Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements

Journal of Microbiological Methods 43 (2001) 153–164

Journal of Microbiological Methods www.elsevier.com / locate / jmicmeth

Invited review

Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements q a b a b, W. William Wilson , Mary Margaret Wade , Steven C. Holman , Franklin R. Champlin * a

b

Department of Chemistry, Mississippi State University, Box 9573, Mississippi State, MS 39762, USA Department of Biological Sciences, Mississippi State University, Box GY, Mississippi State, MS 39762, USA Received 18 September 2000; accepted 27 September 2000

Abstract Surface interfacial physiology is particularly important to unicellular organisms with regard to maintenance of optimal cell function. Bacterial cell surfaces possess net negative electrostatic charge by virtue of ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules which are exposed to the extracellular environment. The degree of peripheral electronegativity influences overall cell surface polarity and can be assessed on the basis of zeta potential which is most often determined by estimating the electrophoretic mobility of cells in an electric field. The purpose of this review is to provide bacteriologists with assistance as they seek to better understand available instrumentation and fundamental principles concerning the estimation of zeta potential as it relates to bacterial surface physiology.  2001 Elsevier Science B.V. All rights reserved. Keywords: Bacterial cell surface charge; Zeta potential; Electrophoretic light scattering

1. Introduction

1.1. Electrostatic properties of the bacterial cell surface Bacteria are unicellular and lack intracellular membranous compartmentalization; therefore, the interface formed between the outer cell envelope and the extracellular environment plays a particularly important role in their overall physiology. The outer cell surface mediates exchange and adhesive pro-

q

Dr. Ronald Doyle is the sponsor of this invited review. *Corresponding author. Tel.: 11-662-325-7595; fax: 11-662325-7939. E-mail address: [email protected] (F.R. Champlin).

cesses, while also influencing interactions with immunological factors and participating in cell growth and division. The ultrastructural components of the surface are comprised of macromolecules containing carboxylate, phosphate, and amino functions which are ionized as a function of the environmental pH, thereby conferring electrostatic charge to the cell periphery (Mozes and Rouxhet, 1990). Bacteria exist in aqueous environments and must accordingly possess hydrated surfaces in order to transport nutrient and waste molecules (Beveridge and Graham, 1991). Surface physicochemical parameters such as electrostatic charge are then fundamentally important with regard to influencing overall polarity in order to confer and maintain the degree of surface hydrophilicity necessary for optimal cell function. Net cell surface charge can be assessed on the basis of zeta

0167-7012 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 00 )00224-4

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potential which is the electrical potential of the interfacial region between the bacterial surface and the aqueous environment (Saito et al., 1997). Zeta potential can be estimated by measuring cellular electrophoretic mobility in an electric field (Mozes and Rouxhet, 1990). Bacterial cells possess a net negative electrostatic surface charge when cultivated at physiological pH values (Mozes and Rouxhet, 1990). Competition between counter ion neutralization and molecular motion results in the establishment of an interfacial electrical double layer. The inner region is referred to as the Stern layer and consists of the surface proper, as well as the ions with which it is electrostatically bound. The outer region protrudes into the aqueous environment and consists of a more diffuse distribution of anions and cations which participate in electrostatic interactions between the cell and other charged surfaces. The zeta potential approximates the potential of the inner portion of the diffuse layer. Molecules comprising the outer cell envelope which contribute to the net electronegativity of the overall bacterial cell surface are structurally disparate and differ somewhat as a function of Gram reactivity. The peptidoglycan cell wall of gram-positive bacteria influences surface electronegativity by virtue of phosphoryl groups located in the substituent teichoic and teichuronic acid residues, as well as unsubstituted carboxylate groups (Beveridge, 1988). In contrast, the peptidoglycan of gram-negative bacteria is sequestered within the periplasmic space by virtue of the outer membrane and is therefore not exposed to the extracellular environment. Negative electrostatic surface charge in these organisms is conferred by the phosphoryl and 2-keto-3-deoxyoctonate carboxylate groups of lipopolysaccharide located in the outer leaflet of the outer membrane. Several surface layers found external to the cell walls of certain gram-positive and gram-negative bacteria (Beveridge and Graham, 1991; Sprott et al., 1994) are also known to affect cell surface charge properties at physiological pH. Extracellular polysaccharides are typically acidic in nature and may exist as relatively compact capsules attached to the cell surface, or as diffuse slime layers which are only loosely associated with the cell surface. Layers consist of symmetric paracrystalline arrays visible

only with the aid of electron microscopy. They are composed of divalent cation-stabilized protein or glycoprotein subunits which associate noncovalently in a lateral manner on the cell wall surface in a wide variety of bacteria.

