A monolayer (π,ΔV) study of the ionic properties of alanylphosphatidylglycerol: Effects of pH and ions

1,2-Didodecanoyl-sn-glycero-3-phosphoryl-1'-(3'-O-L-alanyl)-sn-glycerol (Ala-PG) has been synthesized. Its ionic properties have been studied at the air-water interface through film compressions and surface potential measurements as a function of subphase pH and ionic content (NaCl, Na₂MoO₄, CaCl₂). The existence of the polar head in a loop conformation allowing for interactions between phosphate and amino groups is suggested. Ionic properties of Ala-PG clearly depended on subphase ionic strength but no specific interactions between either cations or anions in the subphase and phosphate or amino groups in the film could be detected. Results are interpreted in terms of ion-pair interactions at the interface between these two groups and anions and cations from the subphase. Occurrence of charge separation between these two groups, induced by increasing subphase ionic strength, is postulated. Since the molecular packing appeared independent of the subphase ionic content over a large domain of pH (3–8) and surface pressure (π > 5 dyne/cm) and since the lipid can be considered as zwitterionic or slightly positive below pH 5–6, it is suggested that in the parent bacteria, grown under acidic conditions, Ala-PG could play a role in maintaining the membrane intergrity and in preventing the passive diffusion of protons.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion

Abstract In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physico-chemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereo-chemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucanbinding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods. during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

Morphology of modified regenerated model cellulose II surfaces studied by atomic force microscopy: effect of carboxymethylation and heat treatment

Model cellulose II surfaces with different surface charge have been prepared from carboxymethylated wood pulp. AFM tapping-mode imaging in air showed that the introduction of charged groups into the film does not appreciably change the surface morphology. However, after a mild heat treatment (heating at 105 degrees C for 6 h), an irreversible surface structure change, from near spherical-type aggregates to a fibrillar structure, was observed. This might be attributed to the formation of strong hydrogen bonds in the crystalline region of the films while the amorphous regions shrank upon drying. The suitability of these charged cellulose films for surface forces studies was also investigated. At pH below the pK(a) of the carboxyl groups present in the film, the interaction force could be fit by a van der Waals force interaction. At higher pH, the interaction was of a purely electrostatic nature with no van der Waals component observable due to the swelling of the surfaces.     

Specific and non-specific interactions in bacterial adhesion to solid substrata

Abstract Based on a literature review, a hypothesis is forwarded on the mechanism of initial bacterial adhesion to solid substrata, which accounts both for the role of specific microscopic surface components as well as for the role of non-specific macroscopic surface properties (surface free energy, zeta potential or hydrophobicity). Three distinct regions in the adhesion process are suggested in which at large and intermediate separation distances adhesion is mediated by the macroscopic surface properties as surface free energy and surface charge, respectively. At small separation distances specific short-range interactions can occur, leading to a strong and irreversible bonding, provided the water film present in between the interaction surfaces can be removed. A major role of hydrophobic groups, supposed to be associated with bacterial surface appendages is suggested to be its dehydrating capacity, enabling the removal of the vicinal water film yielding small areas of direct contact between protruberant parts of the cell surface and the substratum.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion.

In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physicochemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereochemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucan-binding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods, during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

Determination of surface charge of some bacteria by colloid titration

The surface charge of bacteria is always negative except below pH 2. The negative charge depends mainly upon phosphate and carboxyl groups. The charge of six kinds of bacteria was determined by colloid titration between pH 2 and 11. When there is amino group on the cell surface, it is positive as ammonium cation between pH 2 and 10, and it combines with the negative groups to neutralize the negative charge. In the presence of formalin, the colloid titration results show that the free amine is blocked by formalin, and the amino group can be estimated by the difference between the two titration results. Above pH 10, the titration results of Escherichia coli and Salmonella typhi in the presence of formalin implied the existence of guanidyl group. The titration results of living bacterial suspensions were quite the same as those of heat-killed ones.     

