Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

The final precursor for PG synthesis is lipid II, the GlcNAc-MurNAc(pentapepide) building block linked to C 55 -PP. Lipid II is synthesized in two steps at the inner leaflet of the cytoplasmic membrane from UDP-MurNAc-pentapeptide, UDP-GlcNAc and C 55 -P by the enzymes MraY and MurG (Bouhss et al., 2008). PG glycosyltransferases (GTases) polymerize lipid II at the outer leaflet of the membrane to glycan chains. This reaction releases C 55 -PP which is dephosphorylated for new rounds of precursor synthesis and transport.
Peptidoglycan synthesis is a prime target for antibacterial compounds and enzymes. Bacteria and higher organisms often produce antibacterial compounds to target competing bacteria and invading pathogens, respectively (Malanovic and Lohner, 2016). Bacterial competition is particularly fierce in dense populations such as biofilms and soil communities. Whilst the group of actinomycetes are known for their capability to secrete a repertoire of small metabolites that often show antibacterial activity, many Gram-negative bacteria utilize sophisticated type VI secretion systems to target adjacent bacterial cells by antimicrobial enzymes (Russell et al., 2011;. Another type of bacterial toxins are colicins, which are secreted by certain strains of Escherichia coli (Cascales et al., 2007). Colicins use energized nutrient uptake systems to enter the periplasm of susceptible strains of E. coli.
Most colicins kill the target cell by inserting into the cytoplasmic membrane to form pores (Braun and Patzer, 2013). An exception is colicin M, which has a phosphatase activity against lipid II, cleaving the essential peptidoglycan precursor to disaccharide pyrophosphate and undecaprenol (El Ghachi et al., 2006). More recently, it was shown that some Gram-positive species use a type VII secretion system to target other bacteria (Cao et al., 2016). So far the best example is Streptococcus intermedius, which uses a type VII secretion system to deliver an antibacterial toxin, TelC, to target bacteria (Whitney et al., 2017). TelC was shown to degrade lipid II and C 55 -PP to release disaccharide pentapeptide and pyrophosphate, respectively, and undecaprenol. S. intermedius also produces the immunity protein TipC, which inactivates TelC by direct interaction to prevent the lysis of the toxin-producing cell (Whitney et al., 2017). In this methods paper, we provide a detailed description of the TLC and HPLC methods that established the degradation of lipid II and C 55 -PP by TelC (Whitney et al., 2017). These methods can be generally used to assess the activity and specificity of phosphatases against membrane-bound bacterial cell wall precursors.

A.
Enzymatic digestion of lipid II or undecaprenyl pyrophosphate Note: All reactions are carried out in 1.5 ml microtubes and incubated using a microtube shaking incubator at 800 rpm.

1.
Reactions are carried out in a final volume of 50 μl and set up as described below for each substrate. All enzyme substrates are dried under vacuum and subsequently solubilized in the reaction mixture.
Note: Take into account the constituents present in the storage buffer of the assayed proteins to calculate the buffer mixture.

B.
Thin layer chromatography Note: All steps are carried out in a chemical fume hood at room temperature if not indicated otherwise. The basic procedure for thin layer chromatography is shown in the published movie (Cockburn and Koropatkin, 2015).

1.
Pour the mobile phase into the developing chamber and adjust the solvent level to 1 cm.

2.
Close the chamber with the lid and allow for saturation of the gaseous phase with solvent (60 min).

3.
Incubate the TLC plate for at least 1 h at 60 °C to remove any humidity left from storage.

4.
Use a pencil to draw a line 1.5 cm from the bottom of the plate and mark sample spots. Sample spots are separated by 2 cm, and the distance from the outer spots to the edge of the plate should be at least 4 cm.