1.2. Need for analytical methods to assess cell surface charge Beveridge and Graham (1991) emphasized the importance of the bacterial cell surface as they summarized its involvement in disparate physiological functions such as envelope diffusion, shape maintenance, growth and division, turgor support, and protection of the underlying protoplast from both chemical and physical insults. That bacteria invest a major portion of their metabolic energy in the synthesis and maintenance of the macromolecular components of the cell surface further supports the notion that interfacial physiology is profoundly important to the well being of the organism. In order to better understand the molecular mechanisms underlying prokaryotic surface physiology, it is essential that accurate and precise analytical means be available to assess the innate physiochemical properties of the outer cell envelope. The electrostatic charge of relatively small particles such as bacterial cells cannot be ascertained directly (Lytle et al., 1999); it is therefore necessary to employ indirect means. This review represents an effort to provide bacteriologists with a readily accessible collection of information designed to assist them in regard to better understanding the available instrumentation, as well as the fundamental principles underlying measurable electrostatic charge as it relates to bacterial surface physiology.

2. Primary existing methodologies

2.1. Microelectrophoresis A variety of methods has been used in the past to characterize bacterial cell surfaces with regard to overall electrostatic properties. Assessment of cell surface charge has been of interest to bacteriologists since at least 1924, and various analytical methods have subsequently evolved. Microelectrophoresis is

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one such method and involves the placement of a cell suspension in an electrophoresis cell, application of voltage across the cell, and direct microscopic observation of the movement of individual bacteria over a given distance, the velocity of which is then used to calculate electrophoretic mobility (Moyer, 1936a; Brinton and Lauffer, 1959). Electrophoretic mobility can be used to ascertain zeta potential values from which cell surface charge can be estimated by calculation. The direction and rate of the movement is dependent on a variety of factors such as ionic strength, temperature, and pH of the medium, as well as electric field strength and the net surface charge of the bacterium. Several microelectrophoresis apparatuses have been used ranging from very early models such as the Abramson microelectrophoresis instrument described in detail by Moyer (1936a) to more modern instrumentation such as the Zeta Meter (Zeta Meter, New York, NY, USA), FACE Zeta-Potential Meter ZPOM (Kyowa Interface Science, Tokyo, Japan), Rank Microelectrophoresis System (MKII, Rank Bros., Cambridge, UK), and Lazer Zee Meter 501 (Pen Kem, Bedford Hills, NY, USA) and S3000 (Pen Kem). All have certain features in common including an observation chamber located between two electrodes and a microscope for direct viewing of particle movement, as well as a means for delivery of the sample (Brinton and Lauffer, 1959). Microelectrophoresis has been used to assess bacterial cell surface charge in many investigations (Buggs and Green, 1935; Moyer, 1936a,b; Dyar and Ordal, 1945; Einolf and Carstensen, 1967; Reynolds and Wong, 1983; Mozes et al., 1987, 1988; Satou et al., 1988; Weerkamp et al., 1988; van der Mei et al., 1988a,b, 1991, 1993, 1995, 1997; Kambara et al., 1989; Miyake et al., 1989; Uyen et al., 1989; Bayer and Sloyer, 1990; Chang and Hsieh, 1991; Gilbert et al., 1991; Vitaya and Toda, 1991; Collins and Stotzky, 1992; Cowan et al., 1992; Harkes et al., 1992a; Devasia et al., 1993; Granlund-Edstedt et al., 1993; Nicholov et al., 1993; Mangia et al., 1995; van Raamsdonk et al., 1995; Busscher et al., 1997; Chen and Koopman, 1997; Millsap et al., 1997; Nosanchuk and Casadevall, 1997; Saito et al., 1997; Groenink et al., 1998; Honda et al., 1998; Kiely and Olson, 1998; Baldi et al., 1999; Campanha et al., 1999). For example, Moyer (1936b) found electro-