Comparative molecular biological analysis of the microbial community of the holocene and pleistocene deposits of Posol'skaya Shoal, Lake Baikal

The bacterial diversity was studied in sediment layers of Posol'skaya Shoal station (Southern Baikal) belonging to different periods. A set of primers specific to individual bacterial groups was used to analyze the 16S rRNA gene fragments. The bacterial diversity in the Holocene deposits was found to be higher than in the Pleistocene ones. In the upper sediments, a positive PCR reaction with bacterial primers and with specific cyanobacterial and archaebacterial primers was detected. The following phylogenetic groups were revealed in the microbial community of the surface horizon: green nonsulfur bacteria, delta-proteobacteria, beta-proteobacteria (Nitrospirae), alpha-proteobacteria, acidobacteria, crenarchaeota, euryarchaeota, and groups of uncultured bacteria. From the DNA of the Pleistocene deposits, the PCR product was obtained only with bacterial primers. The representatives of the genus Pseudomonas were most closely related to the sequences obtained (95-97% homology).     

Surface Properties of Bifidobacterial Strains of Human Origin

The adherence of Bifidobacterium strains isolated from infant feces and commercial fermented dairy products to enterocyte-like cells was correlated with the autoagglutination and hemagglutination properties of these organisms. These results allowed us to define two groups: (i) cell-adherent bacteria showing hemagglutination and autoagglutination and (ii) non-cell-adherent, nonhemagglutinating, nonautoagglutinating bacteria. Glass adherence was shown to be nonspecific and was discarded as a criterion for selection of adherent cells. Hydrophobicity appeared to be necessary for adhesion to enterocyte-like cells and autoagglutination. Adhesive strains were highly hydrophobic, and the degree of adherence was slightly dependent on the surface potential. Cells autoagglutinated more when the electrostatic negative charges on the cell surface were shielded by a decrease in the pH from 7 to 2. However, in some strains negative charges at the cell surface were adjuvant to adhesion, thus suggesting that specific chemical interactions occurred. The present results provide a method for preliminary selection of bacteria potentially adherent to epithelial cells by means of autoagglutination.     

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346–2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2–5 times higher than that of planktonic bacteria. Consequently, the proton motive force (Δp) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge‐regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter Δp. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultimately the affect the ATP levels of adhered bacteria. The results indicated that the negatively charged glass surface can result in a two‐unit drop in cell surface pH, whereas adhesion to a positively charged amine surface can result in a two‐unit rise in pH. The working hypothesis indicates that the negatively charged surface should enhance Δp and increase cellular ATP, while the positively charged surface should decrease Δp and decrease ATP, and these results of the hypothesis are directly supported by prior experimental results with both negatively and positively charged surfaces. Overall, these results suggest that the nature of charge on the solid surface can have an impact on the proton motive force and cellular ATP levels. Biotechnol. Bioeng. 2010;105: 965–972. © 2009 Wiley Periodicals, Inc.     

Electrochemically induced disintegration of layer-by-layer-assembled thin films composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer assembly composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared on the surface of a platinum (Pt) film-coated quartz resonator, and an electrochemically induced disintegration of the avidin-ib-PEI assembly was studied using a quartz crystal microbalance. The resonance frequency of a five-bilayer (avidin-ib-PEI)5 film-coated quartz resonator was increased upon application of an electric potential to the Pt layer of the quartz resonator, suggesting that the mass on the quartz resonator was decreased as a result of disintegration of the (avidin-ib-PEI)5 film, due to a pH change in the vicinity of the surface of the Pt-coated quartz resonator. It may be that the (avidin-ib-PEI)5 film assembly was decomposed by acidification of the local pH on the surface of the Pt layer, which in turn was induced through electrolysis of water on Pt, because ib-PEI forms complexes with avidin only in basic media. In pH 9 solution, the (avidin-ib-PEI)5 film was decomposed under the influence of an applied potential of 0.6-1.0 V versus Ag/AgCl. The (avidin-ib-PEI)5 film was decomposed almost completely within a minute in a low concentration buffer (1 mM, pH 9), while the decomposition was slower in 10 and 100 mM buffer solutions at the same pH. The decomposition of the assembly was rapid when the electrode potential was applied in pH 9 solutions, while the response was relatively slow in pH 10 and 11 solutions. All the results are rationalized on the basis of an electrochemically induced acidification of the local environment around the (avidin-ib-PEI)5 film on the Pt layer.     

