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma-Aldrich</td><td>C8754</td><td>Also called cocarboxylase</td></tr><tr><td>TPEN</td><td>Sigma-Aldrich</td><td>P4413</td><td>Powder</td></tr><tr><td>Tris</td><td>VWR</td><td>497</td><td>Powder</td></tr><tr><td>Uracil</td><td>Sigma-Aldrich</td><td>U0750</td><td>Powder</td></tr><tr><td>Zinc sulfte heptahydrate</td><td>Sigma-Aldrich</td><td>204986</td><td>Irritant, do not inhale</td></tr></tbody></table>

metal-limited growth of neisseria gonorrhoeae for characterization of metal-responsive genes and metal acquisition from host ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

A specific defined medium in the absence of trace metals for the growth of Neisseria gonorrhoeae was developed and implemented for the characterization of metal-responsive genes and their gene products. In the optimized protocol, the metal profile of media is controlled by adding metals back at the discretion of the investigator, rather than by titrated chelation of a metal target, allowing for increased control and consistency from lab to lab and experiment to experiment. This m...

Watch this Scientific Journal Video about Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands at JoVE.com

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

metal-limited-growth-neisseria-gonorrhoeae-for-characterization-metal

Research

JoVE Journal

Immunology and Infection

5.5K Views.  Georgia State University. We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Video: Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Chemistry

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

Environment

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Biology

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Summary

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Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Measuring the Migration and Biofilm Formation of Various Bacteria