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phoretic mobility of rough and smooth phenotypes of Escherichia coli to continually decrease during the lag and early exponential phases of growth. Buggs and Green (1935) explored the possibility that electrophoretic mobility assessment might prove useful for distinguishing between virulent and avirulent strains of Corynebacterium diphtheriae. More recently, the role cell surface charge might play with regard to bacterial adherence has been investigated (Reynolds and Wong, 1983; Mozes et al., 1987; Satou et al., 1988; Weerkamp et al., 1988; van der Mei et al., 1988b, 1993, 1995, 1997; Miyake et al., 1989; Uyen et al., 1989; Chang and Hsieh, 1991; Gilbert et al., 1991; Vitaya and Toda, 1991; Cowan et al., 1992; Devasia et al., 1993; Granlund-Edstedt et al., 1993; Nicholov et al., 1993; Busscher et al., 1997; Chen and Koopman, 1997; Millsap et al., 1997; Saito et al., 1997; Groenink et al., 1998; Baldi et al., 1999; Campanha et al., 1999). For example, Satou et al. (1988) published work suggesting that cell surface charge influences the binding of Streptococcus mutans to various restorative materials used in dentistry. In contrast, cell surface charge was not found to effect the binding of Staphylococcus aureus to HeLa cells (Miyake et al., 1989). Busscher et al. (1997) reported that binding to negatively-charged silicone rubber is decreased in certain streptococcal and staphylococcal strains exhibiting marked negative zeta potentials and known to colonize devices used in voice restoration. Although microelectrophoresis has proven an effective method for determining zeta potentials of bacterial cells, disadvantages exist in that tracking individual cells over time can be laborious and time consuming (Pedersen, 1981). For this reason, less cumbersome methods have been developed and used for assessing charge properties.

2.2. Electrostatic interaction chromatography The desire to develop novel, less laborious methods for the characterization of cell surface charge resulted in the development of electrostatic interaction chromatography (ESIC). ESIC was originally employed as a method for isolation of microorganisms (Wood, 1980), but has since been applied to a variety of aspects of microbial physiology (Pedersen, 1981; Kabir and Ali, 1983; Dickson and

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Koohmaraie, 1989; Wong et al., 1989; Wilcox and Knox, 1990; Gannon et al., 1991; Harkes et al., 1992b; Flint et al., 1997; Sklodowska and Matlakowska, 1998;). ESIC was first employed as a method for assessing relative cell surface charge in bacteria by Pedersen (1981). This method involves the use of either positively or negatively charged ion-exchange resins with which the bacteria interact depending on their relative cell surface charge. Anion-exchange resins such as Dowex 1 3 8 or DEAE-Sepharose CL-6B and the cation-exchange resins such as Dowex 50 W 3 8 or CM-Sepharose CL-6B are typically employed. Relative cell surface charge of the bacteria is reflected in the affinity of cells for the resin as judged on the basis of the relative retention to elution ratio. Additionally, a hydroxyapatite adherence assay has been employed by other workers as an alternative to conventional ESIC for the purpose of assessing surface charge properties in bacteria (Lachica and Zink, 1984; Takahashi et al., 1993). ESIC has been used to characterize several bacterial species with regard to their respective surface electrostatic properties (Pedersen, 1981; Kabir and Ali, 1983; Wong et al., 1989; Harkes et al., 1992b). It has also been exploited as a means to assess cell surface charge as it relates to bacterial adhesion (Dickson and Koohmaraie, 1989; Wilcox and Knox, 1990; Flint et al., 1997; Sklodowska and Matlakowska, 1998). For example, Dickson and Koohmaraie (1989) reported that attachment to both lean muscle and fat tissue increased as negative charge increased for several bacterial organisms. In contrast, Flint et al. (1997) were unable to demonstrate a positive correlation between bacterial attachment to stainless steel and cell surface charge in thermophilic streptococci known to form biofilms on certain surfaces in dairy manufacturing plants. Finally, Gannon et al. (1991) reported movement of several bacteria through soil was not influenced by their respective cell surface charges. This method has proved to be relatively simple (Pedersen, 1981) as well as efficacious with regard to such considerations as the availability of a variety of different resins having large amounts of surface area which, due to their spherical shape, can be housed within small column volumes (Wood, 1980). However, disadvantages do exist in that we have

found this method to be time consuming given the more rapid alternatives available today, and it does not yield data which can be used to calculate zeta potentials.

