Bacteria Surface Charge

Wilhelm MJ, Sharifian Gh M, Wu T, Li Y, Chang CM, Ma J, Dai HL. Determination of bacterial surface charge density via saturation of adsorbed ions. Biophys J. 2021 Jun 15;120(12):2461-2470. PMC free article

The goal of this post is to introduce lay people to the work of the Michael Wilhelm and colleagues at Temple University in Philadelphia, Pennsylvania.  The question has been asked if Cu⁺ or Cu²⁺ changes the surface of bacteria more than the other.This report is full of insights and one tidbit as to why Cu⁺ might be the better biocide.

The feature image tells the hypothesis this paper inspired. Malachite green is a reporter probe that binds to negative charges on bacteria cell walls just like Cu⁺ and Cu²⁺ do. The hydrated radius of Cu⁺ is predicted to be smaller than that of Cu²⁺and can more easily diffuse into the cell wall. Neutralization of phosphate negative charges should prevent binding of the MG reporter.

 Structures of bacteria cell walls

• OM  The outer membrane of phospholipids.   In gram negative bacteria, lipopolysaccharide, LPS is part of this layer.  The authors estimated 2-8nm wide water channels in this layer.
• PM  Gram positive bacteria have comparatively more piptidoglyan mesh (PM)   Both have an inner cytoplasmic membrane (CM). Note the comparative thickness of PM in Gram positive bacteria.  Pores in these structures are 7-12nm wide.
• CM Gram-positive cells have only a single phospholipid membrane, the inner cell membraneCM.  The PM layer is much thicker. which is surrounded by a substantially thicker PM.  Gram-positive bacteria also have an S-layer of crystalized proteins on their surface.  All of these substances have the potential to bind copper and other antimicrobial compounds.
• SL is a shell of crystallized proteins on the surface layer of  Gram positive bacteria. The bacterial S-layer is a homogeneous protein wall, self-assembled into a crystalline lattice with regularly spaced pores of distinct symmetry (e.g., oblique, square, hexagonal)  Negatively charged amino acids predominate this protein layer.
• MG, malachite green, a 1nm wide hydrophobic dye that is 5-% in the +1 charge state at pH 6.9.  This makes it small enough to diffuse to the cell membrane. The MG cation has an electronic absorption band near 400 nm and consequently its second order nonlinear polarizability is resonantly enhanced when exposed to fundamental light of 800 nm. Neutral MG lacks these electronic transitions.  Only surface bound MG cations can be observed.  MG may diffuse across lipid bilayers.  These appear to be SHG.

All of these layers tend to have a negative charge and therefore the potential of binding mono and divalent cations and the malachite green probe used in these experiments.  There is a time component of diffusion through these layers too. In these experiments Lactobacillus, L. rhamnosus was the representative Gram-positive bacterium and Escherichia, E. coli the representative Gram-negative bacterium.

Three techniques were used

1. SGS/SHG Second Harmonic Light Scattering occurs when a portion of an incident light of frequency ω is converted to 2ω after interacting with a material.  This is a two photon process by which two photons of the same initial energy interact with a nonlinear material and combine to produce a single photon of twice the energy, twice the frequency, and half the wavelength… s new color that is blue shifted from the initial incident light. Probes that generate such signals are SHG.
2. Light phase microscopy to estimate cell surface area
3. Zeta potential was used to measure total charge absorption

On MG binding to cell membranes

Adsorption of MG cations onto bacterial surfaces occurs primarily through an attractive electrostatic interactions with the OM of LPS or the S layer of Gram positive L rhamnosus. Then it migrates inward with the final destination of the cell membrane.  It was not if the peptidoglycan layer is electronically silent.  In Figure 7 we see that there are multiple components of LPS.

The concentration dependent signal response at each interface allowed the authors to construct Langmuir adsorption isotherms, which allowed for the calculation of charge density. Brightfield transmission microscopy was used to estimate the surface area that would ultimately be used to determine the charge density. 

Both cell types exhibit an outer layer responsible for rapid binding.  Diffusion through water filled channels to the cell membrane might represent the drop in the SHS signal.

Saturating negatively charged groups with MG⁺

Once saturation is achieved and no more can be absorbed, there is no further increase in the SHS signal. The authors noted that any SHG-active cation, not just MG ion, can be used as the surface charge probe.  Crystal violet, the basis of staining Gram positive, is SHG active as is propidium.

