Using a multi-faceted approach to determine the changes in bacterial cell surface properties influenced by a biofilm lifestyle

Biofilm formation is a developmental process in which initial reversible adhesion is governed by physico-chemical forces, whilst irreversible adhesion is mediated by biological changes within a cell, such as the production of extracellular polymeric substances. Using two bacteria, E. coli MG1655 and B. cereus ATCC 10987, this study establishes that the surface of the bacterial cell also undergoes specific modifications, which result in biofilm formation and maintenance. Using various surface characterisation techniques and proteomics, an increase in the surface exposed proteins on E. coli cells during biofilm formation was demonstrated, along with an increase in hydrophobicity and a decrease in surface charge. For B. cereus, an increase in the surface polysaccharides during biofilm formation was found as well as a decrease in hydrophobicity and surface charge. This work therefore shows that surface modifications during biofilm formation occur and understanding these specific changes may lead to the formulation of effective biofilm control strategies in the future.     

AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7

The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper. Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-) as a redox couple. AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface. The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion. The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit. It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements. Immunoreaction with E. coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E. coli antibodies as evidenced by AFM imaging and impedance spectroscopy. It was found that the binding of E. coli cells mainly affected the electron transfer resistance and Warburg impedance.     

A patch coating method for preparing biocatalytic films of Escherichia coli

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.     

Microcapsule modification with peroxidase-catalyzed phenol polymerization

A biocatalytic polymer synthesis on a surface of polyelectrolyte microcapsules was studied. Horseradish peroxidase assembled in nanoorganized capsule walls by alternate adsorption with linear polyions retains its activity in reactions of enzyme-catalyzed polymerization of 4-oxyphenols. It allowed controllable synthesis of a phenolic polymer layer on microcapsule walls using an outermost surface peroxidase layer as a template. By varying the phenol type, buffer pH, and reaction component concentrations, the phenolic polymer coating of the capsules with a thickness in the range 20-50 nm was formed. The polymeric products are fluorescent, which provided a good opportunity for confocal image analysis of the capsule wall structure and the attached layer. The influence of a phenolic polymer layer on the permeability of the capsule walls was investigated.     

Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms

The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.     

Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter,GalP EPR of spin-labelled lipids

The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.     

Partitioning of host and recombinant cells in aqueous two-phase polymer systems

The separation of host and recombinant Escherichia coli bacterial cells has been studied using the surface-sensitive technique of partitioning in aqueous two-phase polymer systems. Experiments were designed to probe charge-and hydrophobicity-related property differences of antibiotic-resistant recombinant cells and their antibiotic-sensitive hosts. Differential partitioning was observed in both charge-sensitive and non-charge-sensitive phase systems for three host-recombinant cell systems, but the non-charge-related effects appear to have a greater impact on partitioning behavior. This result suggests that plasmid-encoded products related to antibiotic resistance modify the surface hydrophobicity of the E. coli bacterial cell and that these differences can be exploited for cell separation.     

The pH dependence of polymerization and bundling by the essential bacterial cytoskeletal protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)--it stabilizes associations of protofilaments.     

Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Effect of Glutaraldehyde on the Outer Layers of Escherichia coli

S ummary : Sodium lauryl sulphate (SLS) at pH 3 and 8 lysed cell walls of Escherichia coli. Pretreatment with glutaraldehyde at pH 3 and at pH 8 prevented this lysis. SLS induced maximum lysis of E. coli cells at 40°; pretreatment of cells with glutaraldehyde prevented this lysis also. Electrophoretic studies indicated that glutaraldehyde accumu lated on the surface of E. coli cells more rapidly in acid than in alkaline conditions, and that it blocked amino groups on the surface layer of Bacillus subtilis spores. The relationship of these findings to the bactericidal efficiency of glutaraldehyde in acid and alkaline solution is discussed.     

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.     

PROBING THE SURFACE OF LIVING DIATOMS WITH ATOMIC FORCE MICROSCOPY: THE NANOSTRUCTURE AND NANOMECHANICAL PROPERTIES OF THE MUCILAGE LAYER1

Atomic force microscopy (AFM) is used to investigate the topography and material properties of the mucilage layer of live cells of three benthic diatoms, the marine species Crasepdostauros australis E. J. Cox and Nitzschia navis‐varingica Lundholm et Moestrup and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. Contrary to previous studies, we show that this surface mucilage layer displays unique nanostructural features. In C. australis, tapping mode images revealed a soft mucilage layer encasing the silica cell wall, consisting of a smooth flat surface that was interrupted by regions with groove‐like indentations, whereas force measurements revealed the adhesive binding of polymer chains. The elastic responses of these polymer chains, as they were stretched during force measurements, were successfully fitted to the worm‐like chain model, indicating the stretching of mostly single macromolecules from which quantitative information was extracted. In P. viridis, tapping mode images of cells revealed a mucilage layer that had the appearance of densely packed spheres, whereas force measurements exhibited no adhesion. In N. navis‐varingica, tapping mode images of the outer surface of this cell in the girdle region revealed the absence of a mucilage layer, in contrast to the other two species. In addition to these topographic and adhesion studies, the first quantitative measurement of the elastic properties of microalgal extracellular polymeric substance is presented and reveals significant spatial variation in the C. australis and P. viridis mucilage layers. This study highlights the capacity of AFM in elucidating the topography and mechanical properties of hydrated microalgal extracellular polymeric substance on a nanoscale.     

Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling

The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin-labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed.     