2.3. Aqueous two-phase partitioning Biphasic partitioning is a method that has also been employed to characterize surface charge properties of bacterial cells (Magnusson et al., 1977; Stendahl et al., 1977; Liang et al., 1993). In this procedure, a polyethylene glycol (PEG)-dextran twophase system is established, cells are allowed to partition between the two phases, and the distribution of bacteria between the two phases is determined. Cells will preferentially partition into either the more hydrophobic PEG phase or the less hydrophobic dextran layer depending on their surface polarity as influenced by cell surface charge. Stendahl et al. (1977) used this method to reveal a paucity of electrostatic charge on smooth Salmonella typhimurium strains, whereas rough mutants were found to be more negatively charged. Liang et al. (1993) found cell surface charge to not be involved in the binding of S. aureus strain V8 to various extracellular matrix and serum proteins using this method. Although less laborious than most microelectrophoresis methods, biphasic partitioning does not yield data which can be used to calculate zeta potential values.

2.4. Isoelectric equilibrium analysis Sherbet et al. (1972) modified a method of isoelectric focusing for use as an isoelectric equilibrium analysis protocol for the purpose of measuring electronegative charge on the surfaces of intact cells. This involved the generation of a pH gradient stabilized by linear gradients of either sucrose, glycerol, or Ficoll in a column equipped with platinum electrodes. After addition of ampholines and formation of a pH gradient, cells were loaded onto the column at a specified density, appropriate voltage was applied to maintain the desired current flow, and cells were allowed to migrate to their isoelectric positions within 24 h. Isoelectric equilibrium analysis subsequently allowed Sherbet and

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Lakshmi (1973) to characterize the E. coli cell surface with regard to the ionizable groups present and the physical location of the phospholipid fraction of the outer membrane. This method has seldom been used to assess cell surface charge of bacterial cells, perhaps due to the length of time necessary to generate results. However, the resultant electrophoretic mobility data may be used to derive zeta potential values.

2.5. Electrophoretic light scattering More recent work pertaining to the characterization of bacterial cell surface charge properties has seen the introduction of electrophoretic light scattering (ELS), a technology which has proven to be of substantial value within the context of a variety of physiological applications (van Loosdrecht et al., 1987; de Weger et al., 1989; Vanhaecke et al., 1990; Blake et al., 1994; Morris et al., 1995; Jones et al., 1996; Jones et al., 1997; Swanson et al., 1997; Bengoechea et al., 1998; Li and McLandsborough, 1999; Lytle et al., 1999). The velocity of particles moving in an electric field is directly measured by determining the frequency change of the laser light they scatter, thereby yielding their electrophoretic mobility (Blake et al., 1994). ELS has proven to be a very rapid and relatively easy method for estimating zeta potentials, and therefore, for the purpose of this review, we focus on its pertinent theoretical aspects and experimental methodologies related to the study of bacterial cells.

3. Electrophoretic light scattering as applied to bacterial systems

3.1. Theoretical aspects of electrophoretic light scattering The ELS experiment was first performed by Ware and Flygare (1971,1972). Thorough descriptions of the theory and experimental apparatus have been presented in review articles by Uzgiris (1931), Ware (1974), and Haas and Ware (1976). We consider here the appropriate background and working equations necessary to guide the reader toward practical applications.

Fig. 1. Schematic representations of electrophoretic light scattering fundamentals. (a) Electrophoretic light scattering cell containing a bacterial suspension. (b) Origin of the scattering vector © (K ).

Consider a system comprised of particles, such as bacteria, suspended in a liquid medium as depicted in Fig. 1a. Each particle experiences two types of motion. The first, with electric field off, is a random (Brownian) motion that allows the particles to wander into and out of the incident light beam. As a particle traverses the light beam, it scatters light which can be detected by a photodetector at scattering angle u. The intensity of the scattered light randomly fluctuates about an average value due to the random motion of the particles, which in turn creates a fluctuating detector signal. If spectral analysis is performed on the photodetector signal, a frequency spectrum, S(v ), is obtained with the Lorentzian form (Ware and Flygare, 1971)

G S(v ) ~ ]]] 2 G 1v2

(1)

S(v ) represents the amplitude of the spectrum as a function of the angular frequency, v. Fig. 2 shows a sketch of the function and indicates that the spectrum is centered about zero frequency and has a half-width at half-height (HWHH) represented by G. The magnitude of G depends on scattering particle motion via the relationship