To determine the saturation density of MG cations adsorbed on the external surface of the bacteria, the authors measured Langmuir adsorption isotherms by plotting the SHS peak intensities (deduced from data in Figure 4,

Figure 4  Each point is the average of three experiments ± the standard deviation of the mean.  Note that Gram negative E coli requires significantly more MG to saturate the outer layer of the surface.  These experiments were performed at the pKa of MG, pH 6.9 at which half the MG will be by definition carry a charge of +1.  As the MG cation concentration increases, the magnitude of the measured SHS signal likewise increases. 

• The flat portion of this curve represents all accessible negatively charged sites being occupied by MG.   If Cu+ were present, this saturation would be predicted to occur at a lower MG concentration.  For L. rhamnosus, saturation occurs at 5.8 mM, whereas and70.3 mM for E coli. 
• At saturation, four times more SHS signal is measured for MG cations interacting with L. rhamnosus (~80,000 counts/s) than E. coli (~20,000 counts/s).

Unlike SHG, zeta potential measures the total charge and is not specific to the outermost bacterial surface (e.g., the outer surface of the OM). Zeta-potential results from the cumulative charge of the entire bacterial cell wall complex, including contributions from both sides of both the OM and inner membrane, as well as the peptidoglycan. Cationic MG has access to all anionic-binding sites within the bacterial cell wall complex and is able to neutralize them all.

Calculating charge density

This process was repeated for more than 325 cells of each strain.  E. coli and L. rhamnosus are very similar in size: 2.8 mm long by 1 mm wide.  E. coli has a slightly larger average surface area of 11.0 ± 1.5 mm² compared with 10.1 ± 1.4 mm² for L. rhamnosus.  Mathematical analysis took into consideration the total quantity of MG in the solution and on the bacterial surfaces is constant thus  permitting the  determination of surface density (Nmax) as well as the equilibrium adsorption constant  These parameters allowed for the calculation of the free energy of adsorption (ΔG_ads).

Discussion

In Gram Pos bacteria MG” is restricted to the surface layer of proteins with a lot of negatively charged amino acids, 7b. Gram Neg surface charge is really a 3D matter of vertically packed phosphates on LPS that can bind multiple MG+ cations. In panel 7b MG⁺ is binding to multiple phosphate groups on one small plot of surface area.

MG cation saturation density of 6.6 ±1.3 nm^-2 suggests an average net anionic charge of q = 5.5 ± 1.6 for each of the LPS in the strain of E. coli used in this study.  This agree with results of previous studies that the authors cited.

This value is reasonably consistent with previously deduced

• Salmonella minnesota (q ≥≥ 5),
• Chromobacterium violaceum (q ≥ 4),
• Rhodobacter capsulatus (q ≥ 4.5),
• Paracoccus denitrificans (q ≥ 5)
• E. coli (q ≥ 5) 

Apparent contradictions in these data

• MG bond on the surface of L. rhamnosus yields ~4x the SHS signal  compared with E. coli.
• 7x more MG binds to E coli

MG may simply be more aligned and a better SHG in the Gram positive bacterium.  This adsorption geometry is expected to yield a larger coherent nonlinear polarization from the adsorbed ensemble of MG cations through constructive interference. MG cations are predicted to be more tilted toward the surface plane when bound to LPS resulting in partial cancellation of the nonlinear polarizability and hence a smaller SHS signal.

Surface charge

• Gram positive, one cation per nanometer squared
• Gram negative, seven cations per nanometer squared

The SHS observations also allowed the measurement of Langmuir adsorption isotherms of MG cations on the outer surfaces of the two bacteria were strikingly different.  The S-layer of Gram + has a small charge density compared to Gram negative:   ~1.0 ± 0.2 nm^-2 vs    ~6.6 ± 1.3 nm^-2  .    Mathematical analysis on a biophysical level indicate that charge-charge interaction based on charge density is the driving force for binding.

The inspired hypothesis

The makers of CopperOne claim that in spite of having only one +1 charge, Cu⁺ is more bactericidal than Cu²⁺.  The former is predicted to have a smaller radius of hydration and perhaps more able to navigate the water channels of the cell surface and into the cell membrane where damage may be the most severe.  Can SHS detect changes in the geometry of Gram Pos and Gram Neg bacteria cell walls upon binding to Cu⁺ and Cu²⁺?