5.
Load the complete organic phase (upper phase, see Step A3) in 10 µl aliquots on the sample spots. After the addition of each aliquot, the spot is dried with a heat gun. Alternatively, the plate is left under a fume hood for each drying step.
Note: It is important that the lower (aqueous) phase is not transferred on the plate, as this will result in smearing of the spots. When using a heat gun, it is important to not overheat the spots, as this can lead to degradation of compounds and additional bands.

6.
Place the TLC carefully in the developing chamber such that the solvent does not reach the spots. Optimally, there should be a distance of 0.5 cm between the solvent level and the pencil line.

7.
The TLC plate is incubated in the chamber with the lid on until the solvent front reaches 4/5 of the plate length, which takes 1.5-2 h.

1.
Remove the plate from the chamber and dry it with a heat gun. The plate should be completely dried to avoid the appearance of solvent bands during staining.

Data analysis
Take a high-resolution picture and determine the retention factor (R f ) using commercially available programs (e.g., ImageJ). Bands present in control reactions serve as a standard.
R f = distance of the substance zone from the sample origin [mm] solvent front migration distance [mm] Note: The distance is measured from the application line to the middle of the substance spots. For asymmetric spots (here: undecaprenyl pyrophosphate) measure the distance between the application line and lowest point of the spot. Spots in reaction mixtures should have similar R f values, shape and color as spots derived from the standard compounds.

A.
Lipid II reactions for HPLC assay Note: A control reaction, containing the peptidoglycan synthase PBP1B and its cognate activator LpoB, is assayed to polymerize lipid II into short glycan chains with C 55 -PP at the terminal MurNAc residue.
Note: Take into account the constituents present in the storage buffer of the assayed proteins to calculate the buffer mixture.

5.
Add the reaction mixture to the resuspended lipid II and incubate it for 60 min in a microtube shaking incubator at 37 °C with shaking (800 rpm).

6.
Spin down the condensation using a microcentrifuge.
Reactions with phosphatases (TelCt, TelCt-TipC or Colicin M) are processed as follows: 7. Adjust the pH of the sample to 3.5-4.0 using 20% phosphoric acid and pH indicator stripes.
Note: Measure the pH by putting 0.3 μl sample onto the pH indicator stripe.
8. Centrifuge the sample in a microcentrifuge for 15 min at maximum speed and room temperature. Transfer the supernatant into an HPLC vial containing a 400 µl vial insert.
The control reaction with PBP1B-LpoB requires additional steps to digest the peptidoglycan with a muramidase and reduce the resulting unphosphorylated muropeptides. After Step A6 the reaction must be processed as follows: 9. Incubate samples for 5 min at 100 °C using a dry bath, then spin down the condensation using a microcentrifuge.
10. Let the samples cool down at room temperature for 2 min.
12. Incubate the samples for 70 min in a microtube shaking incubator at 37 °C with shaking (800 rpm).
13. Spin down the condensation using a microcentrifuge.
14. Boil the reaction for 10 min at 100 °C on a dry bath and centrifuge the sample using a microcentrifuge for 15 min at maximum speed and room temperature.
15. Punch a hole in the lid of a new 2 ml microcentrifuge tube using a needle.
Note: The hole will allow releasing the H 2 gas produced during the reduction step. 16. Transfer the supernatant to the 2 ml microcentrifuge tube.
17. Reduce the muropeptides by adding 100 μl of 0.5 M sodium borate, pH 9.0 and a tip of a spatula of solid sodium borohydride (ca. 1 mg).
18. Incubate the sample for 30 min at room temperature in a microcentrifuge at 4,700 x g to prevent spillage due to gas bubbles. 19. Adjust the pH of the sample to 3.5-4.0 using 20% phosphoric acid and pH indicator stripes.
Note: Measure the pH by putting 0.3 μl of sample onto the pH indicator stripe.
20. Centrifuge the sample in a microcentrifuge for 15 min at maximum speed and room temperature. Transfer the supernatant into an HPLC vial containing a 400 µl vial insert.

B.
Detection of lipid II products by HPLC Note: Chloroform should be added step-wise and slowly to prevent phase separation.