Wettability and bacteria attachment evaluation of multilayer proteases films for biosensor application

Multilayer films were prepared through a self-assembly technique of proteases. Solutions of pepsin, lysozyme and trypsin at 10⁻⁵ M (pH 6.4, pH 6.4, and pH 7.6, respectively) were used as precursors for film building. The wettability of the film surfaces were estimated by contact angle measurements indicating a higher hydrophobicity to trypsin. This was in agreement to the calculated surface tension components. The patterns of the films were examined using atomic force microscopic images. Surfaces before and after bacteria (Escherichia coli) interactions were also characterized. The results indicate that the hydrophobicity plays a key role in bacterial adhesion and that roughness can be considered as a secondary factor.     

Molecular signatures for the main phyla of photosynthetic bacteria and their subgroups

The bacterial groups corresponding to different photosynthetic prokaryotes are presently identified mainly on the basis of their branching in phylogenetic trees. The availability of genome sequences is enabling identification of many molecular signatures that are specific for different groups of photosynthetic bacteria. Our recent work has identified large numbers of signatures consisting of conserved inserts or deletions (indels) in widely distributed proteins, as well as whole proteins that are specific for various sequenced species/strains from Cyanobacteria, Chlorobi, and Proteobacteria phyla. Based upon these signatures, it is now possible to identify/distinguish bacteria from these phyla of photosynthetic bacteria as well as their major subclades in clear molecular terms. The use of these signatures in conjunction with phylogenomic analyses, summarized here, is leading to a holistic picture concerning the branching order and evolutionary relationships among the above groups of photosynthetic bacteria. Although detailed studies in this regard have not yet been carried on Chloroflexi and Heliobacteriaceae, we have identified some conserved indels that are specific for these groups. Some of the conserved indels for the photosynthetic bacteria are present in photosynthesis-related proteins. These include a 4 aa insert in the pyruvate flavodoxin/ferridoxin oxidoreductase that is specific for the genus Chloroflexus, a 2 aa insert in magnesium chelatase that is uniquely shared by all Cyanobacteria except the deepest branching Clade A (Gloebacterales), a 6 aa insert in an A-type flavoprotein that is specific for various marine unicellular Cyanobacteria, a 2 aa insert in heme oxygenase that is specific for various Prochlorococcus strains/isolates, and 1 aa deletion in the protein protochlorophyllide oxidoreductase that is commonly shared by various Prochlorococcus strains except the deepest branching isolates MIT 9303 and MIT 9313. The identified CSIs are located in the structures of these proteins in surface loops indicating that they may be important in mediating protein–protein interactions. The cellular functions of these conserved indels, or most of the signature proteins are presently unknown, but they provide valuable means for discovering novel properties that are unique to different groups of photosynthetic bacteria.     

Nanotribological characterization of tooth enamel rod affected by surface treatment

Tooth enamel is a hybrid organic–inorganic bionanocomposite comprised predominantly of enamel rods. Understanding the effects of anti-caries treatment on the biomechanical properties of these rods is essential in developing effective caries prevention strategies. Calcium fluoride-like deposits play an important role in caries prevention and their nanotribological properties have a direct effect upon their long-term effectiveness. Accordingly, this study utilizes a variety of techniques, namely nanoindentation, nanoscratch tests, nanowear tests and atomic force microscopy (AFM), to characterize the mechanical and tribological properties of single enamel rods before and after topical fluoride application. The results show that the CaF₂-like deposits formed on the enamel surface following fluoride application increase the coefficient of friction of the enamel rods, but decrease their critical load and nanohardness. As a result, the nanowear depth of the treated enamel surface is around six times higher than that of the native enamel surface under an applied load of 300 μN. Following the removal of the surface deposits, however, the modulus of elasticity and wear depth of the underlying enamel surface are found to be similar to those of the original enamel surface. However, a notable increase in the surface roughness is observed.     

