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

Summary

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.

Abstract

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.

Introduction

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 host^1,2,3. 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 proteins^4,5. 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 Neisseria^8,9,10,11,12. Furthermore, similar methods have been described for metal characterizations in eukaryotic cell culture work, which may also be considered.¹³

Protocol

1. Preparation of Chelex-treated Defined Medium (CDM) Stock Solutions

1. Stock solution I
   1. 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.
   2. Filter sterilize the solution and aliquot into 50 mL conical tubes.
   3. Store at -20 °C.
2. Stock solution II
   1. 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.
   2. Aliquot into 50 mL conical tubes and store at -20 °C.
3. Stock solution III
   1. 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.
   2. 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.
   3. Adjust the pH of the solution with 10 N NaOH until all particulate is dissolved. The final pH will be 10.0–11.0.
   4. Bring the final volume to 1 L with deionized water.
   5. Filter sterilize the solution and aliquot into 250 mL volumes.
   6. Store at -20 °C.
4. Stock solution IV
   1. 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.
   2. Filter sterilize and aliquot the solution into 50 mL conical tubes.
   3. Store at -20 °C.
5. Stock solution V
   1. Combine hypoxanthine (5.0 g), uracil (5.0 g), and NaOH (4.0 g) in deionized water to a final volume of 1 L.
   2. Filter sterilize and aliquot into 50 mL conical tubes.
   3. Store at -20 °C.
6. Solution VI
   1. Prepare a 1 M CaCl₂-2H₂O (147 g/L) solution in deionized water. Filter sterilize and store at room temperature (RT).
7. Stock solution VII
   1. Prepare a 1 M anhydrous MgSO₄ solution in deionized water. Filter sterilize and store at RT.
8. Stock solution VIII
   1. Prepare a 1 M NaHCO₃ solution in deionized water. Filter sterilize and store at RT.

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.

1. 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.
2. Adjust pH to 7.4, then bring the final volume to 500 mL with deionized water.
3. 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.
4. Add 50 g of resin and slowly stir for exactly 90 min.
5. Remove resin by filter sterilization and store the 4x sterile concentrate at 4 °C.
6. To prepare a 1x working concentration of CDM, first dilute the 4x concentrate with sterile deionized water, then add solution VI (125 µL per 500 mL of 1x solution), solution VII (535 µL 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.

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.

1. Washed agarose
   1. Dissolve 50 g of agarose in 1 L deionized water, stirring for 1 h.
   2. 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.
   3. 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.
   4. 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.
   5. Repeat step 3.1.4.
   6. 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.
   7. Repeat step 3.1.6.
   8. 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.
2. Add 10 g of washed agarose and 5 g of potato starch to 750 mL of deionized water.
3. Autoclave for 30 min at 121 °C, 100 kPa above atmospheric pressure.
4. Allow media to cool to ~65 °C, then add 250 mL of 4X CDM, 250 µL of solution VI, 1.07 mL of solution VII, and 20 mL of solution VIII.
5. If desired, add the metals of choice before pouring plates. The addition of chelators is not necessary to maintain metal-free conditions.
6. Pour into Petri dishes and allow to solidify.

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's supplement I¹⁴ and 12.5 μM Fe(NO₃)₃. If early metal stress is desired, we recommend preparing GC medium base plates without Fe(NO₃)₃ and with 5 μM TPEN (N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine) for zinc chelation or 10 μM deferoxamine for iron chelation. All incubation is conducted at 37 °C with 5% CO_2.

1. 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.
2. On day -1, streak single colonies onto fresh GC medium plates. Try to do this 14–16 h prior to the growth experiment.
3. On the day of the experiment, add 5–10 mL 1x CDM to an acid washed 125 mL baffled sidearm flask (Supplemental Figure 1) and use this to blank a Klett colorimeter.
4. 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 (https://support.hunterlab.com/hc/en-us/articles/214490283-Klett-Color-Scales).
5. Incubate with shaking at 250 rpm until approximately one mass doubling (40 Klett units). This should take between 1–2 h.
6. 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).