The allergenicity of complex cations

A homologous series of eight quaternary ammonium salts (quats) were used as complex cations in a survey of contact hypersensitivity in guinea pigs. Two of the quats tested were found to be strong allergens which was due to stable association with membrane lipids at the surface of epidermal cells. This surface complexation reaction was studied in detail by using a spin-labelled quat of intermediate allergenicity. Electron spin resonance was used to show that stable "ion pairs" are formed between membrane receptor sites and the two strong allergens. Information was obtained on the specificity and kinetics of immunogenic complex formation as well as on the position and orientation of these haptens on epidermal receptor sites in vivo.     

Inhibition of Escherichia coli and Proteus mirabilis adhesion and biofilm formation on medical grade silicone surface

Silicone has been utilized extensively for biomedical devices due to its excellent biocompatibility and biodurability properties. However, its surface is easily colonized by bacteria which will increase the probability of nosocomial infection. In the present work, a hydrophilic antimicrobial carboxymethyl chitosan (CMCS) layer has been grafted on medical grade silicone surface pre-treated with polydopamine (PDA). The increase in hydrophilicity was confirmed from contact angle measurement. Bacterial adhesion tests showed that the PDA-CMCS coating reduced the adhesion of Escherichia coli and Proteus mirabilis by ≥ 90%. The anti-adhesion property was preserved even after the aging of the functionalized surfaces for 21 days in phosphate-buffered saline (PBS), and also after autoclaving at 121°C for 20 min. Both E. coli and P. mirabilis readily form biofilms on the pristine surface under static and flow conditions but with the PDA-CMCS layer, biofilm formation is inhibited. The flow experiments indicated that it is more difficult to inhibit biofilm formation by the highly motile P. mirabilis as compared to E. coli. No significant cytotoxicity of the modified substrates was observed with 3T3 fibroblasts.     

The expression in E. coli of synthetic repeating polymeric genes coding for poly(L-aspartyl-L-phenylalanine)

Two dodecadeoxynucleotides of defined sequence have been synthesised by phosphotriester methodology. They can be polymerised to give a double stranded DNA which codes, when read in the correct phase, for the repeating dipeptide poly(aspartyl-phenylalanine). This polymeric DNA has been cloned in E. coli K12 using as vector a plasmid having a controllable bacterial promoter upstream of the insertion site. Clones containing genes coding for up to 150 repeats of (aspartyl-phenylalanine) have been isolated and characterised. The polymeric inserts appear to be stable over many generations and are expressed in E. coli under the control of the bacterial promoter, to give a polymer of phenylalanine and aspartic acid which may be broken down enzymically to yield aspartyl-phenylalanine.     

Topological distribution of bovine serum albumin in multilamellar and unilamellar liposomes

The topological distribution of bovine serum albumin (BSA) in multilamellar vesicles (MLV) and unilamellar vesicles (ULV) composed of 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DML)/cholesterol (molar ratio, 3:1) was studied by ESR using hydrophobic spin-labelled lecithins and hydrophilic Tempocholine. A spin-labelled BSA was also prepared, characterized and used as a probe. Results with hydrophobic spin-labelled lecithin probes showed no significant phospholipid-albumin interaction, indicating the virtual absence of albumin from the phospholipid bilayer of MLV and ULV. Reduction with L-ascorbic acid showed that MLV contained about 50% and ULV 90% of spin-labelled albumin on the surface. The distribution of Tempocholine in MLV and ULV was similar to that of entrapped BSA. These findings were confirmed by results using liposomes treated with nickel which broadened the ESR spectra of probes on the surface of vesicles. This study and our previous work suggest that the immunoadjuvant effect of liposomes can be mediated by surface antigens and can be maximized by preferential surface distribution as in ULV-associated BSA.     

The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.     

Effect of trace elements on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells

Trace elements have significant effect on the physiology of bacteria. Variation in the concentration of trace elements may affect the expression of virulence by microorganisms. The effect of trace elements on hydrophobicity and adherence of E.coli to uroepithelial cells was studied. Increasing concentrations of Ca2+, Mg2+, Fe3+ and Zn2+ significantly decreased the surface hydrophobicity. Toxic trace elements like Co2+, Cu2+, Mn2+ and Ni2+ did not alter surface hydrophobicity. With regards to adherence of E.coli to uroepithelial cells, only Mg2+ had significant effect. Toxic trace elements decreased the rate of cell adherence. The pathogenic strains of E.coli showed higher surface hydrophobicity and better cell adherence compared to the nonpathogenic strains. There was good correlation between surface hydrophobicity and cell adherence at higher concentrations (0.1 to 0.2mM) of Fe2+ and Zn2+. The results indicated that trace elements can significantly affect surface hydrophobicity and adherence of E.coli to uroepithelial cells. Such effect may have a significant impact on the initial stages of bacterial infection.     

Enzyme-induced Formation of Spheres from Cells and Envelopes of Escherichia coli

Normal and filamentous whole cells and isolated envelopes of Escherichia coli B were exposed to various enzymatic treatments to remove surface layers and to characterize the component(s) conferring rigidity in this organism. Modification of cell rigidity was determined by sphere formation in both whole cells and isolated envelopes. Enzymes capable of converting trypsinized normal or untreated filamentous whole cells and untreated envelopes to spheres included: lysozyme plus ethylenediaminetetraacetic acid, clostridial phospholipase C, and phospholipase D from cabbage. These data suggest that there are at least two components essential for maintenance of cell rigidity in E. coli B. The first is the peptidoglycan (mucopeptide), which is susceptible to lysozyme. The second is a phospholipid which is either covalently linked to the mucopeptide or in close association with it. This phospholipase C-sensitive component is protected more completely in normal than in filamentous whole cells by a protein layer which is easily modified by trypsin treatment to allow enzymatically induced sphere formation to occur.