Bacterial biofilm in seawater: cell surface properties of early-attached marine bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6 h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.     

Bacterial Biofilm in Seawater: Cell Surface Properties of Early-attached Marine Bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6?h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.     

Structural Proteins Involved in Emergence of Microbial Aerial Hyphae

Filamentous fungi and filamentous bacteria (i.e., the streptomycetes) belong to different kingdoms that diverged early in evolution. Yet, they adopted similar lifestyles. After a submerged feeding mycelium has been established, hyphae grow into the air and form aerial structures from which (a)sexual spores can develop. These spores are dispersed and can give rise to a new mycelium. Some of the key processes involved in the formation of aerial hyphae by these microbes appear to be very similar. In both cases molecules that lower the surface tension are secreted into the aqueous environment, thereby enabling hyphae to grow into the air. Aerial hyphae are then covered with a hydrophobic film. In fungi, this film is characterized by a mosaic of parallel rodlets, while similar rodlets have also been observed on aerial structures of filamentous bacteria. Although the erection of aerial hyphae in both filamentous fungi and filamentous bacteria is dependent upon (poly)peptides that are structurally unrelated, they can, at least partially, functionally substitute for each other.     

Comparative molecular biological analysis of the microbial community of the Holocene and Pleistocene deposits of Posol’skaya Shoal,Lake Baikal

The bacterial diversity was studied in sediment layers of Posol’skaya Shoal station (Southern Baikal) belonging to different periods. A set of primers specific to individual bacterial groups was used to analyze the 16S rRNA gene fragments. The bacterial diversity in the Holocene deposits was found to be higher than in the Pleistocene ones. In the upper sediments, a positive PCR reaction with bacterial primers and with specific cyanobacterial and archaebacterial primers was detected. The following phylogenetic groups were revealed in the microbial community of the surface horizon: green nonsulfur bacteria, δ-proteobacteria, β-proteobacteria (Nitrospirae), α-proteobacteria, acidobacteria, crenarchaeota, euryarchaeota, and groups of uncultured bacteria. From the DNA of the Pleistocene deposits, the PCR product was obtained only with bacterial primers. The representatives of the genus Pseudomonas were most closely related to the sequences obtained (95–97% homology).     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H⁺-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na⁺- instead of H⁺-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H⁺ transfers to ATP synthases via membrane-associated microcircuits between the H⁺ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.     

Dextran-induced Agglutination of Streptococcus mutans, and Its Potential Role in the Formation of Microbial Dental Plaques

Glucose-grown washed cells of streptococci similar to Streptococcus mutans, which contain cell-bound dextransucrase, have been observed to agglutinate upon the addition of high molecular weight dextran. Low molecular weight dextran or unrelated polysaccharides were ineffective. Agglutination also occurred upon addition of sucrose, which can be converted into dextran, but not with other mono- and disaccharides. Other bacteria, including species capable of synthesizing dextrans, were not observed to exhibit this phenomenon. Cells of S. mutans agglutinated upon addition of dextran over a wide pH range, but maximal sensitivity to dextran occurred at pH 8.5. At this pH, such cells can be used for a simple, specific, and exquisitely sensitive qualitative assay for high molecular weight dextran, for addition of 6 ng of dextran with a molecular weight of 2 x 10(6) (i.e., approximately three molecules per cell) caused detectable agglutination. High concentrations of glucose, levan, and dextran of molecular weight of 2 x 10(4) inhibited the reaction. Fluorescein-labeled cells of S. mutans were observed to adhere to dextran-containing plaques and dextran-treated teeth, suggesting that this phenomenon may be of importance in the formation of streptococcal dental plaques. The mechanism responsible for dextraninduced agglutination appears to involve the affinity of a receptor site, possibly dextransucrase, on the surface of several cells for common dextran molecules.