G 5 DK 2

(2)

where D is the translational diffusion coefficient of

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G S(v ) ~ ]]]]]] © © 2 G 1 (v 1K ? v d )2

Fig. 2. Frequency spectrum of scattered light intensity with the electric field off and on.

the particle and K is called the scattering vector with magnitude 4p n u uKu 5 ]] sin ] l0 2

(3)

In Eq. (3), n is the refractive index of the scattering medium (usually an aqueous solution for bacterial cells), l0 is the wavelength of the incident light in vacuum and u is the scattering angle. The scattering vector is defined as ©

©

(5)

The electrophoretic frequency spectrum is also a Lorentzian function (Fig. 2), but the spectrum has been shifted away from zero frequency by an amount © © Dv 5 K ? v d . Referring to Fig. 1b, the frequency shift is given by © © u Dv 5K ? v d 5 Kvd cos ] (6) 2 Thus, the magnitude of the particle drift velocity due to directed motion in an electric field can be determined by measuring the frequency shift of the frequency spectrum. Using v 5 2py, we have 2p Dy vd 5 ]]] K cos u / 2

(7)

For a typical frequency shift of 50 Hz with uKu5 3.45310 4 cm 21 and u 5158, the drift velocity is calculated to be 92 ms 21 .

3.2. Relationship to electrophoretic mobility

©

K 5k 0 2k s

(4)

and is pictorially represented in Fig. 1b. In this © diagram, k 0 is the wave vector of the incident light © and k s is the wave vector of the scattered light at angle u. For example, using a helium–neon laser with l0 5633 nm and detecting the scattered light at u 5158 in aqueous solution (n51.33) gives uKu5 3.45310 4 cm 21 . Thus, for a given experimental configuration, the spectral broadening given by G is determined by the translational diffusion coefficient, D. Small, fast moving particles result in a broad spectrum whereas large, slow moving particles give a more narrow spectrum. When an electric field is applied to the solution, there will be a directed motion of the particles superimposed on their Brownian motion. In Fig. 1a the case is shown for a particle, such as a bacterial cell, with a net negative charge. With the electric field turned on, the particle will migrate towards the positive electrode. The direction and magnitude©of this motion is given by a drift velocity vector, v d . In this case, the photodetector frequency spectrum becomes (Ware and Flygare, 1971)

The magnitude of the drift velocity is proportional to the electric field strength vd ~ E (8) and the constant of proportionality is defined to be the electrophoretic mobility, m, of the particle. vd 5 m 3 E

(9)

Using Eq. (7), we find that the mobility can be calculated to be 2p Dy m 5 ]]]] (10) KE cos u / 2 Using the parameter values from above, a measured frequency shift of 50 Hz with an electric field of 25 V/ cm gives m 53.7310 24 cm 2 / V s. Electrophoretic mobilities in the range of 1310 24 to 43 10 24 cm 2 / Vs are typical for macromolecules, colloidal particles, and bacterial cells.

3.3. Electrophoretic mobility related to zeta potential The zeta potential is the electric potential of a charged particle at the plane of shear. Fig. 3 illus-

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159

where h is the viscosity of the medium, ´0 is the permittivity of vacuum and ´ is the dielectric constant of the medium. For example, if the electrophoretic mobility in water is determined to be 1.0310 24 cm 2 / V s at 258C, then using ´0 58.845310 27 g cm / V 2 s 2 , ´ 5 78.54, and h 50.0089 Poise (g / cm s) gives z 512.8 mV. The Smoluchowski equation is generally accepted for large particles with k r values larger than about five (Henry, 1931) where 1 /k is the Debye length, the distance from the surface to the bulk solution, and r is the particle radius. For bacteria and viruses, the size is such that the Smoluchowski approximation is valid for most cases.