5. Western Analysis of Metal Responsive Gene Products

1. 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.
   1. Iron-sensing genes can be derepressed with 12.5 μM Fe(NO₃)₃. Zinc-responsive genes may be derepressed with 10 μM ZnSO₄.
   2. 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 1–2 μM of deferoxamine or TPEN, as excessive stress will prevent cultures from growing.
2. 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.
3. Standardize cultures to a suitable density and prepare lysates.
   1. 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.
4. Follow standard SDS-PAGE and Western blotting procedures¹⁵ to probe for proteins of interest.

6. Metal-limited Growth Assays

NOTE: These assays describe premade growth premixes. The preparation of these mixes is described in section 8.

1. 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 µL of 10x premix and 90 µL of CDM.
2. Once the cultures in the sidearm flasks have doubled, add 100 µL 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).
   1. While measuring the OD, place the flasks back in the incubator to ensure the gonococci remain viable.
3. 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.
4. 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 °C while working.
5. Incubate the plate for 8–12 h with shaking in a plate reader, taking OD₆₀₀ measurements at desired intervals.

7. Detection of Ligand Binding by Outer Membrane Metal Transporters

1. Treat cultures as desired as in step 5.1, then incubate with shaking for 4 h.
2. 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.
3. 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.
4. Pipet cell cultures onto the nitrocellulose and allow sufficient time for the filter paper to absorb all the liquid.
5. 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.
6. Reassemble the dot blot apparatus, replacing the filter paper with paraffin film to create a leak-proof seal under the nitrocellulose.
7. Dilute the metal-binding ligand of interest to 0.2 μM in blocker and probe cells for 1 h.
8. 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.

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.

1. Dissolve human transferrin at 10 mg/mL (125 μM) 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).
   1. 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 µL of ferration solution is suitable for 5 mL transferrin if made at 10 mg/mL).
   2. 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 μM S100A7 or calprotectin with 50 μM ZnSO₄ can be used.
   3. For both cases, allow end-over-end mixing for at least 1 h for metal loading.
2. 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 °C.
3. Add the metal loaded proteins to a dialysis cassette using a syringe and dialyze against the first buffer volume for 4 h at RT.
4. Move the cassette to the second buffer volume and dialyze overnight at 4 °C. After these steps, any unbound metals should be removed.
   NOTE: We advise a bicinchoninic acid assay to determine protein concentrations after dialysis.
5. Use a 10x transferrin premix for transferrin utilization as a sole iron source.
   1. Prepare this premix by diluting 30% human Fe-transferrin and bovine apo-transferrin (prepared at 125 μM as described for human transferrin, without the iron loading step) to 75 μM and 30 μM, respectively, in PBS. A positive control premix replaces 30% Fe-transferrin with 75 μM Fe(NO₃)₃, and a negative control premix omits any added iron, retaining only the bovine apo-transferrin. In the growth assay, dilute 10 µL of these concentrates with 90 µL of culture. Final concentrations are 7.5 μM 30% human Fe-transferrin and 3 μM bovine apo-transferrin.
6. Use a modified version of the transferrin 10x premix for S100A7 or calprotectin utilization as a sole zinc source.
   1. For a positive control premix, incorporate 50 μM ZnSO₄ into the transferrin premix. For a negative control, incorporate 50 μM TPEN and omit the zinc. Then, take 10 µL 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 µL of premix, move it to a new tube, and add 50 µL of PBS. Do the same for the negative control.
   2. To make the S100A7 or calprotectin premix, take 10 µL 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 µL of premix and dilute with 85 µL of culture. Final concentrations for the transferrins remain the same as in step 8.5, with an added 5 μM of Zn, TPEN, or S100A7/calprotectin.

Results

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...

Discussion

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...

Materials

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