Name: metal-limited growth of neisseria gonorrhoeae for characterization of metal-responsive genes and metal acquisition from host ligands
Uploaded: 2020-03-04T14:30:00
Description: 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

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), thiam...

<ol>
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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+zinc-responsive+regulon+of+Neisseria+meningitidis+comprises+17+genes+under+control+of+a+Zur+element.">The zinc-responsive regulon of Neisseria meningitidis comprises 17 genes under control of a Zur element.</a> Journal of Bacteriology. 194 (23), 6594-6603 (2012).</li><li>Andreini, C., Banci, L., Bertini, I., Rosato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+through+the+three+domains+of+life.">Zinc through the three domains of life.</a> Journal of Proteome Research. 5 (11), 3173-3178 (2006).</li><li>Frawley, E. R., Fang, F. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+immunity+beyond+iron:+a+role+for+manganese+and+zinc.">Nutritional immunity beyond iron: a role for manganese and zinc.</a> Current Opinion in Chemical Biology. 14 (2), 218-224 (2010).</li><li>Seib, K. L., 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=Defenses+against+oxidative+stress+in+Neisseria+gonorrhoeae:+a+system+tailored+for+a+challenging+environment.">Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment.</a> Microbiology and Molecular Biology Reviews. 70 (2), 344-361 (2006).</li><li>Wu, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PerR+controls+Mn-dependent+resistance+to+oxidative+stress+in+Neisseria+gonorrhoeae.">PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae.</a> Molecular Microbiology. 60 (2), 401-416 (2006).</li><li>Rayner, M. H., Suzuki, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+the+removal+of+trace+metal+cations+from+a+mammalian+culture+medium+supplemented+with+10%+fetal+calf+serum.">A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum.</a> Biometals. 8 (3), 188-192 (1995).</li><li>Kellogg, D. S., Peacock, W. L., Deacon, W. E., Brown, L., Pirkle, D. 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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=Iron+piracy:+acquisition+of+transferrin-bound+iron+by+bacterial+pathogens.">Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.</a> Molecular Microbiology. 14 (5), 843-850 (1994).</li><li>Quillin, S. J., Seifert, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+gonorrhoeae+host+adaptation+and+pathogenesis.">Neisseria gonorrhoeae host adaptation and pathogenesis.</a> Nature Reviews Microbiology. 16 (4), 226-240 (2018).</li><li>Platt, D. 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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;filter paper&#34; 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 &#38; 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 ...

This protocol enables researchers to analyze bacteria grown under conditions that more closely mimic those found in a mammalian host, which are metal limited through a process called nutritional immunity. This protocol facilitates generation of consistent reproducible bacterial growth under metal restricted conditions and can be adapted for growth of many different types of bacteria. Two days before beginning the experiment, streak gonococcide from freezer stocks onto GC medium plates for a no more than 24 hour incubation at 37 degrees Celsius.14 to 16 hours before the growth experiment, steak single colonies onto fresh GC medium plates. On the day of the experiment, add five to 10 milliliters CDM to an acid-washed 125 milliliter baffled side-arm flask and use this medium to blank a Klett colorimeter. Use a sterile cotton tipped swab to inoculate 20 Klett units of CDM from healthy single colonies.Generation of a healthy Neisseria gonorrhoeae inoculum for subculture into the 96 well plates for liquid growth assays is critical to the success of all downstream experiments. Incubate the cultures at 37 degrees Celsius in 250 revolutions per minute for one to two hours until approximate a one mass doubling before diluting the cultures with a sufficient volume of CDM to reach half of the initial culture density. Then, return the culture to the shaking incubator.To prepare metal loaded proteins, add phoration solution to a 10 milligram per milliliter human transferrin solution to achieve 30%iron saturation. Add the metal of interest to the protein solution at a 15%molar ratio to obtain a 25%saturation. Use a syringe to add the metal loaded protein to a dialysis cassette and dialyze against two liters of dialysis buffer for four hours at room temperature.Then, move the cassette to two liters of four degrees Celsius dialysis buffer and dialyze overnight at four degrees Celsius to remove any unbound metals. During the mass doubling, slightly dilute 10X pre-mixes of interest with PBS. Pre-treat the wells of a 96 well microplate with 15 microliters of the diluted pre-mixes and add 10 microliters of 10X pre-mix and 90 microliters of CDM to each of three wells for the blank controls.Once the cultures in the side-arm flask have doubled, add 100 microliters of each culture to a unused well in the microplate and measure the optical density at 600 nanometers. After calculating the correct amount of dilution required to bring the cultures to an optical density at 600 nanometers of 0.02, dilute the cultures with CDM in small culture tubes and add a sufficient volume to dilute the 10X pre-mixes to 1X in the plate. Then, place the plate in a plate reader for eight to 12 hours with shaking to obtain optical density at 600 nanometers readings at the desired experimental intervals.After the mass doubling incubation, back dilute the cultures with three volumes of CDM and add the metal treatments of interest. Then, incubate the cultures at 37 degrees Celsius with shaking for four hours. Shortly before the four hour mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot-blot apparatus.Pre-soak the nitrocellulose in deionized water for about 10 minutes before assembling the apparatus with filter paper below the nitrocellulose. At the end of the incubation, record the cell densities and standardize the densities to an appropriate final density. Then, pipette the cell cultures onto the nitrocellulose.When the filter paper has absorbed all of the liquid disassemble the apparatus and allow the blot to dry. Block the nitrocellulose membrane for one hour with an appropriate blocking solution and reassemble the dot-blot apparatus, replacing the filter paper with paraffin film to create a leak proof seal under the nitrocellulose. Dilute the metal binding ligand of interest to 0.2 micromolar in blocker and probe the cells for one hour with moderate shaking.At the end of the incubation, siphon off the liquid with a vacuum, wash the blot, and follow standard immunological procedures to develop the signal. Western blot analysis of protein production in variable metal concentrations reveals up regulation of the zinc responsive outer membrane tdfJ in response to zinc chelation by TPEN. TdfJ is essentially undetectable when zinc is added back to the medium.N.gonorrhoeae grows in the presence of zinc loaded calprotectin, which requires the action of the zinc responsive tdfH. When no usable zinc source is available, growth is restricted. Similar results are observed in the presence of zinc S100 A7, which can serve as a sole zinc source when the outer membrane transporter, tdfJ, is produced.Gonococcide grown in CDM are able to bind calprotectin when producing tdfH and as a result of zinc scarcity. Increased binding of transferrin is also observed in cultures grown in CDM, indicative of higher levels of protein expression due to the more iron depleted nature of CDM compared to chelated GC broth. This protocol's been optimized for use with the described water sources and additional titration with other chelators or metals will be needed if other types of water are used.For additional analysis, one could perform quantitative metal uptake assays or quantitative RTPCR for gene expression studies under metal deplete or metal replete conditions.

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