3.4. Instrumentation

Fig. 3. Cross-section representation depicting the various solvent layers surrounding a bacterial cell.

trates the various layers that are often referred to in describing particles in solution. The zeta potential is shown as being somewhere close to the shear plane, at the boundary of the diffuse layer. The zeta potential represents the ‘‘effective location’’ of the solid–liquid interface (Hunter, 1993a). Hunter explains that the exact position of these planes is still a subject of debate. Suffice it to say that a charged molecule in motion produces an electric field. The shear plane, or the plane of slip, is the distance from the surface to the distance in solution where the solvent molecules are not bound to the surface and are not moving as a unit with the particle. At this boundary, the zeta potential can be determined and a charge density can be inferred (Adamson, 1982). As shown in the previous section, the frequency shift is measured experimentally, and the electrophoretic mobility is calculated from the frequency shift. The zeta potential is then calculated from the electrophoretic mobility. The model dependent relation most often used for large particles is the Smoluchowski equation (Hunter, 1993b): hm z 5] (11) ´0 ´

The instrumentation utilized for zeta potential measurements has various geometries, laser powers, scattering angles, etc. However, the basic configurations are the same. A typical instrument layout is shown in Fig. 4. The incident laser beam (10–100 mW) is split into two beams, typically referred to as the main beam and the reference beam. The main beam is the one from which particle scattering is detected while the reference beam, after attenuation, passes directly to the detector. Fig. 4 shows that the main beam is frequency modulated, typically by 250–1000 Hz. The modulation allows the sign of the electrophoretic mobility to be determined and also helps to reduce low frequency noise. The two beams then pass through a lens that

Fig. 4. Schematic representation of an electrophoretic light scattering unit with the main beam frequency modulated.

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focuses and converges them at the center of the scattering cell. The scattered light and the reference beam pass through an aperture and are focused onto the surface of a photodetector which is mounted at a fixed scattering angle. The detectors used range from photodiodes to photomultipliers to avalanche photodiodes. The method of mixing the scattered light with reference beam light on the detector surface is usually referred to as heterodyne detection. For the case depicted in Fig. 4, the frequency of the scattered light differs from the frequency of the reference beam by an amount equal to the modulation frequency plus the frequency shift, Dy, due to particle motion. Spectral analysis of the photodetector signal allows an accurate determination of Dy with subsequent determination of m via Eq. (10). Instruments available to determine electrophoretic mobility based on scattering of laser light include the DELSA 440SX (Beckman Coulter, Inc., Hialeah, FL, USA), the ZetaPlus and ZetaPals (Brookhaven Instrument Corp., Holsville, NY, USA), various Zetasizer apparatuses (Malvern Instruments, Inc., Southborough, MA, USA) and the NICOMP 380 / 2 (Particle sizing systems, Santa Barbara, CA, USA).

4. Utility of electrophoretic light scattering for bacterial systems

4.1. Recent applications Many of the more recent applications of ELS in the field of bacterial physiology have involved investigating the role cell surface charge plays in bacterial adhesion to disparate solid surfaces (van Loosdrecht et al., 1987; de Weger et al., 1989; Vanhaecke et al., 1990; Jones et al., 1997; Li and McLandsborough, 1999). van Loosdrecht et al. (1987) found cell surface charge influences binding of bacterial cells to negatively-charged polystyrene, while Li and McLandsborough (1999) reported no electrostatic influence on binding of E. coli to beef muscle. Vanhaecke et al. (1990) reported the effect of cell surface charge on binding of several Pseudomonas aeruginosa strains to stainless steel was negligible. Recent investigations in our own laboratories have benefited from the use of ELS technology as we

sought to obtain a better understanding of outer cell envelope physiology in pathogenic gram-negative bacteria. Hart and Champlin (1988) speculated that Pasteurella multocida, an organism otherwise susceptible to lipophilic molecules due to its atypically permeable outer membrane, exhibits intrinsic resistance to the lipopeptide daptomycin due to electrostatic repulsion between the polyanionic antibiotic and the negatively-charged outer membrane surface. This notion was tested by Morris et al. (1995) as they attempted to sensitize P. aeruginosa, E. coli, and P. multocida to daptomycin with the use of the polycationic outer membrane permeabilizer polymyxin B nonapeptide (PMBN). They hypothesized that PMBN would act to mitigate the electronegativity of the cell surface in the process of disorganizing the outer membrane, thereby allowing the antibiotic to associate with the cell surface and subsequently, to traverse the outer membrane. Net cell surface electrostatic charge properties were determined by measuring the electrophoretic mobility of control and PMBN-treated cells in terms of zeta potential as assessed on the basis of light scattering using a DELSA 440SX (Beckman Coulter). Results from this study suggested that sensitization to daptomycin by growth in the presence of PMBN is in fact dependent on diminution of overall cell surface electronegativity, thereby rendering the outer membrane permeable to the polyanionic antibiotic. More recently, we have employed similar technology using a Zetasizer 3000 (Malvern Instruments) to investigate the influence of hyaluronic acid capsulation on the cell surface physiology of serotype A P. multocida strains within the context of overall surface physiology and susceptibility to phagocytosis (unpublished data). Capsulated strains were found to possess cell surfaces which are approximately twice as electronegative as those of naturally-occurring noncapsulated strains. Experimental decapsulation of capsulated strains using either hyaluronidase or dissociation by serial subculturing resulted in concomitant decreases in surface electronegativity to levels comparable to those of noncapsulated strains. Partial removal of capsular material using mechanical shearing reduced surface electronegativity significantly, but to a lesser degree. Naturally-occurring noncapsulated strains were found to be readily phagocytized

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by murine peritoneal phagocytes, while capsulated and experimentally decapsulated strains were not. It was concluded that while serotype A capsulation and the inherent electronegativity it confers to the bacterial cell surface preclude association with the surfaces of phagocytic cells, one or more additional factor(s) present on the surface of capsulated strains, yet absent on the surface of naturally-occurring noncapsulated strains, act to facilitate phagocytic evasion.

4.2. Representative experiment The electrostatic charge properties of the bacterial cell surface can be expected to change as a function of environmental pH fluctuation and recent studies have employed ELS analysis in efforts to better understand this relationship (Li and McLandsborough, 1999; Lytle et al., 1999). Moreover, previous work from our laboratory has established that serotype A capsular material, which is composed largely of hyaluronic acid, confers both enhanced hydrophilic (Thies and Champlin, 1989) and electronegative (unpublished data) properties to the otherwise relatively hydrophobic cell surface of P. multocida. In order to better understand the influence of serotype A capsulation on peripheral electrostatic charge as it relates to cell surface physiology, zeta potential values were determined for a capsulated parental strain of P. multocida (ATCC 11039) and a noncapsulated derivative strain (11039 / iso) obtained by in vitro serial subculturing of strain ATCC 11039 (Champlin et al., 1999). Zeta potential measurements were obtained by suspending capsulated and noncapsulated cells to a concentration of approximately 10 8 cfu / ml in potassium phosphate magnesium sulfate buffer (PPMS; 6.97 g of K 2 HPO 4 , 2.99 g KH 2 PO 4 , and 0.2 g of MgSO 4 ?7H 2 O per liter of deionized, glass-distilled water) at pH values ranging from 2.0 to 9.0 (adjustment with 1.0 M HCl or 5.0 M KOH). Zeta potential values were determined for each sample using a Doppler Electrophoretic Light Scattering Analyzer (DELSA 440SX; Beckman Coulter). As can be seen in Fig. 5, cell surface electronegativity increases concomitantly with increasing pH to values of about 5.6 to 6.0 for both the capsulated and noncapsulated strains, after which no

Fig. 5. Zeta potentials of capsulated parental P. multocida ATCC 11039 (s) and noncapsulated derivative P. multocida ATCC 11039 / iso (d) as a function of pH in PPMS buffer. Each value represents the mean of data obtained from three independent cultures.

further effect on surface charge can be seen. Regardless of pH, however, the cell surface of strain ATCC 11039 exhibits a lower isoelectric point and is more electronegative over the entire pH range than that of the noncapsulated derivative strain 11039 / iso. These data strongly support the notion that serotype A hyaluronic acid extracellular polysaccharide confers a degree of hydrophilicity to the cell due at least in part to its electrostatic properties owing to its acidic nature.

5. Conclusion The determination of surface charge properties of bacterial cells is of paramount importance for modeling cell function and behavior in various environmental conditions. Although no method exists for directly determining surface charge, an indirect determination of the zeta potential has proven to be a useful alternative. Of the various analytical / physical methods that have been developed for estimating zeta potential, the technique of electrophoretic light scattering offers distinct advantages with regard to

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accuracy, measurement time, and ease of use. Zeta potentials can be reproducibly measured on a single bacterial cell sample of about 1.0 ml to within 65% in no longer than about 5 min using technician level personnel.

Acknowledgements We gratefully acknowledge partial financial support for this work by NASA grant NAG8-1566. The assistance of Mr. Joe Fanguy in the preparation of the illustrations is also appreciated.

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